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33 gesichtete, geschützte Fragmente: Plagiat

[1.] Sj/Fragment 001 01 - Diskussion
Bearbeitet: 30. May 2015, 21:12 Hindemith
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1. INTRODUCTION

1.1. Diabetes Mellitus

Human bodies need to maintain a glucose concentration level in a narrow range (70 - 109 mg/dl or 3.9 - 6.04 mmol/l). If the glucose concentration level is significantly out of the normal range (70 - 110 mg/dl), this person is considered to have hyperglycaemia (140 mg/dl or 7.8 mmol/l after an oral glucose tolerance test, or 100 mg/dl or 5.5 mmol/l after a fasting glucose tolerance test) or hypoglycaemia (less than 40 mg/dl or 2.2 mmol/l). Diabetes mellitus is a disease in the glucose-insulin endocrine metabolic regulatory system, in which the pancreas either does not release insulin or does not properly use insulin to uptake glucose in the plasma (1) (2) which is referred as hyperglycaemia. The consequences are that the body does not metabolize the glucose and builds up hyperglycaemia which eventually damages the regulatory system. Complications of diabetes mellitus include retinopathy, nephropathy, peripheral neuropathy and blindness (3). Diabetes mellitus is one of the worst diseases with respect to size of the affected population. The world wide diabetics affected population is much higher, especially in underdeveloped countries.

Diabetes mellitus is currently classified as type 1 or type 2 diabetes (2). Type 1 diabetes was previously called insulin-dependent diabetes mellitus (IDDM) or juvenile-onset diabetes. It develops when the body’s immune system destroys pancreatic beta cells, the only cells in the body that synthesize the hormone insulin, which regulates blood glucose. This form of diabetes usually strikes children and young adults, although disease onset can occur at any age. Type 1 diabetes may account for 5% to 10% of all diagnosed cases of diabetes. Risk factors for type 1 diabetes include autoimmune, genetic, and environmental factors. Type 2 diabetes is adult onset or non-insulin-dependent diabetes mellitus (NIDDM) as this is due to a deficit in the mass of β cells, reduced insulin secretion (4), and resistance to the action of insulin. The relative contribution and interaction of these defects in the pathogenesis of this disease remains to be clarified (5). About 90% to 95% of all diabetics diagnose type 2 diabetes. Type 2 diabetes is associated with older age, obesity, family history of diabetes, prior history of gestational diabetes, impaired glucose tolerance, physical inactivity, and race/ethnicity. African Americans, Hispanic/Latino Americans, native Americans, some Asian Americans, native Hawaiian, and other Pacific Islanders are at particularly high risk for type 2 diabetes. Type 2 diabetes is increasingly being diagnosed [in children and adolescents.]


1. Topp,B, Promislow,K, deVries,G, Miura,RM, Finegood,DT: A model of beta-cell mass, insulin, and glucose kinetics: pathways to diabetes. J.Theor.Biol. 206:605-619, 2000

2. Bergman,RN, Ider,YZ, Bowden,CR, Cobelli,C: Quantitative estimation of insulin sensitivity. Am.J.Physiol 236:E667-E677, 1979

3. Derouich,M, Boutayeb,A: The effect of physical exercise on the dynamics of glucose and insulin. J.Biomech. 35:911-917, 2002

4. Kloppel,G, Lohr,M, Habich,K, Oberholzer,M, Heitz,PU: Islet pathology and the pathogenesis of type 1 and type 2 diabetes mellitus revisited. Surv.Synth.Pathol.Res. 4:110-125, 1985

5. Cerasi,E: Insulin deficiency and insulin resistance in the pathogenesis of NIDDM: is a divorce possible? Diabetologia 38:992-997, 1995

[Page 1]

CHAPTER 1

Introduction and Physiological Background

1. Diabetes Mellitus

Human bodies need to maintain a glucose concentration level in a narrow range (70 - 109 ml/dl or 3.9 - 6.04 mmol/l). If one’s glucose concentration level is significantly out of the normal range (70 - 110 ml/dl), this person is considered to have a the plasma glucose problem: hyperglycemia (≥140 mg/dl or 7.8 mmol/l after an Oral Glucose Tolerance Test, or ≥100 mg/dl or 5.5 mmol/l after a Fasting Glucose Tolerance Test) or hypoglycemia (less than 40 mg/dl or 2.2 mmol/l) ([89], [96]).

Diabetes mellitus is a disease in the glucose-insulin endocrine metabolic regulatory system, in which the pancreas either does not release insulin or does not properly use insulin to uptake glucose in the plasma ([9], [85]), which is referred as hyperglycemia.

The consequences are that the body does not metabolize the glucose and builds up hyperglycemia which eventually damages the regulatory system. Complications of diabetes mellitus include retinopathy, nephropathy, peripheral neuropathy and blindness ([25]).

Diabetes mellitus is one of the worst diseases with respect to size of the affected population. [...]

[Page 2]

[...] The world wide diabetics population is much higher, especially in underdeveloped countries.

Diabetes mellitus is currently classified as type 1 diabetes or type 2 diabetes ([9], [85]). Type 1 diabetes was previously called insulin-dependent diabetes mellitus (IDDM) or juvenile-onset diabetes. It develops when the body’s immune system destroys pancreatic beta cells, the only cells in the body that make the hormone insulin, which regulates blood glucose. This form of diabetes usually strikes children and young adults, although disease onset can occur at any age. Type 1 diabetes may account for 5% to 10% of all diagnosed cases of diabetes. Risk factors for type 1 diabetes include autoimmune, genetic, and environmental factors. Type 2 diabetes is adult onset or non-insulin-dependent diabetes mellitus (NIDDM) as this is due to a deficit in the mass of β cells, reduced insulin secretion [53], and resistance to the action of insulin [32]. The relative contribution and interaction of these defects in the pathogenesis of this disease remains to be clarified [17]. About 90% to 95% of all diabetics diagnose type 2 diabetes. Type 2 diabetes is associated with older age, obesity, family history of diabetes, prior history of gestational diabetes, impaired glucose tolerance, physical inactivity, and race/ethnicity. African Americans, Hispanic/Latino Americans, Native Americans, some Asian Americans, Native Hawaiian, and other Pacific Islanders are at particularly high risk for type 2 diabetes. Type 2 diabetes is increasingly being diagnosed in children and adolescents ([93]).


[9] R. N. Bergman, D. T. Finegood, S. E. Kahn, The evolution of beta-cell dysfunction and insulin resistance in type 2 diabetes, Eur. J. Clin. Invest., 32 (2002), (Suppl. 3), 35–45.

[10] R. N. Bergman, Y. Z. Ider, C. R. Bowden and C. Cobelli, Quantitative estimation of insulin sensitivity, Am. J. Physiol., 236 (1979), E667–E677.

[17] E. Cerasi, Insulin deficiency and insulin resistance in the pathogenesis of NIDDM: is a dovorce Possible?, Diabetologia 38, 992–997.

[25] M. Derouich, A. Boutayeb, The effect of physical exercise on the dynamics of glucose and insulin, J. Biomechanics, 35 (2002), 911–917.

[32] The report of the Expert Committee on the diagnosis and classification of diabetes mellitus, Diabetes Care, 20 (1997), 1183–1197.

[53] G. Kloppel, M. Lohr, K. Habich, M. Oberholzer and P. U. Heitz, Islet pathology and the pathogenesis of type 1 and type 2 diabetes mellitus revisited, Surv. Synth. Path. Res. 4, 110–125.

[85] B. Topp, K. Promislow, G. De Vries, R. M. Miura and D. T. Finegood, A Model of β-cell mass, insulin, and glucose kinetics: pathways to diabetes, J. Theor. Biol. 206 (2000), 605–619.

[89] http://arbl.cvmbs.colostate.edu/hbooks /pathphys/endocrine/pancreas/index.html

[93] http://www.diabetes.org

[96] http://www.endocrineweb.com/insulin.html

Anmerkungen

Nothing has been marked as a citation.

Sichter
(Graf Isolan), SleepyHollow02

[2.] Sj/Fragment 002 01 - Diskussion
Bearbeitet: 30. May 2015, 21:12 Hindemith
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In addition to type 1 and type 2 diabetes, gestational diabetes is a form of glucose intolerance that is diagnosed in some women during pregnancy (1).

Gestational diabetes occurs more frequently among African Americans, Hispanic/Latino Americans, and native Americans. It is also more common among obese women and women with a family history of diabetes. During pregnancy, gestational diabetes requires treatment to normalize maternal blood glucose levels to avoid complications in the infant. After pregnancy, 5% to 10% of women with gestational diabetes are found to have type 2 diabetes. Women who have had gestational diabetes have a 20% to 50% chance of developing diabetes in the next 5-10 years. Other specific types of diabetes result from specific genetic conditions such as maturity-onset diabetes of youth, surgery, drugs, malnutrition, infections, and other illnesses. Such types of diabetes may account for 1% to 5% of all diagnosed cases of diabetes. The relative contribution and interaction of these defects in the pathogenesis of this disease remains to be clarified (5). Due to the large population of diabetes patients in the world and the big health expenses, many researchers are motivated to study the glucose-insulin endocrine metabolic regulatory system so that we can better understand how the mechanism functions (6;7) , what causes the dysfunctions of the system, how to detect the onset of the either type of diabetes including the so called prediabetes , (2;7) (8) and eventually provide more reasonable, more effective, more efficient and more economic treatments to diabetics.

1.2 Glucose-insulin endocrine metabolic regulatory system

Metabolism is the process of extracting useful energy from chemical bounds. A metabolic pathway is a sequence of enzymatic reactions that take place in order to transfer chemical energy from one form to another. The chemical adenosine-triphosphate (ATP) is a common carrier of energy in a cell. There are two different ways to form ATP by adding one inorganic phosphate group to the adenosine-diphosphate (ADP), or adding two inorganic phosphate groups to the adenosine-monophosphate (AMP). The process of inorganic phosphate group addition is referred to phosphorylation. Due to the fact that the three phosphate groups in ATP carry negative charges, it requires lots of energy to overcome the natural repulsion of like-charged phosphates when additional groups are added to AMP. So considerable amount of energy is released during the hydrolysis of ATP to ADP. In the glucose-insulin endocrine metabolic regulatory system, the two pancreatic endocrine hormones, insulin and glucagon, are the primary dynamic factors that regulate the system. Glucose stimulates insulin secretion from β-cells by activating two pathways that require [metabolism of the sugar: the triggering and the amplifying pathway (9) .]


1. Topp,B, Promislow,K, deVries,G, Miura,RM, Finegood,DT: A model of beta-cell mass, insulin, and glucose kinetics: pathways to diabetes. J.Theor.Biol. 206:605-619, 2000

2. Bergman,RN, Ider,YZ, Bowden,CR, Cobelli,C: Quantitative estimation of insulin sensitivity. Am.J.Physiol 236:E667-E677, 1979

5. Cerasi,E: Insulin deficiency and insulin resistance in the pathogenesis of NIDDM: is a divorce possible? Diabetologia 38:992-997, 1995

6. Porksen,N: The in vivo regulation of pulsatile insulin secretion. Diabetologia 45:3-20, 2002

7. Bergman,RN, Finegood,DT, Kahn,SE: The evolution of beta-cell dysfunction and insulin resistance in type 2 diabetes. Eur.J.Clin.Invest 32 Suppl 3:35-45, 2002

8. Toffolo,G, Bergman,RN, Finegood,DT, Bowden,CR, Cobelli,C: Quantitative estimation of beta cell sensitivity to glucose in the intact organism: a minimal model of insulin kinetics in the dog. Diabetes 29:979-990, 1980

9. Henquin,JC: Triggering and amplifying pathways of regulation of insulin secretion by glucose. Diabetes 49:1751-1760, 2000

In addition to Type 1 and Type 2 diabetes, gestational diabetes is a form of glucose intolerance that is diagnosed in some women during pregnancy ([9], [85], [97]). Gestational diabetes occurs more frequently among African Americans, Hispanic/Latino Americans, and Native Americans. It is also more common among obese women and

[page 3]

women with a family history of diabetes. During pregnancy, gestational diabetes re- quires treatment to normalize maternal blood glucose levels to avoid complications in the infant. After pregnancy, 5% to 10% of women with gestational diabetes are found to have type 2 diabetes. Women who have had gestational diabetes have a 20% to 50% chance of developing diabetes in the next 5-10 years. Other specific types of dia- betes result from specific genetic conditions (such as maturity-onset diabetes of youth), surgery, drugs, malnutrition, infections, and other illnesses. Such types of diabetes may account for 1% to 5% of all diagnosed cases of diabetes ([97]).

The relative contribution and interaction of these defects in the pathogenesis of this disease remains to be clarified ([17]).

Due to the large population of diabetes patients in the world and the big health expenses, many researchers are motivated to study the glucose-insulin endocrine metabolic regulatory system so that we can better understand how the mechanism functions ([79], [84], [85], [67], [74], [31], [85], [4] and their references), what cause the dysfunctions of the system ([9] and its rich references), how to detect the onset of the either type of diabetes including the so called prediabetes ([10], [83], [8], [97], [23], [57], [6], [63] and their references), and eventually provide more reasonable, more effective, more efficient and more economic treatments to diabetics.

[page 4]

2. Glucose-Insulin Endocrine Metabolic Regulatory System

Metabolism is the process of extracting useful energy from chemical bounds. A metabolic pathway is a sequence of enzymatic reactions that take place in order to transfer chemical energy from one form to another. The chemical adenosine triphos-phate (ATP) is a common carrier of energy in a cell. There are two different ways to form ATP:

1. adding one inorganic phosphate group (HPO2− 4 ) to the adenosine diphosphate (ADP), or

2. adding two inorganic phosphate groups to the adenosine monophosphate (AMP).

The process of inorganic phosphate group addition is referred to phosphorylation. Due to the fact that the three phosphate groups in ATP carry negative charges, it requires lots of energy to overcome the natural repulsion of like-charged phosphates when addi- tional groups are added to AMP. So considerable amount of energy is released during the hydrolysis of ATP to ADP ([51], [89] and [91]).

In the glucose-insulin endocrine metabolic regulatory system, the two pancre- atic endocrine hormones, insulin and glucagon, are the primary dynamic factors that regulate the system.

[page 11]

Glucose stimulates insulin secretion from β-cells by activating two pathways that require metabolism of the sugar as follows ([47]).


[9] R. N. Bergman, D. T. Finegood, S. E. Kahn, The evolution of beta- cell dysfunction and insulin resistance in type 2 diabetes, Eur. J. Clin. Invest., 32 (2002), (Suppl. 3), 35–45.

[10] R. N. Bergman, Y. Z. Ider, C. R. Bowden and C. Cobelli, Quantitative estimation of insulin sensitivity, Am. J. Physiol., 236 (1979), E667–E677.

[17] E. Cerasi, Insulin deficiency and insulin resistance in the pathogenesis of NIDDM: is a dovorce Possible?, Diabetologia 38, 992–997.

[47] J. C. Henquin, Triggering and amplifying pathways of regulation of in- sulin secretion by glucose, Diabetes, 49:17511760, 2000.

[83] G. Toffolo, R. N. Bergman, D. T. Finegood, C. R. Bowden, C. Cobelli, Quantitative estimation of beta cell sensitivity to glucose in the intact organism: a minimal model of insulin kinetics in the dog, Diabetes, 29 (1980), No. 12, 979–990.

[85] B. Topp, K. Promislow, G. De Vries, R. M. Miura and D. T. Finegood, A Model of β-cell mass, insulin, and glucose kinetics: pathways to diabetes, J. Theor. Biol. 206 (2000), 605–619.

Anmerkungen

Nothing has been marked as a citation.

Note: not all references to the literature of the source have been documented.

Sichter
(Graf Isolan), (Hindemith), SleepyHollow02

[3.] Sj/Fragment 003 01 - Diskussion
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In the triggering pathway, the facilitative glucose transporter GLUT2 transports the glucose into the β cell. It causes the rise in the ratio of ATP/ADP which leads ATP-sensitive K+ channels (KATP channels) in the plasma membrane to close. The decreased K+ permeability leads to membrane depolarization, opening of voltage-dependent Ca2+ channels, Ca2+ influx, and the eventual rise of the cytosolic Ca2+ concentration ([Ca2+]c) that triggers exocytosis of insulin containing vesicles. This pathway is also called KATP channel-dependent pathway. Please see Figure 1 for an illustration. The amplifying pathway which is a KATP channel-independent pathway, simply increases the efficiency of the Ca2+ on exocytosis when the concentration of Ca2+ has been elevated. Triggering Pathway The GLUT2 transports the glucose into the β cell. It causes the rise in the ratio of ATP/ADP which causes ATP-sensitive K+ channels (KATP channels) in the plasma membrane to close. The decreased K+ permeability leads to membrane depolarization, opening of voltage-dependent Ca2+ channels, Ca2+ influx, and the eventual rise of the cytosolic Ca2+ concentration ([Ca2+]c) that triggers exocytosis. This pathway is also called KATP channel-dependent pathway. See Figure 1.3.2 for an illustration.

Amplifying Pathway The KATP channel-independent pathway simply increases the efficiency of the Ca2+ on exocytosis when the concentration of Ca2+ has been elevated.

Anmerkungen

The two described figures are not identical.

The source is not mentioned.

Sichter
(Hindemith) LieschenMueller

[4.] Sj/Fragment 003 12 - Diskussion
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Figure 1. Pancreatic beta cells secrete insulin when glucose concentration levels are elevated.

The facilitated glucose transporter GLUT2 transports the glucose into the cell where it is phosphorylated by glucokinase. The glucose metabolism causes ATP-sensitive K+ channels to close, the membrane to depolarize and the Ca2+ channels to open. This triggers a cascade of protein phosphorylations leading to insulin exocytosis.

The insulin receptor is a transmembrane glycoprotein that belongs to the large class of tyrosine kinase receptors. Two α subunits and two β subunits make up the insulin receptor. The β subunits pass through the cellular membrane and are linked by disulfide bonds (10) .


10. Ackermann,AM, Gannon,M: Molecular regulation of pancreatic beta-cell mass development, maintenance, and expansion. J.Mol.Endocrinol. 38:193-206, 2007

Figure 1.3.2. The β cells secrete insulin when glucose concentration level elevated

The facilitated GLUT2 transport the glucose into the β cell and the glucose is phosphorylated by glucokinase. The ratio of ATP:ADP is elevated. The glucose metabolism causes ATP-sensitive K+ channels to close, the membrane to depolarize and the Ca2+ channels to open. This triggers a cascade of protein phosphorylations and leads to insulin exocytosis [68]. (The figure is partially adapted from [68].)

[page 13]

3.4. Insulin Receptors. In molecular biology, the insulin receptor is a transmembrane glycoprotein that is activated by insulin. It belongs to the large class of tyrosine kinase receptors. Two α subunits and two β subunits make up the insulin receptor. The β subunits pass through the cellular membrane and are linked by disulfide bonds ([90]).


[68] V. Poitout, An integrated view of β-cell dysfunction in type-II diabetes, Annu. Rev. Med. 1996. 47:6983.

[89] http://arbl.cvmbs.colostate.edu/hbooks/pathphys/endocrine/pancreas/index.html

Anmerkungen

The source is not given.

The two figures with nearly identical captions are in fact quite different.

Sichter
(Hindemith), PlagProf:-)

[5.] Sj/Fragment 004 01 - Diskussion
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The insulin receptors are embedded in the plasma membrane of myocytes and adipocytes (11). The binding of insulin to the receptors is the initial step in a signal transduction pathway, triggering the consumption and metabolism of glucose. Bound by insulin , the insulin receptor phosphorylates several proteins in the cytoplasm, including insulin receptor substrates (IRS-1 and IRS-2) that activate Phosphatidylinositol 3-kinase (PI-3-K) leading to an increase in the facilitative glucose transporters (GLUT4 and GLUT1) molecules in the outer membrane of muscle cells and adipocytes, and therefore to an increase in the uptake of glucose from blood into muscle and adipose tissue. Figure 2 elucidates this signaling pathway (12).

[FIGURE]

Figure 2. Insulin signals cells to utilize glucose. Insulin binds to its receptors on the membrane of the cells and induces phosphorylation of several proteins in the cytoplasm, including insulin receptor substrates (IRS-1 and IRS-2) which activate Phosphatidylinositol 3-kinase (PI-3-K) thereby leading to an increase in glucose transporter (GLUT1 and GLUT4) molecules in the plasma membrane. GLUT1 and GLUT4 transport the glucose into the cells efficiently.

The kinetics of insulin receptor binding is complex. The number of insulin receptors of each cell changes opposite to the circulating insulin concentration level. Increased insulin circulating level reduces the number of insulin receptors per cell and the decreased [circulating level of insulin triggers the number of receptors to increase (13).]


11. Sesti,G: Pathophysiology of insulin resistance. Best.Pract.Res.Clin.Endocrinol.Metab 20:665-679, 2006

12. Wardzala,LJ, Jeanrenaud,B: Potential mechanism of insulin action on glucose transport in the isolated rat diaphragm. Apparent translocation of intracellular transport units to the plasma membrane. J.Biol.Chem. 256:7090-7093, 1981

13. Grunberger,G, Ryan,J, Gorden,P: Sulfonylureas do not affect insulin binding or glycemic control in insulin-dependent diabetics. Diabetes 31:890-896, 1982

The insulin receptors are embedded in the plasma membrane of hepatocytes and myocytes. The binding of insulin to the receptors is the initial step in a signal

[page 14]

transduction pathway, triggering the consumption and metabolism of glucose ([89], [86]). Bound by insulin, the insulin receptor phosphorylates from ATP to several proteins in the cytoplasm, including insulin receptor substrates (IRS-1 and IRS-2) containing signaling molecules, activates Phosphatidylinositol 3-kinase (PI-3-K) and leads to an increase in glucose transporter (GLUT4) molecules ([98]) in the outer membrane of muscle cells and adipocytes, and therefore to an increase in the uptake of glucose from blood into muscle and adipose tissue ([89]). GLUT4 will transport the glucose to the cells efficiently. Figure 1.3.3 elucidates this signaling pathway.

[...]

However, the kinetics of insulin receptor binding are complex. The number of insulin receptors of each cell changes opposite to the circulating insulin concentration level. Increased insulin circulating level reduces the number of insulin receptors per cell and the decreased circulating level of insulin triggers the number of receptors to increase.

[page 15]

[FIGURE]

Figure 1.3.3. Insulin signals cells to utilize glucose

Insulin binds to its receptors on the membrane of the cells and phosphorylates several proteins in the cytoplasm, including insulin receptor substrates (IRS-1 and IRS-2) containing signaling molecules, activates Phosphatidylinositol 3-kinase (PI-3-K) and leads to an increase in glucose transporter (GLUT4) molecules. This leads to an increase in glucose transporter (GLUT4) molecules. GLUT4 will transport the glucose to the cells efficiently.


[86] S.Wanant and M. J. Quon, Insulin Receptor Binding Kinetics: Modeling and Simulation Studies, J. Theor. Bio., 205 (2000), 355-364.

[89] http://arbl.cvmbs.colostate.edu/hbooks/ pathphys/endocrine/pancreas/index.html

[98] E. Y. Skolnik, Insulin receptor signaling pathways, http://www.med.nyu.edu/research/skolne01.html

Anmerkungen

The source is not given.

The two figures are not identical.

Sichter
(Hindemith), LieschenMueller

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The number of receptors is increased during starvation and decreased in obesity and acromegaly (14). The affinity of the receptor for the second insulin molecule is significantly lower than for the first bound molecule. This may explain the negative cooperative interactions observed at high insulin concentrations. That is, as the concentration of insulin increases and more receptors become occupied, the affinity of the receptors for insulin decreases (15). Conversely, at low insulin concentrations, positive cooperation has been recorded. That is, the binding of insulin to its receptor at low insulin concentrations seems to enhance further binding.

The alpha cells in the pancreas release glucagon, a protein hormone that has important effects in the regulation of carbohydrate metabolism. Glucagon mobilizes glucose, fatty acids and amino acids from storage into the blood. When the glucose concentration level in the plasma is low, the liver converts glucagon to glucose. Both insulin and glucagon are important in the regulation of carbohydrate, protein and lipid metabolism.


14. Pav,J, Marek,J, Sramkova,J: [The effect of acromegaly treatment on glucose tolerance]. Cas.Lek.Cesk. 125:1451-1454, 1986

15. Niessen,M, Jaschinski,F, Item,F, McNamara,MP, Spinas,GA, Trub,T: Insulin receptor substrates 1 and 2 but not Shc can activate the insulin receptor independent of insulin and induce proliferation in CHO-IR cells. Exp.Cell Res. 313:805-815, 2007

[page 14]

The number of receptors is increased during starvation and decreased in obesity and acromegaly. But, the receptor affinity is decreased by excess glucocorticoids. The affinity of the receptor for the second insulin molecule is significantly lower than for the first bound molecule. This may explain the negative cooperative interactions observed at high insulin concentrations. That is, as the concentration of insulin increases and more receptors become occupied, the affinity of the receptors for insulin decreases. Conversely, at low insulin concentrations, positive cooperation has been recorded. That

[page 15]

is, the binding of insulin to its receptor at low insulin concentrations seems to enhance further binding (([89]), [86]).

[page 9]

The α cells release glucagon, a protein hormone that has important effects in the regulation of carbohydrate metabolism. Glucagon is a catabolic hormone, that is, it mobilizes glucose, fatty acids and amino acids from storage into the blood. When the glucose concentration level in the plasma is low, the liver will convert the glucagon to glucose.

Both insulin and glucagon are important in the regulation of carbohydrate, protein and lipid metabolism.


[86] S.Wanant and M. J. Quon, Insulin Receptor Binding Kinetics: Modeling and Simulation Studies, J. Theor. Bio., 205 (2000), 355-364.

[89] http://arbl.cvmbs.colostate.edu/hbooks/ pathphys/endocrine/pancreas/index.html

Anmerkungen

The source is not mentioned.

Sichter
(Hindemith), LieschenMueller

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[FIGURE]

Figure 3. Glucose- insulin endocrine metabolic regulatory system. The dashed lines indicate that exercises and fasting consume glucose and lower the glucose concentration, which signals the pancreas to release glucagon in the liver converts glycogen to glucose. The solid lines indicate that the glucose infusion elevates the plasma glucose concentration level which signals the pancreas to secrete insulin and decrease plasma glucose.

[FIGURE]

Figure 1.2.1. Glucose-Insulin Regulatory System

The dashed lines indicate that exercises and fasting consume glucose and lower the glucose concentration, which signals the pancreas to release glucagon and the liver converts the glucagon and glycogen to glucose. The solid lines indicate that the glucose infusion elevate the plasma glucose concentration level which signals the pancreas to secrete insulin and consume the glucose. (This figure is adapted from [96].)


[96] http://www.endocrineweb.com/insulin.html

Anmerkungen

The two figures are similar, but not identical.

Neither the source Li (2004) is mentioned, or the original source [1], from where Li has taken some inspiration.

The figure caption cannot be found in the original source.

Sichter
(Hindemith) LieschenMueller

[8.] Sj/Fragment 006 04 - Diskussion
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Facilitated diffusion of glucose and related hexoses across biological membranes is catalysed by members of the SLC2 family, referred to as glucose transporters or GLUTs. These transporters function as simple carriers and the movement of hexose across the plasma membrane proceeds in the direction imposed by its electrochemical gradient. A common structural feature of the SLC2 family members is the presence of 12 transmembrane domains (TM) with both the amino and carboxy-terminal ends present on the cytosolic side and a unique N-linked oligosaccharide side-chain present either in the first or the fourth extracellular loop (16).

16. Mueckler,M, Makepeace,C: Transmembrane segment 12 of the Glut1 glucose transporter is an outer helix and is not directly involved in the transport mechanism. J.Biol.Chem. 281:36993-36998, 2006

Facilitated diffusion of glucose and related hexoses across biological membranes is catalysed by members of the SLC2 family, referred to as glucose transporters or GLUTs. These transporters function as simple carriers and the movement of hexose across the plasma membrane proceeds in the direction imposed by its electrochemical gradient. A common structural feature of the SLC2 family member is the presence of 12 transmembrane domains (TM) with both the amino and carboxy-terminal ends present on the cytosolic side and a unique N-linked oligosaccharide side-chain present either in the first or the fourth extracellular loop.
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[Signature sequences conserved between the] different members of the SLC2 family are present at distinct locations in the primary structure. The presence of these sequences, however, does not predict the substrate specificity of these transporters.

Glucose transporters are expressed in every cell of the body, as might be anticipated from the key role of glucose in providing metabolic energy and building blocks for the synthesis of biomolecules. The specific physiological role of the isoforms expressed in tissues involved in the control of glucose homeostasis, i.e. muscle, adipose tissue, liver, pancreatic beta-cells and brain, has been studied in greatest detail. Indeed, in these tissues glucose transporters play important roles in the control of glucose utilization, glucose production and glucose sensing and their dysregulated expression may underlie pathogenetic mechanisms leading to development of diabetes mellitus, but also other specific monogenic diseases (17).

Facilitated diffusion of glucose across plasma membranes has been studied for several decades (18). The recognition that human erythrocytes have a high density of glucose transporters allowed the initial biochemical purification of this transporter and the preparation of specific antibodies. These were then used for initial cloning of a human glucose transporter by screening an expression library prepared from a human hepatoma cell line (HepG2) (4).This glucose transporter, named GLUT1 (SLC2A1) , was then used for subsequent cloning, by low-stringency screening, of GLUT2–5 (SLC2A2, SLC2A3, SLC2A4, SLC2A5)


4. Kloppel,G, Lohr,M, Habich,K, Oberholzer,M, Heitz,PU: Islet pathology and the pathogenesis of type 1 and type 2 diabetes mellitus revisited. Surv.Synth.Pathol.Res. 4:110-125, 1985

17. Fernandez,EB: [Monogenic forms of diabetes mellitus]. An.R.Acad.Nac.Med.(Madr.) 123:211-217, 2006

18. Gylfe,E, Grapengiesser,E, Hellman,B: Propagation of cytoplasmic Ca2+ oscillations in clusters of pancreatic beta-cells exposed to glucose. Cell Calcium 12:229-240, 1991

Signature sequences conserved between the different members of the SLC2 family are present at distinct locations in the primary structure (Fig. 2). The presence of these sequences, however, does not predict the substrate specificity of these transporters.

Glucose transporters are expressed in every cell of the body, as might be anticipated from the key role of glucose in providing metabolic energy and building blocks for the synthesis of biomolecules. The specific physiological role of the isoforms expressed in tissues involved in the control of glucose homeostasis, i.e. muscle, adipose tissue, liver, pancreatic beta- cells and brain, has been studied in greatest detail. Indeed, in these tissues glucose transporters play important roles in the control of glucose utilization, glucose production and glucose sensing and their dysregulated expression may underlie pathogenetic mechanisms leading to development of diabetes mellitus, but also other specific monogenic diseases (see below).

Facilitated diffusion of glucose across plasma membranes has been studied for several decades [43]. The recognition that human erythrocytes have a high density of glucose transporters allowed the initial biochemical purification of this transporter and the preparation of specific antibodies. These were then used for initial cloning of a human glucose transporter by screening an expression library prepared from a human hepatoma cell line (HepG2) [53]. This glucose transporter, GLUT1, was then used for subsequent cloning, by low-stringency screening, of GLUT2–5.


43. Lieb WR, Stein WD (1971) New theory for glucose transport across membranes. Nat New Biol 230:108–109

53. Mueckler M, Caruso C, Baldwin SA, Panico M, Blench I, Morris HR, Allard WJ, Lienhard GE, Lodish HF (1985) Sequence and structure of a human glucose transporter. Science 229:941–945

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Class I is comprised of the extensively characterized glucose transporters GLUT1 to GLUT4, which can be distinguished on the basis of their distinct tissue distributions (GLUT1, erythrocytes, brain micro vessels; GLUT2 , liver, pancreatic islets; GLUT3, neuronal cells; GLUT4, muscle, adipose tissue) and their hormonal regulation (e.g., insulin sensitivity of GLUT4) (19). Class II is comprised of the fructose-specific transporter GLUT5 (20) and three related proteins, GLUT7 (SLC1A7), GLUT9 (SLC1A9), and GLUT11 (SLC1A11) (21) For GLUT11, fructose-inhibitable glucose transport activity has been demonstrated in a system of reconstituted vesicles. Class III is characterized by the lack of a glycosylation site in the first extra cellular linker domain and by the presence of such a site in loop 9. HMIT1 (SLC1A13), can be included in the class III GLUTs . Glucose transport activity has been demonstrated for GLUT6 and GLUT8. It should be emphasized, however, that the designation of the family does not necessarily reflect the substrate specificity of its members, which may transport sugars or polyols other than glucose (e.g. GLUT5-fructose, MIT1-myoinositol) (20).

19. Davey,KA, Garlick,PB, Warley,A, Southworth,R: Immunogold labeling study of the distribution of GLUT-1 and GLUT-4 in cardiac tissue following stimulation by insulin or ischemia. Am.J.Physiol Heart Circ.Physiol 292:H2009-H2019, 2007

20. Drozdowski,LA, Woudstra,TD, Wild,GE, Clandinin,MT, Thomson,AB: Ageassociated changes in intestinal fructose uptake are not explained by alterations in the abundance of GLUT5 or GLUT2. J.Nutr.Biochem. 15:630-637, 2004

21. Stuart,CA, Yin,D, Howell,ME, Dykes,RJ, Laffan,JJ, Ferrando,AA: Hexose transporter mRNAs for GLUT4, GLUT5, and GLUT12 predominate in human muscle. Am.J.Physiol Endocrinol.Metab 291:E1067-E1073, 2006

Class I is comprised of the extensively characterized glucose transporters GLUT1 to GLUT4, which can be distinguished on the basis of their distinct tissue distributions (GLUT1, erythrocytes, brain microvessels; GLUT2, liver, pancreatic islets; GLUT3, neuronal cells; GLUT4, muscle, adipose tissue) and their hormonal regulation (e.g., insulin sensitivity of GLUT4). Class II is comprised of the fructose-specific transporter GLUT5 and three related proteins, GLUT7, GLUT9, and GLUT11. For GLUT11, fructose-inhibitable glucose transport activity has been demonstrated in a system of reconstituted vesicles (4). Class III is characterized by the lack of a glycosylation site in the first extracellular linker domain and by the presence of such a site in loop 9.

[page E976]

[...] (HMIT1, Ref. 18) can be included in the class III GLUTs (10). Glucose transport activity has been demonstrated for GLUT6 and GLUT8. It should be emphasized, however, that the designation of the family does not necessarily reflect the substrate specificity of its members, which may transport sugars or polyols other than glucose (e.g., GLUT5, fructose; HMIT1, myoinositol).


4. Doege H, Bocianski A, Scheepers A, Axer H, Eckel J, Joost HG, and Schürmann A. Characterization of the human glucose transporter GLUT11, a novel sugar transport facilitator specifically expressed in heart and skeletal muscle. Biochem J 359: 443–449, 2001.

10. Joost HG and Thorens B. The extended GLUT-family of sugar/polyol transport facilitators—nomenclature, sequence characteristics, and potential function of its novel members. Molec Membr Biol 18: 247–256, 2001.

18. Uldry M, Ibberson M, Riederer B, Chatton JY, Horisberger JD, and Thorens B. Identification of a novel H+-myoinositol symporter expressed predominantly in the brain. EMBO J 20: 4467–4477, 2001.

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1.3.1 The facilitative glucose transporters in the CNS and blood brain barrier

Glucose is the preferred energy substrate of the brain. Due to its expression in the endothelial cells forming the blood brain barrier, the glucose transporter GLUT1 is essential for glucose delivery to the brain (22).Given the fact that the abluminal surface of brain capillaries is covered by specialized astrocytic end-feet that also express GLUT1, the astrocytes probably constitute a major site of glucose uptake (23) In astrocytes, glucose is catabolized by glycolysis to lactate, which may be delivered to neurons through a glialspecific monocarboxylate transporter (MCT1) and a neuron-specific one (MCT2). In neurons, lactate is converted to pyruvate, which enters the tricarboxylic acid cycle to generate ATP. Glucose can also be taken up directly by neurons, which express the GLUT3 isoform (24). GLUT2 is also expressed in the brain in specific regions such as the hypothalamus and the brain stem where it may participate in the mechanisms of glucose sensing involved in the control of glucose homeostasis.

The role of GLUT8 in some specific neurons remains unclear. It is localized to intracellular vesicles and may possibly move to the cell surface upon as yet unidentified stimuli (25) . Finally, HMIT is expressed in astrocytes and in neurons. In astrocytes, HMIT is both intracellular and at the plasma membrane, whereas its subcellular localization in neurons is under investigation (26).

1.3.2 The facilitative glucose transporters as pharmaceutical targets

Elevation of blood glucose is the main symptom of types 1 and 2 diabetes. The GLUT isoforms that transport glucose represent therefore a potential therapeutic target for normalizing glycaemia. A compound that increases the Vmax maximal velocity of GLUT1 would increase whole-body glucose utilization. Given the fact that this isoform is almost ubiquitous, such activation could, however, also lead to severe hypoglycaemia. Another possible site of action for limiting the blood glucose level would be inhibition of glucose absorption in the intestine or reabsorption in the kidney. In the intestine, this could be possible by blocking both GLUT2 and the alternative membrane-traffic-based pathway of basolateral glucose release. In the kidney, GLUT2 deficiency results in glucose excretion in the urine, which decreases glycaemia (27). Inhibition of GLUT2 specifically in the kidney could thus treat hyperglycaemia.


22. Klepper,J, Scheffer,H, Leiendecker,B, Gertsen,E, Binder,S, Leferink,M, Hertzberg,C, Nake,A, Voit,T, Willemsen,MA: Seizure control and acceptance of the ketogenic diet in GLUT1 deficiency syndrome: a 2- to 5-year follow-up of 15 children enrolled prospectively. Neuropediatrics 36:302-308, 2005

23. Belanger,M, Desjardins,P, Chatauret,N, Butterworth,RF: Selectively increased expression of the astrocytic/endothelial glucose transporter protein GLUT1 in acute liver failure. Glia 53:557-562, 2006

24. Pellerin,L, Bonvento,G, Chatton,JY, Pierre,K, Magistretti,PJ: Role of neuron-glia interaction in the regulation of brain glucose utilization. Diabetes Nutr.Metab 15:268-273, 2002

25. Ibberson,M, Riederer,BM, Uldry,M, Guhl,B, Roth,J, Thorens,B: Immunolocalization of GLUTX1 in the testis and to specific brain areas and vasopressin-containing neurons. Endocrinology 143:276-284, 2002

26. Uldry,M, Ibberson,M, Horisberger,JD, Chatton,JY, Riederer,BM, Thorens,B: Identification of a mammalian H(+)-myo-inositol symporter expressed predominantly in the brain. EMBO J. 20:4467-4477, 2001

27. Guillam,MT, Hummler,E, Schaerer,E, Yeh,JI, Birnbaum,MJ, Beermann,F, Schmidt,A, Deriaz,N, Thorens,B: Early diabetes and abnormal postnatal pancreatic islet development in mice lacking Glut-2. Nat.Genet. 17:327-330, 1997

GLUTs in the CNS and blood brain barrier

Glucose is the preferred energy substrate of the brain. Due to its expression in the endothelial cells forming the blood brain barrier, GLUT1 is essential for glucose delivery to the brain. Given the fact that the abluminal surface of brain capillaries is covered by specialized astrocytic end-feet that also express GLUT1, the astrocytes probably constitute a major site of glucose uptake. In astrocytes, glucose is catabolized by glycolysis to lactate, which may be delivered to neurons through a glial-specific monocarboxylate transporter (MCT1) and a neuron-specific one (MCT2). In neurons, lactate is converted to pyruvate, which enters the tricarboxylic acid cycle to generate ATP. Glucose can also be taken up directly by neurons, which express the GLUT3 isoform [57]. GLUT2 is also expressed in the brain in specific regions such as the hypothalamus and the brain stem where it may participate in the mechanisms of glucose sensing involved in the control of glucose homeostasis.

The role of GLUT8 in some specific neurons remains unclear. It is localized to intracellular vesicles and may possibly move to the cell surface upon as yet unidentified stimuli [33]. Finally, HMIT is expressed in astrocytes and in neurons. In astrocytes, HMIT is both intracellular and at the plasma membrane, whereas its subcellular localization in neurons is under investigation [74].

Pharmaceutical relevance

Elevation of blood glucose is the main symptom of types-1 or -2 diabetes. The GLUT isoforms that transport glucose represent therefore a potential therapeutic target for normalizing glycaemia. A compound that increases the Vmax of GLUT1 would increase whole-body glucose utilization. Given the fact that this isoform is almost ubiquitous, such activation could, however, also lead to severe hypoglycaemia. Another possible site of action for limiting the blood glucose level would be inhibition of glucose absorption in the intestine or reabsorption in the kidney. In the intestine, this could be possible by blocking both GLUT2 and the alternative membrane-traffic-based pathway of basolateral glucose release. In the kidney, GLUT2 deficiency results in glucose excretion in the urine, which decreases glycaemia [23]. Inhibition of GLUT2 specifically in the kidney could thus treat hyperglycaemia.


23. Guillam MT, Hummler E, Schaerer E, Yeh JI, Birnbaum MJ, Beermann F, Schmidt A, Deriaz N, Thorens B, Wu JY (1997) Early diabetes and abnormal postnatal pancreatic islet development in mice lacking Glut-2. Nat Genet 17:327–330

33. Ibberson M, Riederer BM, Uldry M, Guhl B, Roth J, Thorens B (2002) Immunolocalization of GLUTX1 in the testis and to specific brain areas and vasopressin-containing neurons. Endocrinology 143:276–284

57. Pellerin L, Bonvento G, Chatton JY, Pierre K, Magistretti PJ (2002) Role of neuron-glia interaction in the regulation of brain glucose utilization. Diabetes Nutr Metab 15:268–273; discussion 273

74. Uldry M, Ibberson M, Horisberger JD, Chatton JY, Riederer BM, Thorens B (2001) Identification of a mammalian H+-myoinositol symporter expressed predominantly in the brain. EMBO J 20:4467–4477

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[However, the sodium dependent glucose transporter] SGLT2 (SLC5A1) seems to be a more interesting target in the kidney for this purpose since its expression is more limited.

Type 2 diabetes is characterized by the loss of insulin sensitivity that leads to a decrease in GLUT1 translocation to the plasma membrane in response to a high blood glucose. To compensate the resulting reduced flux of glucose into muscle and adipocytes, it would be useful to find a pharmacological compound that increases the Vmax of GLUT1 for glucose, or stimulate its translocation to the cell surface.

An impaired brain inositol metabolism has been linked to psychiatric diseases, in particular bipolar disorders (28). Indeed, current treatments of these mood disorders relies on the use of Li+ salts, valproic acid and carbamazepine, drugs whose action may interfere with inositol metabolism.(28) It is well established that one mechanism of action of Li+ is the inhibition of inositol monophosphate phosphatase and polyphosphoinositide 1-phosphate phosphatase (29), which blocks recycling of inositol phosphate and reduces the availability of inositol for subsequent cycles of intracellular signal transduction. Inhibition of HMIT could also lead to such beneficial effects for bipolar disorders by decreasing the intracellular inositol concentration.

Some members of the GLUT family (GLUT1, 2 and 4) can transport glucosamine, which is important in the biosynthesis of glycoproteins and, in particular, glycosaminoglycan synthesis in cartilage (30). In association with collagen fibres, these molecules are responsible for the resilience of the cartilage to deformation. Destruction of joint cartilage occurs in osteoarthritis, and several studies have shown that glucosamine is beneficial for this disease . Given the fact that GLUT1 is expressed in chondrocytes, the cells that synthesize cartilage, glucosamine has favourable effects for osteoarthritis are probably mediated by transport across GLUT1 into these cells. Furthermore, glucosamine absorption seems to be mediated in part by GLUT2. This provides an example of the use of GLUT isoforms to deliver therapeutic molecules to their site of action.

1.3.3 The facilitative glucose transporter GLUT1

GLUT1 cDNA was isolated from an expression library using antibodies against the human´s erythrocyte glucose transporter(4). Although cloned from a hepatoma cDNA library, GLUT1 is not expressed in normal hepatocytes. It is, however, induced during oncogenic transformation of most cell types and its expression correlates with the increase in glucose [metabolism observed in tumour cells (31).]


4. Kloppel,G, Lohr,M, Habich,K, Oberholzer,M, Heitz,PU: Islet pathology and the pathogenesis of type 1 and type 2 diabetes mellitus revisited. Surv.Synth.Pathol.Res. 4:110-125, 1985

28. Shaldubina,A, Buccafusca,R, Johanson,RA, Agam,G, Belmaker,RH, Berry,GT, Bersudsky,Y: Behavioural phenotyping of sodium-myo-inositol cotransporter heterozygous knockout mice with reduced brain inositol. Genes Brain Behav. 6:253-259, 2007

29. Berridge,MJ, Downes,CP, Hanley,MR: Neural and developmental actions of lithium: a unifying hypothesis. Cell 59:411-419, 1989

30. Uldry,M, Ibberson,M, Hosokawa,M, Thorens,B: GLUT2 is a high affinity glucosamine transporter. FEBS Lett. 524:199-203, 2002

31. Haber,PS, Pirola,RC, Wilson,JS: Clinical update: management of acute pancreatitis. J.Gastroenterol.Hepatol. 12:189-197, 1997

[page 481]

GLUT1

Glut 1 was the first transporter to be characterized by molecular cloning, and its cDNA was isolated from an expression library using antibodies against the humans erythrocyte glucose transporter [53]. Although cloned from a hepatoma cDNA library, GLUT1 is not expressed in normal hepatocytes. It is, however, induced during oncogenic transformation of most cell types and its expression correlates with the increase in glucose metabolism observed in tumour cells [20].

[page 487]

However, SGLT2 seems to be a more interesting target in the kidney for this purpose since its expression is more limited.

Type-2 diabetes is characterized by the loss of insulin sensitivity that leads to a decrease in GLUT4 translocation to the plasma membrane in response to a high blood glucose. To compensate the resulting reduced flux of glucose into muscle or adipocytes, it would be useful to find a pharmacological compound that increases the Vmax of GLUT4 for glucose, or stimulate its translocation to the cell surface.

An impaired brain inositol metabolism has been linked to psychiatric diseases, in particular bipolar disorders. Indeed, current treatments of these mood disorders relies on the use of lithium salts, valproic acid and carbamazepine, drugs whose action may interfere with inositol metabolism. It is well established that one mechanism of action of Li+ is the inhibition of inositol monophosphate phosphatase and polyphosphoinositide 1-phosphate phosphatase [6], which blocks recycling of inositol phosphate and reduces the availability of inositol for subsequent cycles of intracellular signal transduction. Inhibition of HMIT could also lead to such beneficial effects for bipolar disorders by decreasing the intracellular inositol concentration.

Some members of the GLUT family (GLUT1, 2 and 4) can transport glucosamine, which is important in the biosynthesis of glycoproteins and, in particular, glycosaminoglycan synthesis in cartilage [75]. In association with collagen fibres, these molecules are responsible for the resilience of the cartilage to deformation. Destruction of joint cartilage occurs in osteoarthritis, and several studies have shown that glucosamine is beneficial for this disease. Given the fact that GLUT1 is expressed in chondrocytes, the cells that synthesize cartilage, glucosamine’s favourable effects for osteoarthritis are probably mediated by transport across GLUT1 into these cells. Furthermore, glucosamine absorption seems to be mediated in part by GLUT2. This provides an example of the use of GLUT isoforms to deliver therapeutic molecules to their site of action.


6. Berridge MJ, Downes CP, Hanley MR (1989) Neural and developmental actions of lithium: a unifying hypothesis. Cell 59:411–419

20. Flier JS, Mueckler MM, Usher P, Lodish HF (1987) Elevated levels of glucose transport and transporter messenger RNA are induced by ras or src oncogenes. Science 235:1492–1495

53. Mueckler M, Caruso C, Baldwin SA, Panico M, Blench I, Morris HR, Allard WJ, Lienhard GE, Lodish HF (1985) Sequence and structure of a human glucose transporter. Science 229:941–945

75. Uldry M, Ibberson M, Hosokawa M, Thorens B (2002) GLUT2 is a high affinity glucosamine transporter. FEBS Lett 524:199–203

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GLUT1 is found in almost every tissue with different levels of expression in different cell types. The expression level usually correlates with the rate of cellular glucose metabolism. As mentioned above, it is also expressed highly in blood-tissue barriers, in particular in the endothelial cells forming the blood-brain barrier. Several heterozygous mutations resulting in GLUT1 haploinsufficiency have been identified. These cause hypoglycorrachia, a condition characterized by seizures, developmental delay, acquired microcephaly, and hypotonia, and which is due to a decrease rate of glucose transport from the blood into cerebrospinal fluid.

The topological arrangement of GLUT1 within the plasma membrane has been confirmed using several experimental approaches. Recently, two models have been proposed for the tertiary structure of GLUT1. The first is based on data obtained from cysteine scanning mutagenesis of five of the α-helices of GLUT1 together with information from site-directed mutagenesis (32). The second is based primarily on the proposed helical bundle arrangement of the Lac permease and has been refined using energy minimization algorithm (33) These two models describe a key role for helix 7 in the formation of a water-filled channel which may form the path for glucose across the plasma membrane.

The transport of glucose may be described as an alternating confirmation model in which the transporter has mutually exclusive binding sites located on the extracellular (import site) and on the intracellular face (export site) of the transporter (Figure 4). Binding of glucose to one site induces the transporter to switch to the opposite conformation, a process that is accompanied by a movement of the substrate across the plasma membrane (34). In human erythrocytes, GLUT1 is thought to be present as homodimers or homotetramers, with the conversion between both oligomeric forms being dependent on the redox state, . GLUT1 transports glucose with an affinity constant (Km) of ~3 mM. Other transported substrates are galactose (30mM) (34), mannose (35) (20mM) and glucosamine 2.1±0.5mM (36). Glucose transport by GLUT1 is sensitive to several inhibitors that also block transport by other isoforms. Many of them are competitive inhibitors of sugar binding, either to the extracellular or the cytosolic sugar binding sites. Cytochalasin B binds to the inner surface of GLUT1 and inhibits its glucose transport activity with an IC50 of 0.44 μM. Binding of cytochalasin B is to a site which contains tryptophan 388 and 412. Also acting on the same intracellular site is the diterpene toxin forskolin. Forskolin has been used as a photoaffinity label with some specificity for the glucose transporter and its affinity is increased in the 3-iodo4-azidophenethylamido-7-O-succinyldeacetyl (IAPS) derivative.


32. Keymeulen,B, Ling,Z, Gorus,FK, Delvaux,G, Bouwens,L, Grupping,A, Hendrieckx,C, Pipeleers-Marichal,M, Van Schravendijk,C, Salmela,K, Pipeleers,DG: Implantation of standardized beta-cell grafts in a liver segment of IDDM patients: graft and recipients characteristics in two cases of insulin-independence under maintenance immunosuppression for prior kidney graft. Diabetologia 41:452-459, 1998

33. Fischbarg,J, Cheung,M, Li,J, Iserovich,P, Czegledy,F, Kuang,K, Garner,M: Are most transporters and channels beta barrels? Mol.Cell Biochem. 140:147-162, 1994

34. Joost,HG, Thorens,B: The extended GLUT-family of sugar/polyol transport facilitators: nomenclature, sequence characteristics, and potential function of its novel members (review). Mol.Membr.Biol. 18:247-256, 2001

35. Palfreyman,RW, Clark,AE, Denton,RM, Holman,GD, Kozka,IJ: Kinetic resolution of the separate GLUT1 and GLUT4 glucose transport activities in 3T3-L1 cells. Biochem.J. 284 ( Pt 1):275-282, 1992

36. Robinson,KA, Sens,DA, Buse,MG: Pre-exposure to glucosamine induces insulin resistance of glucose transport and glycogen synthesis in isolated rat skeletal muscles. Study of mechanisms in muscle and in rat-1 fibroblasts overexpressing the human insulin receptor. Diabetes 42:1333-1346, 1993

[page 481]

GLUT1 is found in almost every tissue with different levels of expression in different cell types. The expression level usually correlates with the rate of cellular glucose metabolism. It is also expressed highly in blood-tissue barriers, in particular in the endothelial cells forming the blood-brain barrier [45].

The topological arrangement of GLUT1 within the plasma membrane has been confirmed using several experimental approaches. Recently, two models have been proposed for the tertiary structure of GLUT1. The first is based on data obtained from cysteine scanning mutagenesis of five of the α-helices of GLUT1 together with information from site-directed mutagenesis [52]. The second is based primarily on the proposed helical bundle arrangement of the Lac permease and has been refined using energy minimization algorithm [79]. These two models describe a key role for helix 7 in the formation of a water-filled channel which may form the path for glucose across the plasma membrane.

The transport of glucose may be described as an alternating conformer model in which the transporter has mutually exclusive binding sites located on the extracellular (import site) and on the intracellular face (export site) of the transporter. Binding of glucose to one site induces the transporter to switch to the opposite conformation, a process that is accompanied by a movement of the substrate across the plasma membrane. In human erythrocytes, GLUT1 is thought to be present as homodimers or homotetramers, with the conversion between both oligomeric forms being dependent on the redox state [27, 28]. GLUT1 transports glucose with a Km of ~3 mM. Other transported substrates are galactose, mannose and glucosamine [75].

Glucose transport by GLUT1 is sensitive to several inhibitors that also block transport by other isoforms. Many of them are competitive inhibitors of sugar binding, either to the extracellular or the cytosolic sugar binding sites. Cytochalasin B binds to the inner surface of GLUT1 [4] and inhibits its glucose transport activity with an IC50 of 0.44 μM. Binding of cytochalasin B is to a site which contains tryptophan 388 and 412 (see Fig. 2). Also acting on the same intracellular site is the diterpene toxin forskolin. Forskolin has been used as a photoaffinity label with some specificity for the glucose transporter and its affinity is increased in the 3-iodo4-azidophenethylamido-7-O-succinyldeacetyl (IAPS) derivative. [...]

[...]

Several heterozygous mutations resulting in GLUT1 haploinsufficiency have been identified. These cause

[page 482]

hypoglycorrachia, a condition characterized by seizures, developmental delay, acquired microcephaly, and hypotonia, and which is due to a decrease rate of glucose transport from the blood into cerebrospinal fluid [42, 67].


4. Baldwin SA, Lienhard GE (1989) Purification and reconstitution of glucose transporter from human erythrocytes. Methods Enzymol 174:39–50

27. Hamill S, Cloherty EK, Carruthers A (1999) The human erythrocyte sugar transporter presents two sugar import sites. Biochemistry 38:16974–16983

28. Hebert DN, Carruthers A (1992) Glucose transporter oligomeric structure determines transporter function. Reversible redoxdependent interconversions of tetrameric and dimeric GLUT1. J Biol Chem 267:23829–23838

42. Klepper J, Voit T (2002) Facilitated glucose transporter protein type 1 (GLUT1) deficiency syndrome: impaired glucose transport into brain—a review. Eur J Pediatr 161:295–304

45. Maher F, Vannucci SJ, Simpson IA (1994) Glucose transporter proteins in brain. FASEB J 8:1003–1011

52. Mueckler M, Makepeace C (2002) Analysis of transmembrane segment 10 of the Glut1 glucose transporter by cysteinescanning mutagenesis and substituted cysteine accessibility. J Biol Chem 277:3498–3503

67. Seidner G, Alvarez MG, Yeh JI, O’Driscoll KR, Klepper J, Stump TS, Wang D, Spinner NB, Birnbaum MJ, De Vivo DC (1998) GLUT-1 deficiency syndrome caused by haploinsufficiency of the blood-brain barrier hexose carrier. Nat Genet 18:188–191

75. Uldry M, Ibberson M, Hosokawa M, Thorens B (2002) GLUT2 is a high affinity glucosamine transporter. FEBS Lett 524:199–203

79. Zuniga FA, Shi G, Haller JF, Rubashkin A, Flynn DR, Iserovich P, Fischbarg J (2001) A three-dimensional model of the human facilitative glucose transporter Glut1. J Biol Chem 276:44970–44975

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(Hindemith) LieschenMueller

[14.] Sj/Fragment 011 01 - Diskussion
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[An iodinated derivative] of forskolin (7-aminoalkylcarbamate) with a very high affinity (IC50 200 nM) has also been described (37).

Glucose transport activity of GLUT1 is inhibited by HgCl2 (IC50 3.5 μM), phloretin (IC50 49 μM), phlorizin (IC50 355 μM) and 4,6-O-ethylidene-D-glucose (IC50 12 mM), which bind to the external glucose binding site where glutamine 161 appears to be critical for inhibitor binding (16).


16. Mueckler,M, Makepeace,C: Transmembrane segment 12 of the Glut1 glucose transporter is an outer helix and is not directly involved in the transport mechanism. J.Biol.Chem. 281:36993-36998, 2006

37. Harrison,SA, Buxton,JM, Czech,MP: Suppressed intrinsic catalytic activity of GLUT1 glucose transporters in insulin-sensitive 3T3-L1 adipocytes. Proc.Natl.Acad.Sci.U.S.A 88:7839-7843, 1991

An iodinated derivative of forskolin (7-aminoalkylcarbamate) with a very high affinity (IC50 200 nM) has also been described [51].

Glucose transport activity of GLUT1 is inhibited by HgCl2 (IC50 3.5 μM), phloretin (IC50 49 μM) phlorizin (IC50 355 μM) [37] and 4,6-O-ethylidene-d-glucose (IC50 12 mM), which binds to the external glucose binding site where glutamine 161 appears to be critical for inhibitor binding [54]


37. Kasahara T, Kasahara M (1996) Expression of the rat GLUT1 glucose transporter in the yeast Saccharomyces cerevisiae. Biochem J 315:177–182

51. Morris DI, Robbins JD, Ruoho AE, Sutkowski EM, Seamon KB (1991) Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter. J Biol Chem 266:13377–13384

54. Mueckler M, Weng W, Kruse M (1994) Glutamine 161 of Glut1 glucose transporter is critical for transport activity and exofacial ligand binding. J Biol Chem 269:20533–20538

Anmerkungen

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(Hindemith) LieschenMueller

[15.] Sj/Fragment 012 03 - Diskussion
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Earlier it was found that, in rat adipocytes insulin triggers the movement of the sugar transporter that is found in these cells from cytoplam to the plasma membrane. That was later confirmed when GLUT4 was identified as the main glucose transporter in these cells. GLUT4 primarily found in muscle and fat cells, is found in a complex intracellular tubulo–vesicular network that is connected to the endosomal–trans-Golgi network (TGN) system. In 1980, it was reported that, in rat adipocytes, insulin triggers the movement of the sugar transporter that is found in these cells from an intracellular store to the plasma membrane2,3.This translocation hypothesis was later confirmed when GLUT4 was identified as the main glucose transporter in these cells. GLUT4, which is expressed primarily in muscle and fat cells, is found in a complex intracellular tubulo–vesicular network that is connected to the endosomal–trans-Golgi network (TGN) system.

2. Suzuki, K. & Kono, T. Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site. Proc. Natl Acad. Sci. USA 77, 2542–2545 (1980).

3. Cushman, S. W. & Wardzala, L. J. Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. Apparent translocation of intracellular transport systems to the plasma membrane. J. Biol. Chem. 255, 4758–4762 (1980).

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(Hindemith), WiseWoman

[16.] Sj/Fragment 012 09 - Diskussion
Bearbeitet: 18. November 2016, 23:22 WiseWoman
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In the basal state, GLUT4 cycles continuously between the plasma membrane and one or more intracellular compartments, with 90-95 percent of the transporter residing within the cell interior, tightly packaged into vesicles. The overall insulin-induced GLUT4 vesicle trafficking results in a >10-fold increase of GLUT4 protein at the cell surface (38). GLUT4 is found within large tubulo-vesicular structures in the perinuclear region of the cell and within small vesicles dispersed throughout the cytoplasm, also known as GLUT4 storage vesicles (GSVs) (39) (40). Perinuclear GLUT4 is likely localized in endosomes. GSVs on the other hand co-localize with insulin-responsive aminopeptidase (IRAP) and vesicle-associated membrane protein 2 (VAMP2). Upon insulin stimulation, it appears that GSVs are translocated to the cell surface, as there is an increase of GLUT4 at the plasma membrane that is proportional to the reduction in GLUT4-containing vesicles from the cytosolic compartment (41) (42), while the level of perinuclear GLUT4-containing vesicles remains relatively the same. Furthermore, total internal reflection fluorescence microscopy has revealed that in basal adipocytes, GLUT4-containing vesicles are located near the plasma membrane and are recruited to the cell surface with insulin stimulation (43) (Figure 2).

1.3.5 Role of insulin signalling in GLUT4 regulation

Activation of the insulin receptor triggers a cascade of phosphorylation events that ultimately promote GLUT4 vesicle exocytosis. The classical insulin signaling pathway (Figure 2) involves docking of the insulin receptor substrate (IRS) to the insulin receptor (IR), activation of phosphatidylinositol 3-kinase (PI3K) which leads to formation of plasma membrane phosphatidylinositol 3,4,5-trisphosphate (PI -P3), subsequent PI -P3-mediated activation of Akt and atypical protein kinase C (aPKC). Additionally, a PI3K-independent pathway involving c-Cbl, c-Cbl associated protein (CAP), and the GTPase TC10 may also regulate GLUT4 translocation. Insulin action is initiated when this peptide hormone binds to its receptor.


38. Malide,D, Ramm,G, Cushman,SW, Slot,JW: Immunoelectron microscopic evidence that GLUT4 translocation explains the stimulation of glucose transport in isolated rat white adipose cells. J.Cell Sci. 113 Pt 23:4203-4210, 2000

39. Kandror,KV, Pilch,PF: Compartmentalization of protein traffic in insulin-sensitive cells. Am.J.Physiol 271:E1-14, 1996

40. Rea,S, James,DE: Moving GLUT4: the biogenesis and trafficking of GLUT4 storage vesicles. Diabetes 46:1667-1677, 1997

41. Sehgal,SN, Baker,H, Vezina,C: Rapamycin (AY-22,989), a new antifungal antibiotic. II. Fermentation, isolation and characterization. J.Antibiot.(Tokyo) 28:727-732, 1975

42. Shi,J, Kandror,KV: Sortilin is essential and sufficient for the formation of Glut4 storage vesicles in 3T3-L1 adipocytes. Dev.Cell 9:99-108, 2005

43. Lizunov,VA, Matsumoto,H, Zimmerberg,J, Cushman,SW, Frolov,VA: Insulin stimulates the halting, tethering, and fusion of mobile GLUT4 vesicles in rat adipose cells. J.Cell Biol. 169:481-489, 2005

[Page 374]

In the basal state, GLUT4 cycles continuously between the plasma membrane and one or more intracellular compartments, with 90-95 per cent of the transporter residing within the cell interior, tightly packaged into vesicles3,4. [...] The overall insulin-dependent shift in the cellular dynamics of GLUT4 vesicle trafficking results in a >10-fold increase of GLUT4 protein at the cell surface7. [...]

GLUT4 is found within large tubulo-vesicular structures in the perinuclear region of the cell and within small vesicles dispersed throughout the cytoplasm, also known as GLUT4 storage vesicles (GSVs). Perinuclear GLUT4 is likely localized in endosomes and trans-Golgi network (TGN) structures, as evidence has shown that it co-localizes with endosomal and TGN markers, including transferrin receptor (TfR) and syntaxin 16, respectively9. GSVs on the other hand co-localize with insulin-responsive aminopeptidase (IRAP) and vesicle-associated membrane protein 2 (VAMP2). Upon insulin stimulation, it appears that GSVs are mobilized to the cell surface, as there is an increase of GLUT4 at the plasma membrane that is proportional to the reduction in GLUT4-containing vesicles from the cytosolic compartment10,11, while the level of perinuclear GLUT4-containing vesicles remains relatively unaffected10,12. Furthermore, total internal reflection fluorescence microscopy has revealed that in basal adipocytes, GLUT4-containing vesicles are located near the plasma membrane and are recruited to the cell surface with insulin stimulation13. [...]

[Page 375]

[...]

Insulin signaling systems regulating GLUT4

Activation of the insulin receptor triggers a cascade of phosphorylation events that ultimately promote GLUT4 vesicle exocytosis (Fig.). The classical insulin signaling pathway involves docking of the insulin receptor substrate (IRS) to the insulin receptor (IR), activation of phosphatidylinositol 3-kinase (PI3K) which leads to formation of plasma membrane phosphatidylinositol 3,4,5-trisphosphate (PI 3,4,5-P3), subsequent PI 3,4,5-P3-mediated activation of Akt and atypical protein kinase C (aPKC), and phosphorylation of AS160 (Akt substrate of 160 kDa) by Akt (Fig. A). Additionally, a PI3K-independent pathway involving c-Cbl, c-Cbl associated protein (CAP), and the GTPase TC10 may also regulate GLUT4 translocation (Fig. B), although this pathway appears to be exclusive to adipocytes. Here, we detail steps within both insulin signaling pathways.

IR and its substrates: Insulin action is initiated when this peptide hormone binds to its receptor.


3. Kandror KV, Pilch PF. Compartmentalization of protein traffic in insulin-sensitive cells. Am J Physiol 1996; 271 : E1-14.

4. Rea S, James DE. Moving GLUT4: the biogenesis and trafficking of GLUT4 storage vesicles. Diabetes 1997; 46 : 1667-77.

7. Malide D, Ramm G, Cushman SW, Slot JW. Immunoelectron microscopic evidence that GLUT4 translocation explains the stimulation of glucose transport in isolated rat white adipose cells. J Cell Sci 2000; 113 : 4203-10.

9. Larance M, Ramm G, Stockli J, van Dam EM, Winata S, Wasinger V, et al. Characterization of the role of the Rab GTPase-activating protein AS160 in insulin-regulated GLUT4 trafficking. J Biol Chem 2005; 280 : 37803-13.

10. Ramm G, Slot JW, James DE, Stoorvogel W. Insulin recruits GLUT4 from specialized VAMP2-carrying vesicles as well as from the dynamic endosomal/trans-Golgi network in rat adipocytes. Mol Biol Cell 2000; 11 : 4079-91.

11. Shi J, Kandror KV. Sortilin is essential and sufficient for the formation of Glut4 storage vesicles in 3T3-L1 adipocytes. Dev Cell 2005; 9 : 99-108.

12. Martin S, Millar CA, Lyttle CT, Meerloo T, Marsh BJ, Gould GW, et al. Effects of insulin on intracellular GLUT4 vesicles in adipocytes: evidence for a secretory mode of regulation. J Cell Sci 2000; 113 : 3427-38.

13. Lizunov VA, Matsumoto H, Zimmerberg J, Cushman SW, Frolov VA. Insulin stimulates the halting, tethering, and fusion of mobile GLUT4 vesicles in rat adipose cells. J Cell Biol 2005; 169 : 481-9.

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(Graf Isolan), WiseWoman

[17.] Sj/Fragment 013 01 - Diskussion
Bearbeitet: 24. November 2016, 20:42 Schumann
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Insulin binding to the two high-affinity extracellular α subunits leads to activation of intrinsic tyrosine kinase activity of the transmembrane β subunits and autophosphorylation of specific tyrosine residues (44), (45). Phosphorylation enhances tyrosine kinase activity of the b subunits towards a host of proteins including members of the insulin receptor substrate family (IRS-1, -2, -3, -4, -5, and -6), Cbl, SIRP (signal regulatory protein) family members, and APS [adapter protein containing a pleckstrin homology (PH) and Src-homology 2 (SH2) domain , (46). [...]

[...]

Insulin-dependent tyrosine phosphorylation of IRS-1/2 creates docking sites for downstream effector molecules including Class IA PI3K. Activated PI3K catalyzes the phosphorylation of PI-P2 on the 3 position of the inositol ring, forming PI-P3. Increased membrane PI-P3 is crucial for insulin-stimulated GLUT4 translocation, as this phospholipid provides docking sites for downstream molecules via their pleckstrin homology (PH) domains. Inhibition of PI-P3 formation with wortmannin or LY29004 effectively blocks insulin-stimulated GLUT4 translocation and glucose transport (47), (48) . Phosphatase regulators of PI3K generated lipids include PTEN and SHIP2. The PTEN catalyzes the dephosphorylation of phosphatidylinositols at the D3 position, while the latter catalyzes dephosphorylation of PI - P3 to yield PI -P2 (49). PI -P3 formation mediates the plasma membrane translocation of two PH domain containing proteins which is important for insulin-regulated glucose uptake: Akt (protein kinase B, PKB) and phosphoinositide-dependent-kinase-1 (PDK1). PDK2 is recently identified as the protein kinase mTOR (mammalian target of rapamycin) complexed to the regulatory protein rictor (50). There are three isoforms of protein kinase AKT (1-3) (51) in which GLUT4 translocation is dependent on AKT2 (52). AKT2 acts through it downstream molecule AS160 (53). AS160 contains a GTPase activating domain for Rabs, small G proteins which is involved in the vesicle trafficking (54), (55). Along with recruiting and activating Akt, PI 3,4,5-P3 formation in concert with PDK1 also leads to the activation of atypical protein kinase C (aPKC), and both aPKC isoforms z and l [sic] have been implicated in GLUT4 translocation (56). PKC knockout models and expression of kinase-inactive PKC-z/l [sic] inhibits GLUT4 translocation and glucose uptake in a variety of cell types, and this phenotype can be reversed in PKC-l–/– [sic] cells by expressing wild-type aPKC.


44. Bjornholm,M, Zierath,JR: Insulin signal transduction in human skeletal muscle: identifying the defects in Type II diabetes. Biochem.Soc.Trans. 33:354-357, 2005

45. Luo,RZ, Beniac,DR, Fernandes,A, Yip,CC, Ottensmeyer,FP: Quaternary structure of the insulin-insulin receptor complex. Science 285:1077-1080, 1999

46. Rocchi,S, Tartare-Deckert,S, Murdaca,J, Holgado-Madruga,M, Wong,AJ, Van Obberghen,E: Determination of Gab1 (Grb2-associated binder-1) interaction with insulin receptor-signaling molecules. Mol.Endocrinol. 12:914-923, 1998

47. Okada,T, Kawano,Y, Sakakibara,T, Hazeki,O, Ui,M: Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. Studies with a selective inhibitor wortmannin. J.Biol.Chem. 269:3568- 3573, 1994

48. Clarke,JF, Young,PW, Yonezawa,K, Kasuga,M, Holman,GD: Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3- kinase inhibitor, wortmannin. Biochem.J. 300 ( Pt 3):631-635, 1994

49. Scheid,MP, Woodgett,JR: Unravelling the activation mechanisms of protein kinase B/Akt. FEBS Lett. 546:108-112, 2003

50. Sarbassov,DD, Guertin,DA, Ali,SM, Sabatini,DM: Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307:1098-1101, 2005

51. Bae,SS, Cho,H, Mu,J, Birnbaum,MJ: Isoform-specific regulation of insulin-dependent glucose uptake by Akt/protein kinase B. J.Biol.Chem. 278:49530-49536, 2003

52. Calera,MR, Martinez,C, Liu,H, Jack,AK, Birnbaum,MJ, Pilch,PF: Insulin increases the association of Akt-2 with Glut4-containing vesicles. J.Biol.Chem. 273:7201-7204, 1998

53. Kane,S, Sano,H, Liu,SC, Asara,JM, Lane,WS, Garner,CC, Lienhard,GE: A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPase-activating protein (GAP) domain. J.Biol.Chem. 277:22115-22118, 2002

54. Sano,H, Kane,S, Sano,E, Miinea,CP, Asara,JM, Lane,WS, Garner,CW, Lienhard,GE: Insulin-stimulated phosphorylation of a Rab GTPase-activating protein regulates GLUT4 translocation. J.Biol.Chem. 278:14599-14602, 2003

55. Jordens,I, Marsman,M, Kuijl,C, Neefjes,J: Rab proteins, connecting transport and vesicle fusion. Traffic. 6:1070-1077, 2005

56. Farese,RV, Sajan,MP, Standaert,ML: Insulin-sensitive protein kinases (atypical protein kinase C and protein kinase B/Akt): actions and defects in obesity and type II diabetes. Exp.Biol.Med.(Maywood.) 230:593-605, 2005

Insulin binding to the two high-affinity extracellular a [sic] subunits leads to activation of intrinsic tyrosine kinase activity of the transmembrane β subunits and autophosphorylation of specific tyrosine residues1,16. Phosphorylation enhances tyrosine kinase activity of the β subunits towards a host of proteins including members of the insulin receptor substrate family (IRS-1, -2, -3, -4, -5, and -6), Gab-1, Shc, p62dok, Cbl, SIRP (signal regulatory protein) family members, and APS [adapter protein containing a pleckstrin homology (PH) and Src-homology 2 (SH2) domain]17-23.

[...]

PI3K-dependent signaling: Insulin-dependent tyrosine phosphorylation of IRS-1/2 creates docking sites for downstream effector molecules including Class IA PI3K32.

[page 377]

[...] Activated PI3K catalyzes the phosphorylation of PI 4,5-P2 on the 3 position of the inositol ring, forming PI 3,4,5-P3. Increased membrane PI 3,4,5-P3 is essential for insulin-stimulated GLUT4 translocation, as this phospholipid provides docking sites for downstream molecules via their pleckstrin homology (PH) domains. Inhibiting the formation of PI 3,4,5-P3 with wortmannin or LY29004 effectively blocks insulin-stimulated GLUT4 translocation and glucose transport33,34. Phosphatase regulators of PI3K generated lipids include PTEN and SHIP2. The former catalyzes the dephosphorylation of phosphatidylinositols at the D3 position, while the latter catalyzes dephosphorylation of PI 3,4,5-P3 to yield PI 3,4-P2 35.

PI 3,4,5-P3 formation mediates the plasma membrane translocation of two PH domaincontaining proteins crucial for insulin-regulated glucose uptake: Akt (protein kinase B, PKB) and phosphoinositide-dependent-kinase-1 (PDK1)35. Following membrane recruitment, the 60 kDa serine/threonine kinase Akt is phosphorylated at two key sites, threonine 308 (Thr308) and serine 473 (Ser473), resulting in enzyme activation. Ser473 phosphorylation occurs first and is accomplished by the once elusive PDK2, which was recently identified as the protein kinase mTOR (mammalian target of rapamycin) complexed to the regulatory protein rictor36-38.[...]

There are three existing Akt isoforms (Akt1-3)41, and studies have identified Akt2 as the relevant isoform in insulin-stimulated GLUT4 translocation. [...]

[...] Also, a number of key properties of the protein have since been elucidated. First, AS160 contains a GTPaseactivating domain (GAP) for Rabs, small G proteins involved in vesicle trafficking49,53.

[page 378]

Along with recruiting and activating Akt, PI 3,4,5-3 formation in concert with PDK1 also leads to the activation of atypical protein kinase C (aPKC), and both aPKC isoforms ζ and λ have been implicated in GLUT4 translocation. As recently reviewed58, aPKC knockout models and expression of kinase-inactive PKC-ζ/λ inhibits GLUT4 translocation and glucose uptake in a variety of cell types, and this phenotype can be reversed in PKC-λ–/– cells by expressing wild-type aPKC.


1. Bjornholm M, Zierath JR. Insulin signal transduction in human skeletal muscle: identifying the defects in Type II diabetes. Biochem Soc Trans 2005; 33 : 354-7.

16. Luo RZ, Beniac DR, Fernandes A, Yip CC, Ottensmeyer FP. Quaternary structure of the insulin-insulin receptor complex. Science 1999; 285 : 1077-80.

17. Pelicci G, Lanfrancone L, Grignani F, McGlade J, Cavallo F, Forni G, et al. A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction. Cell 1992; 70 : 93-104.

18. Holgado-Madruga M, Emlet DR, Moscatello DK, Godwin AK, Wong AJ. A Grb2-associated docking protein in EGF- and insulin-receptor signalling. Nature 1996; 379 : 560-4.

19. Fujioka Y, Matozaki T, Noguchi T, Iwamatsu A, Yamao T, Takahashi N, et al. A novel membrane glycoprotein, SHPS-1, that binds the SH2-domain-containing protein tyrosine phosphatase SHP-2 in response to mitogens and cell adhesion. Mol Cell Biol 1996; 16 : 6887-99.

20. White MF. The IRS-signalling system: a network of docking proteins that mediate insulin action. Mol Cell Biochem 1998; 182 : 3-11.

21. Rocchi S, Tartare-Deckert S, Murdaca J, Holgado-Madruga M, Wong AJ, Van Obberghen E. Determination of Gab1 (Grb2-associated binder-1) interaction with insulin receptor-signaling molecules. Mol Endocrinol 1998; 12 : 914-23.

22. Moodie SA, Alleman-Sposeto J, Gustafson TA. Identification of the APS protein as a novel insulin receptor substrate. J Biol Chem 1999; 274 : 11186-93.

23. Cai D, Dhe-Paganon S, Melendez PA, Lee J, Shoelson SE. Two new substrates in insulin signaling, IRS5/DOK4 and IRS6/DOK5. J Biol Chem 2003; 278 : 25323-30.

32. Shepherd PR. Mechanisms regulating phosphoinositide 3-kinase signalling in insulin-sensitive tissues. Acta Physiol Scand 2005; 183 : 3-12.

33. Okada T, Kawano Y, Sakakibara T, Hazeki O, Ui M. Essential role of phosphatidylinositol 3-kinase in insulininduced glucose transport and antilipolysis in rat adipocytes. Studies with a selective inhibitor wortmannin. J Biol Chem 1994; 269 : 3568-73.

34. Clarke JF, Young PW, Yonezawa K, Kasuga M, Holman GD. Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin. Biochem J 1994; 300 : 631-5.

35. Scheid MP, Woodgett JR. Unravelling the activation mechanisms of protein kinase B/Akt. FEBS Lett 2003; 546: 108-12.

36. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation of Akt/PKB by the rictormTOR complex. Science 2005; 307 : 1098-101.

37. Sarbassov dos D, Ali SM, Sabatini DM. Growing roles for the mTOR pathway. Curr Opin Cell Biol 2005; 17 : 596-603.

38. Wullschleger S, Loewith R, Hall MN. TOR signaling in growth and metabolism. Cell 2006; 124 : 471-84.

41. Bae SS, Cho H, Mu J, Birnbaum MJ. Isoform-specific regulation of insulin-dependent glucose uptake by Akt/ protein kinase B. J Biol Chem 2003; 278 : 49530-6.

49. Sano H, Kane S, Sano E, Miinea CP, Asara JM, Lane WS, et al. Insulin-stimulated phosphorylation of a Rab GTPaseactivating protein regulates GLUT4 translocation. J Biol Chem 2003; 278 : 14599-602.

53. Jordens I, Marsman M, Kuijl C, Neefjes J. Rab proteins, connecting transport and vesicle fusion. Traffic 2005; 6 : 1070-7.

58. Farese RV, Sajan MP, Standaert ML. Insulin-sensitive protein kinases (atypical protein kinase C and protein kinase B/Akt): actions and defects in obesity and type II diabetes. Exp Biol Med (Maywood) 2005; 230 : 593-605.

Anmerkungen

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Note that in the dissertation one finds "z and l" instead of "ζ and λ", and sub-/superscripts that have disappeared. Copying and pasting from a PDF file often leads to this kind of a mistake.

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(Hindemith), WiseWoman

[18.] Sj/Fragment 014 01 - Diskussion
Bearbeitet: 25. November 2016, 21:45 WiseWoman
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[Furthermore, expression of] constitutively active aPKC recapitulates the effects of insulin on GLUT4 translocation and glucose transport.

1.3.7 PI3-K independent pathway

It is well established that activation of GLUT4 translocation by insulin requires a PI3K signal involving the upstream IR and IRS activators and the downstream Akt and PKC target enzymes and AS160 protein. Some studies over the past decade have also suggested that a second pathway occurs as a consequence of Cbl tyrosine phosphorylation (57), (58). Cbl and the adaptor protein CAP are recruited to the insulin receptor by APS (59). Once tyrosine phosphorylated by the receptor, Cbl can recruit the adaptor protein CrkII to lipid rafts, along with the guanyl nucleotide exchange factor C3G (60). C3G can then activate the GTP-binding protein TC10, which resides in lipid rafts . The correct spatial compartmentalization of these signaling molecules in the lipid raft microdomain appears to be essential or insulinstimulated GLUT4 translocation and glucose transport, as these insulin-mediated events are abolished by dominant-interfering mutants of CAP that prevent the localization of Cbl to lipid rafts (61). Nevertheless, investigation suggests a role of TC10 in the regulation of actin dynamics and phosphoinositides. Cellular cortical actin exists in two forms: monomeric globular actin (G-actin) and filamentous actin (F-actin). In the case of cytosketal fusion, microtubules and cortical actin plays the important role. When the actin network (62) in skeletal muscle is treated with actin depolymerizing agent cytochalasin D or the actin monomer binding red sea sponge toxins Latrunculin A or B, it leads to the inhibition of glucose uptake and GLUT4 translocation (63). Two potentially overlapping models hypothesizing the role of actin in glucose uptake. According to the first one, insulin causes cortical actin remodeling, such that incoming vesicles can travel through the peripheral actin mesh to fuse with the plasma membrane (64) . The second suggests that actin filaments function as “highways,” upon which vesicles travel to reach the plasma membrane. Regardless of the exact actin function, it is apparent that insulin signaling to rearrange cortical actin represents a required pathway for optimal movement or fusion of GLUT4-containing vesicles and plasma membranes.


57. Ribon,V, Saltiel,AR: Insulin stimulates tyrosine phosphorylation of the protooncogene product of c-Cbl in 3T3-L1 adipocytes. Biochem.J. 324 ( Pt 3):839-845, 1997

58. Liu,J, DeYoung,SM, Hwang,JB, O'Leary,EE, Saltiel,AR: The roles of Cbl-b and c-Cbl in insulin-stimulated glucose transport. J.Biol.Chem. 278:36754-36762, 2003

59. Liu,J, Kimura,A, Baumann,CA, Saltiel,AR: APS facilitates c-Cbl tyrosine phosphorylation and GLUT4 translocation in response to insulin in 3T3-L1 adipocytes. Mol.Cell Biol. 22:3599-3609, 2002

60. Chiang,SH, Baumann,CA, Kanzaki,M, Thurmond,DC, Watson,RT, Neudauer,CL, Macara,IG, Pessin,JE, Saltiel,AR: Insulin-stimulated GLUT4 translocation requires the CAP-dependent activation of TC10. Nature 410:944-948, 2001

61. Baumann,CA, Ribon,V, Kanzaki,M, Thurmond,DC, Mora,S, Shigematsu,S, Bickel,PE, Pessin,JE, Saltiel,AR: CAP defines a second signalling pathway required for insulin-stimulated glucose transport. Nature 407:202-207, 2000

62. Omata,W, Shibata,H, Li,L, Takata,K, Kojima,I: Actin filaments play a critical role in insulin-induced exocytotic recruitment but not in endocytosis of GLUT4 in isolated rat adipocytes. Biochem.J. 346 Pt 2:321-328, 2000

63. Brozinick,JT, Jr., Hawkins,ED, Strawbridge,AB, Elmendorf,JS: Disruption of cortical actin in skeletal muscle demonstrates an essential role of the cytoskeleton in glucose transporter 4 translocation in insulin-sensitive tissues. J.Biol.Chem. 279:40699-40706, 2004

64. Jiang,ZY, Chawla,A, Bose,A, Way,M, Czech,MP: A phosphatidylinositol 3-kinaseindependent insulin signaling pathway to N-WASP/Arp2/3/F-actin required for GLUT4 glucose transporter recycling. J.Biol.Chem. 277:509-515, 2002

Furthermore, expression of constitutively active aPKC recapitulates the effects of insulin on GLUT4 translocation and glucose transport. [...]

PI3K-independent signaling: It is well established that activation of GLUT4 translocation by insulin requires a PI3K signal involving the upstream IR and IRS activators and the downstream Akt and PKC target enzymes and AS160 protein as presented above (Fig.A). Some studies over the past decade have also suggested that a second pathway (Fig.B) occurs as a consequence of Cbl tyrosine phosphorylation59,60. Cbl and the adaptor protein CAP are recruited to the insulin receptor by APS61. Once tyrosine phosphorylated by the receptor, Cbl can recruit the adaptor protein CrkII to lipid rafts, along with the guanyl nucleotide exchange factor C3G62. C3G can then activate the GTP-binding protein TC10, which resides in lipid rafts63. The correct spatial compartmentalization of these signaling molecules in the lipid raft microdomain appears to be essential for insulin-stimulated GLUT4 translocation and glucose transport, as these insulin-mediated events are abolished by dominant-interfering mutants of CAP that prevent the localization of Cbl to lipid rafts64. [...]

Nevertheless, investigation suggests a role of TC10 in the regulation of actin dynamics69-74 and phosphoinositides75.

[page 379]

Cortical actin: Cellular cortical actin exists in two forms: monomeric globular actin (G-actin) and filamentous actin (F-actin).[...] Disrupting the actin network in cultured cells or in intact rat skeletal muscle with the actin-depolymerizing agent cytochalasin D, or the actin monomer binding Red Sea Sponge toxins Latrunculin A or B, inhibits insulin-stimulated GLUT4 translocation and glucose uptake77,80,81. [...] Overall, these studies have given rise to two potentially overlapping models hypothesizing the role of actin in glucose uptake. The first proposes that insulin causes cortical actin remodeling, such that incoming vesicles can travel through the peripheral actin mesh to fuse with the plasma membrane. The second suggests that actin filaments function as “highways,” upon which vesicles travel to reach the plasma membrane71. Regardless of the exact actin function, it is apparent that insulin signaling to rearrange cortical actin represents a required pathway for optimal movement or fusion of GLUT4-containing vesicles and plasma membranes.


59. Ribon V, Saltiel AR. Insulin stimulates tyrosine phosphorylation of the proto-oncogene product of c-Cbl in 3T3-L1 adipocytes. Biochem J 1997; 324 : 839-45.

60. Liu J, DeYoung SM, Hwang JB, O’Leary EE, Saltiel AR. The roles of Cbl-b and c-Cbl in insulin-stimulated glucose transport. J Biol Chem 2003; 278 : 36754-62.

61. Liu J, Kimura A, Baumann CA, Saltiel AR. APS facilitates c-Cbl tyrosine phosphorylation and GLUT4 translocation in response to insulin in 3T3-L1 adipocytes. Mol Cell Biol 2002; 22 : 3599-609.

62. Chiang SH, Baumann CA, Kanzaki M, Thurmond DC, Watson RT, Neudauer CL, et al. Insulin-stimulated GLUT4 translocation requires the CAP-dependent activation of TC10. Nature 2001; 410 : 944-8.

63. Watson RT, Shigematsu S, Chiang SH, Mora S, Kanzaki M, Macara IG, et al. Lipid raft microdomain compartmentalization of TC10 is required for insulin signaling and GLUT4 translocation. J Cell Biol 2001; 154 : 829-40.

64. Baumann CA, Ribon V, Kanzaki M, Thurmond DC, Mora S, Shigematsu S, et al. CAP defines a second signalling pathway required for insulin-stimulated glucose transport. Nature 2000; 407 : 202-7.

69. Chunqiu Hou J, Pessin JE. Lipid Raft targeting of the TC10 amino terminal domain is responsible for disruption of adipocyte cortical actin. Mol Biol Cell 2003; 14 : 3578-91.

70. Inoue M, Chang L, Hwang J, Chiang SH, Saltiel AR. The exocyst complex is required for targeting of Glut4 to the plasma membrane by insulin. Nature 2003; 422 : 629-33.

71. Jiang ZY, Chawla A, Bose A, Way M, Czech MP. A phosphatidylinositol 3-kinase-independent insulin signaling pathway to N-WASP/Arp2/3/F-actin required for GLUT4 glucose transporter recycling. J Biol Chem 2002; 277 : 509-15.

72. Kanzaki M, Pessin JE. Insulin-stimulated GLUT4 translocation in adipocytes is dependent upon cortical actin remodeling. J Biol Chem 2001; 276 : 42436-44.

73. Kanzaki M, Pessin JE. Caveolin-associated filamentous actin (Cav-actin) defines a novel F-actin structure in adipocytes. J Biol Chem 2002; 277 : 25867-9.

74. Kanzaki M, Watson RT, Hou JC, Stamnes M, Saltiel AR, Pessin JE. Small GTP-binding protein TC10 differentially regulates two distinct populations of filamentous actin in 3T3L1 adipocytes. Mol Biol Cell 2002; 13 : 2334-46.

75. Maffucci T, Brancaccio A, Piccolo E, Stein RC, Falasca M. Insulin induces phosphatidylinositol-3-phosphate formation through TC10 activation. EMBO J 2003; 22 : 4178-89.

77. Tsakiridis T, Vranic M, Klip A. Disassembly of the actin network inhibits insulin-dependent stimulation of glucose transport and prevents recruitment of glucose transporters to the plasma membrane. J Biol Chem 1994; 269 : 29934-42.

80. Omata W, Shibata H, Li L, Takata K, Kojima I. Actin filaments play a critical role in insulin-induced exocytotic recruitment but not in endocytosis of GLUT4 in isolated rat adipocytes. Biochem J 2000; 346 : 321-8.

81. Brozinick JT, Jr., Hawkins ED, Strawbridge AB, Elmendorf JS. Disruption of cortical actin in skeletal muscle demonstrates an essential role of the cytoskeleton in glucose transporter 4 translocation in insulin-sensitive tissues. J Biol Chem 2004; 279 : 40699-706.

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1.3.8 Role of SNARE proteins in GLUT4 regulation

After the insulin-mediated arrival of GLUT4-containing vesicles from intracellular storage sites to the plasma membrane, regulated fusion of these vesicles ensues. Exocytosis of GLUT4-containing vesicles is mediated by interactions between specific vesicular and plasma membrane protein complexes known as SNAREs. Vesicle SNAREs (v-SNARES, vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptors) bind target membrane SNAREs (t-SNAREs) in company with numerous accessory proteins. Syntaxin4 and SNAP23 (23 kDa synaptosomal-associated protein) are the t-SNARES and VAMP2 is the v-SNARE involved in GLUT4 vesicle fusion (67) While SNAREs are essential in GLUT4 exocytosis, they themselves do not appear to be the direct targets of insulin action. Rather, studies suggest that the accessory proteins Munc18 and Synip may be regulated by insulin to accomplish fusion events. Three Munc18 isoforms (Munc18ac) have been identified in mammalian cells: Munc18a (67) is a neuronal isoform and Munc18b and Munc18c are expressed in muscle and adipose tissues. In addition to Munc18, the accessory protein Synip may also play a role in insulin stimulated GLUT4 vesicle fusion (68), although data are conflicting. Synip was first identified by Min and colleagues, who determined that this protein dissociates from syntaxin4 in an insulin-dependent manner and is directly involved in GLUT4 translocation. Recently, it was reported that Akt2 phosphorylates Synip on serine 99 and this phosphorylation mediates the Synip-syntaxin4 dissociation necessary for GLUT4 vesicle exocytosis (69). However, recent studies argue against this possibility and show that a serine-to-alanine Synip mutant (S99A) does not impair GLUT4 translocation.

1.3.9 Role of GLUT4 dysfunctions in obesity and type 2 diabetes

Insulin resistance is significantly caused by both genetic and environmental components. Mutations in the insulin receptor are rare but result in extremely severe insulin resistance. These include Leprechaunism, Rabson-Mendenhall Syndrome, and the type A syndrome of insulin resistance (70). Type 2 diabetes is polygenic, probably involving defects at numerous points in the glucose regulatory system. For example, skeletal muscle analyzed from type 2 diabetic subjects displays diminished insulin-stimulated IRS-1 tyrosine phosphorylation and decreased PI3K activity coupled to impaired glucose transport. These defects could not be explained by alterations in protein expression. Likewise, skeletal muscle and adipocytes from obese, Type 2 diabetic patients demonstrate impaired insulin-triggered IRS-1 associated [PI3K activity (71), (44).]


44. Bjornholm,M, Zierath,JR: Insulin signal transduction in human skeletal muscle: identifying the defects in Type II diabetes. Biochem.Soc.Trans. 33:354-357, 2005

67. Thurmond,DC, Pessin,JE: Molecular machinery involved in the insulin-regulated fusion of GLUT4-containing vesicles with the plasma membrane (review). Mol.Membr.Biol. 18:237-245, 2001

68. Min,J, Okada,S, Kanzaki,M, Elmendorf,JS, Coker,KJ, Ceresa,BP, Syu,LJ, Noda,Y, Saltiel,AR, Pessin,JE: Synip: a novel insulin-regulated syntaxin 4-binding protein mediating GLUT4 translocation in adipocytes. Mol.Cell 3:751-760, 1999

69. Yamada,E, Okada,S, Saito,T, Ohshima,K, Sato,M, Tsuchiya,T, Uehara,Y, Shimizu,H, Mori,M: Akt2 phosphorylates Synip to regulate docking and fusion of GLUT4- containing vesicles. J.Cell Biol. 168:921-928, 2005

70. Saltiel,AR, Kahn,CR: Insulin signalling and the regulation of glucose and lipid metabolism. Nature 414:799-806, 2001

71. Krook,A, Bjornholm,M, Galuska,D, Jiang,XJ, Fahlman,R, Myers,MG, Jr., Wallberg- Henriksson,H, Zierath,JR: Characterization of signal transduction and glucose transport in skeletal muscle from type 2 diabetic patients. Diabetes 49:284-292, 2000

SNAREing GLUT4

Following the insulin-mediated arrival of GLUT4-containing vesicles from intracellular storage sites to the plasma membrane, regulated fusion of these vesicles ensues. Exocytosis of GLUT4-containing vesicles is mediated by interactions between specific vesicular and plasma membrane protein complexes known as SNAREs (Fig. C). Vesicle SNAREs (v-SNARES, vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptors) bind target membrane SNAREs (t-SNAREs) in company with numerous accessory proteins. Syntaxin4 and SNAP23 (23 kDa synaptosomal-associated protein) are the t-SNARES and VAMP2 is the v-SNARE involved in GLUT4 vesicle fusion106. While SNAREs are essential in GLUT4 exocytosis, they themselves do not appear to be direct targets of insulin action. Rather, studies suggest that the accessory proteins Munc18 and Synip may be regulated by insulin to accomplish fusion events. Three Munc18 isoforms (Munc18ac) have been identified in mammalian cells: Munc18a is a neuronal isoform and Munc18b and Munc18c are expressed in muscle and fat. [...] In addition to Munc18c, the accessory protein Synip may also play a role in insulin-stimulated GLUT4 vesicle fusion, although data are conflicting. Synip was first identified by Min and colleagues112, who determined that this protein dissociates from syntaxin4 in an insulin-dependent manner and is directly involved in GLUT4 translocation. Recently, it was reported that Akt2 phosphorylates Synip on serine 99 and this phosphorylation mediates the Synip-syntaxin4 dissociation necessary for GLUT4 vesicle exocytosis113. However, recent studies argue against this possibility and show that a serine-to-alanine Synip mutant (S99A) does not impair GLUT4 translocation114.

[page 381]

[...]

GLUT4 dysregulation in obesity and type 2 diabetes

Abundant studies of insulin resistance demonstrate defects at numerous levels in the insulin-regulated glucose transport pathway. Insulin sensitivity is profoundly affected by both genetic and environmental components. Mutations in the insulin receptor are rare but result in extremely severe insulin resistance. These include Leprechaunism, Rabson-Mendenhall Syndrome, and the type A syndrome of insulin resistance117. Type 2 diabetes is polygenic, probably involving defects at numerous points in the glucose regulation system. For example, skeletal muscle analyzed from type 2 diabetic subjects versus lean controls displays diminished insulin-stimulated IRS-1 tyrosine phosphorylation and decreased PI3K activity coupled to impaired glucose transport118. These defects could not be explained by alterations in protein expression. Likewise, skeletal muscle and adipocytes from obese, insulin-resistant individuals demonstrate impaired insulin-triggered IRS-1 associated PI3K activity compared to matching tissue from lean individuals119,120.


106. Thurmond DC, Pessin JE. Molecular machinery involved in the insulin-regulated fusion of GLUT4-containing vesicles with the plasma membrane. Mol Membr Biol 2001; 18 : 237-45.

112. Min J, Okada S, Kanzaki M, Elmendorf JS, Coker KJ, Ceresa BP, et al. Synip: a novel insulin-regulated syntaxin 4-binding protein mediating GLUT4 translocation in adipocytes. Mol Cell 1999; 3 : 751-60.

113.Yamada E, Okada S, Saito T, Ohshima K, Sato M, Tsuchiya T, et al. Akt2 phosphorylates Synip to regulate docking and fusion of GLUT4-containing vesicles. J Cell Biol 2005; 168 : 921-8.

114. Sano H, Kane S, Sano E, Lienhard GE. Synip phosphorylation does not regulate insulin-stimulated GLUT4 translocation. Biochem Biophys Res Commun 2005; 332 : 880-4.

117. Saltiel AR, Kahn CR. Insulin signalling and the regulation of glucose and lipid metabolism. Nature 2001; 414 : 799-806.

118. Krook A, Bjornholm M, Galuska D, Jiang XJ, Fahlman R, Myers MG, Jr., et al. Characterization of signal transduction and glucose transport in skeletal muscle from type 2 diabetic patients. Diabetes 2000; 49 : 284-92.

119. Bjornholm M, Al-Khalili L, Dicker A, Naslund E, Rossner S, Zierath JR, et al. Insulin signal transduction and glucose transport in human adipocytes: effects of obesity and low calorie diet. Diabetologia 2002; 45 : 1128-35.

120. Brozinick JT, Jr., Roberts BR, Dohm GL. Defective signaling through Akt-2 and -3 but not Akt-1 in insulinresistant human skeletal muscle: potential role in insulin resistance. Diabetes 2003; 52 : 935-41.

Anmerkungen

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Impaired insulin signaling through downstream Akt2 and AS160 proteins has also been reported in skeletal muscle. Furthermore, the fatty acid metabolite ceramide causes insulin resistance that is coupled to impaired membrane recruitment and phosphorylation of Akt (72). Knowledge on mechanisms of such defects has remained underdeveloped. Increased IRS-1 serine phosphorylation may also help explain insulin resistance, as phosphorylated serine residues are thought to sterically hinder interactions with downstream PI3K. Dysregulated PKC activity in insulin resistance could increase serine phosphorylation (73) and PKC knockout mice are protected from insulin resistance (74).

Membrane and cytoskeletal defects are also a possible basis of insulin resistance. We now know that moderate increase in plasma membrane fluidity increase glucose transport . Furthermore, it has been shown that basal glucose transport is not fully active in fat cells and that it can be increased further by augmenting membrane fluidity. Consistent with membrane fluidity influencing insulin responsiveness, insulin-stimulated glucose transport is decreased when fluidity diminishes (75). Recent data suggest that the anti-diabetic drug metformin enhances insulin action by increasing membrane fluidity (76). Interestingly, the beneficial effects of chromium supplementation on insulin responsiveness may also be linked to membrane fluidity (77). With regards to cytoskeletal defects, recent study of various cell culture models of insulin resistance suggests that an underlying basis of reduced cellular insulin sensitivity may be perturbations in phosphoinositide-regulated cortical F-actin structure. In particular, PI -P2 control of cortical F-actin is disturbed by hyperinsulinaemic (78) and hyperendothelinaemic insulin-resistant conditions (79). Furthermore, isolated adipocytes from ethanol-induced insulin resistant Wistar rats (80) and skeletal muscle from obese insulin-resistant Zucker rats display altered actin polymerization. These findings agree with the necessity of an intact cytoskeleton for proper glucose regulation and suggest a membrane/cytoskeletal component of insulin resistance. Finally, some study has also revealed that insulin-resistant conditions are associated with defects in the SNARE pathway. As future research continues to expand our understanding of the signaling pathways of insulinregulated GLUT4 translocation and glucose transport, our ability to develop interventions to prevent, reverse, and ameliorate insulin resistance in obesity and type 2 diabetes will be favourably reached.


72. Teruel,T, Hernandez,R, Lorenzo,M: Ceramide mediates insulin resistance by tumor necrosis factor-alpha in brown adipocytes by maintaining Akt in an inactive dephosphorylated state. Diabetes 50:2563-2571, 2001

73. De Fea,K, Roth,RA: Protein kinase C modulation of insulin receptor substrate-1 tyrosine phosphorylation requires serine 612. Biochemistry 36:12939-12947, 1997

74. Kim,JK, Fillmore,JJ, Sunshine,MJ, Albrecht,B, Higashimori,T, Kim,DW, Liu,ZX, Soos,TJ, Cline,GW, O'Brien,WR, Littman,DR, Shulman,GI: PKC-theta knockout mice are protected from fat-induced insulin resistance. J.Clin.Invest 114:823-827, 2004

75. Czech,MP: Insulin action and the regulation of hexose transport. Diabetes 29:399-409, 1980

76. Wiernsperger,NF: Membrane physiology as a basis for the cellular effects of metformin in insulin resistance and diabetes. Diabetes Metab 25:110-127, 1999

77. Chen,G, Liu,P, Pattar,GR, Tackett,L, Bhonagiri,P, Strawbridge,AB, Elmendorf,JS: Chromium activates glucose transporter 4 trafficking and enhances insulinstimulated glucose transport in 3T3-L1 adipocytes via a cholesterol-dependent mechanism. Mol.Endocrinol. 20:857-870, 2006

78. Chen,G, Raman,P, Bhonagiri,P, Strawbridge,AB, Pattar,GR, Elmendorf,JS: Protective effect of phosphatidylinositol 4,5-bisphosphate against cortical filamentous actin loss and insulin resistance induced by sustained exposure of 3T3-L1 adipocytes to insulin. J.Biol.Chem. 279:39705-39709, 2004

79. Strawbridge,AB, Elmendorf,JS: Endothelin-1 impairs glucose transporter trafficking via a membrane-based mechanism. J.Cell Biochem. 97:849-856, 2006

80. Sebastian,BM, Nagy,LE: Decreased insulin-dependent glucose transport by chronic ethanol feeding is associated with dysregulation of the Cbl/TC10 pathway in rat adipocytes. Am.J.Physiol Endocrinol.Metab 289:E1077-E1084, 2005

Impaired insulin signaling through downstream Akt2 and AS160 proteins has also been reported in skeletal muscle51,120. Furthermore, the fatty acid metabolite ceramide causes insulin resistance that is coupled to impaired membrane recruitment and phosphorylation of Akt121. Knowledge on mechanisms of such defects has remained underdeveloped. [...] Increased IRS-1 serine phosphorylation may also help explain insulin resistance, as phosphorylated serine residues are thought to sterically hinder interactions with downstream PI3K. Dysregulated PKC activity in insulin resistance could increase serine phosphorylation124, and PKC knockout mice are protected from insulin resistance125.

Membrane and cytoskeletal defects are also a possible basis of insulin resistance. We now know that moderate increases in plasma membrane fluidity increase glucose transport126-128. Furthermore, it has been shown that basal glucose transport is not fully active in fat cells and that it can be increased further by augmenting membrane fluidity126. Consistent with membrane fluidity influencing insulin responsiveness, insulin-stimulated glucose transport is decreased when fluidity diminishes127. Recent data suggest that the anti-diabetic drug metformin enhances insulin action by increasing membrane fluidity129,130. Interestingly, the beneficial effects of chromium supplementation on insulin responsiveness may also be linked to membrane fluidity131-133. With regards to cytoskeletal defects, recent study of various cell culture models of insulin resistance suggests that an underlying basis of reduced cellular insulin sensitivity may be perturbations in phosphoinositide-regulated cortical F-actin structure. In particular, PI 4,5-P2 control of cortical F-actin is disturbed by hyperinsulinaemic134 and hyperendothelinaemic135,136 insulin-resistant conditions and reversal of these changes by experimental manipulation of PI 4,5-P2 corresponds with a restoration in insulin sensitivity. Furthermore, isolated adipocytes from ethanol-induced insulinresistant Wistar rats82 and skeletal muscle from obese insulin resistant Zucker rats137 display altered actin polymerization. These findings agree with the necessity of an intact cytoskeleton for proper glucose regulation and suggest a membrane/cytoskeletal

[page 382]

component of insulin resistance. Finally, some study has also revealed that insulin-resistant conditions are associated with defects in the SNARE machinery138. As future research continues to expand our understanding of the signaling pathways of insulinregulated GLUT4 translocation and glucose transport, our ability to develop interventions to prevent, reverse, and ameliorate insulin resistance in obesity and type 2 diabetes will be favourably impacted.


51. Karlsson HK, Zierath JR, Kane S, Krook A, Lienhard GE, Wallberg-Henriksson H. Insulin-stimulated phosphorylation of the Akt substrate AS160 is impaired in skeletal muscle of type 2 diabetic subjects. Diabetes 2005; 54 : 1692-7.

82. Sebastian BM, Nagy LE. Decreased insulin-dependent glucose transport by chronic ethanol feeding is associated with dysregulation of the Cbl/TC10 pathway in rat adipocytes. Am J Physiol Endocrinol Metab 2005; 289 : E1077-84.

120. Brozinick JT, Jr., Roberts BR, Dohm GL. Defective signaling through Akt-2 and -3 but not Akt-1 in insulinresistant human skeletal muscle: potential role in insulin resistance. Diabetes 2003; 52 : 935-41.

121.Teruel T, Hernandez R, Lorenzo M. Ceramide mediates insulin resistance by tumor necrosis factor-alpha in brown adipocytes by maintaining Akt in an inactive dephosphorylated state. Diabetes 2001; 50 : 2563-71.

124.De Fea K, Roth RA. Protein kinase C modulation of insulin receptor substrate-1 tyrosine phosphorylation requires serine 612. Biochemistry 1997; 36 : 12939-47.

125. Kim JK, Fillmore JJ, Sunshine MJ, Albrecht B, Higashimori T, Kim DW, et al. PKC-theta knockout mice are protected from fat-induced insulin resistance. J Clin Invest 2004; 114 : 823-7.

126.Czech MP. Insulin action and the regulation of hexose transport. Diabetes 1980; 29 : 399-409.

127. Pilch PF, Thompson PA, Czech MP. Coordinate modulation of D-glucose transport activity and bilayer fluidity in plasma membranes derived from control and insulin-treated adipocytes. Proc Natl Acad Sci USA 1980; 77 : 915-8.

128. Whitesell RR, Regen DM, Beth AH, Pelletier DK, Abumrad NA. Activation energy of the slowest step in the glucose carrier cycle: break at 23 degrees C and correlation with membrane lipid fluidity. Biochemistry 1989; 28 : 5618-25.

129. Muller S, Denet S, Candiloros H, Barrois R, Wiernsperger N, Donner M, et al. Action of metformin on erythrocyte membrane fluidity in vitro and in vivo. Eur J Pharmacol 1997; 337 : 103-10.

130.Wiernsperger NF. Membrane physiology as a basis for the cellular effects of metformin in insulin resistance and diabetes. Diabetes Metab 1999; 25 : 110-27.

131.Evans GW, Bowman TD. Chromium picolinate increases membrane fluidity and rate of insulin internalization. J Inorg Biochem 1992; 46 : 243-50.

132. Chen G, Liu P, Pattar GR, Tackett L, Bhonagiri P, Strawbridge AB, et al. Chromium activates GLUT4 trafficking and enhances insulin-stimulated glucose transport in 3T3-L1 adipocytes via a cholesterol-dependent mechanism. Mol Endocrinol 2006; 20 : 857-70.

133. Pattar GR, Tackett L, Liu P, Elmendorf JS. Chromium picolinate positively influences the glucose transporter system via affecting cholesterol homeostasis in adipocytes cultured under hyperglycemic diabetic conditions. Mutat Res 2006; 610 : 93-100.

134. Chen G, Raman P, Bhonagiri P, Strawbridge AB, Pattar GR, Elmendorf JS. Protective effect of phosphatidylinositol 4,5-bisphosphate against cortical filamentous actin loss and insulin resistance induced by sustained exposure of 3T3-L1 adipocytes to insulin. J Biol Chem 2004; 279 : 39705-9.

135. Strawbridge AB, Elmendorf JS. Phosphatidylinositol 4,5-bisphosphate reverses endothelin-1-induced insulin resistance via an actin-dependent mechanism. Diabetes 2005; 54 : 1698-705.

136. Strawbridge AB, Elmendorf JS. Endothelin-1 impairs glucose transporter trafficking via a membrane-based mechanism. J Cell Biochem 2006; 97 : 849-56.

137. McCarthy AM, Spisak KO, Brozinick JT, Jr., Elmendorf JS. Phosphatidylinositol 4,5-bisphosphate and cortical Factin abnormalities in insulin resistant skeletal muscle. Diabetes 2005; 54 : A319.

138.Chen G, Liu P, Thurmond DC, Elmendorf JS. Glucosamine-induced insulin resistance is coupled to Olinked glycosylation of Munc18c. FEBS Lett 2003; 534 : 54-60.

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1.4 The Serum and Glucocorticoid inducible Kinase SGK1

The Serum and Glucocorticoid inducible protein Kinase 1 (SGK1) was originally cloned in 1993 as an immediate early gene transcriptionally stimulated by serum or glucocorticoids in rat and mammary tumor cells (81;82). Transcription of SGK1 was also shown to occur rapidly in response to many agonists like mineralocorticoids(83) , follicle stimulating hormone (FSH) (84), transforming growth factor (TGF- β) (85;86), thrombin (87), hypertonicity (88-90), high glucose (85;88) and neuronal injury or excitotoxicity (88;91;92)

SGK1 belongs to the ‘AGC’ subfamily of serine/threonine protein kinases, which include protein kinase A (PKA) or adenosine 3’, 5’ monophophate (cAMP)-dependent protein kinase, protein kinase G (PKG) or guanosine 3’, 5’ monophosphate (cGMP)-dependent protein kinase and isoforms of protein kinase C (PKC). SGK1 is present in the genomes of all eukaryotic organisms examined so far, including Caenorhabditis elegans, Drosphila, fish and mammals. Structure of SGK1 has been highly conserved through evolution like many other protein kinases (90;93;94).

Two other isoforms of SGK1 that have been identified in mammals and are named as SGK2 and SGK3. The catalytic domains of SGK2 and SGK3 isoforms share 80% amino acid sequence identity with one another and with SGK1 (89). The human gene encoding SGK1 was found in chromosome 6q23 (94) whereas the gene encoding SGK2 was identified in chromosome 20q12. SGK-like gene which encodes a protein having predicted amino acid sequence identical to that of human SGK3 (95) was found in chromosome 8q12.2.

SGK1 is expressed in humans that have been studied including the pancreas, liver, heart, lung, skeletal muscle, placenta, kidney and brain (90) but SGK1 is not expressed in all cell types within those tissues. For example, SGK1 transcript levels are found high in acinar cells in the pancreas (96). High transcript levels of SGK1 are also found in the distal tubule and collecting duct of the kidney and in thick ascending limb epithethial cells (85). The expression of SGK2 mRNA is restricted in human tissues. It expresses most abundantly in liver, kidney and pancreas (89). As like SGK1, SGK3 mRNA is present in all human and murine tissues examined but expression is particularly high in the mouse, heart and spleen and in the embryo (89;97).


81. Lang,F, Cohen,P: Regulation and physiological roles of serum- and glucocorticoid-induced protein kinase isoforms. Sci.STKE<. 2001:RE17, 2001

82. Webster,MK, Goya,L, Ge,Y, Maiyar,AC, Firestone,GL: Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum. Mol.Cell Biol. 13:2031-2040, 1993

83. Brennan,FE, Fuller,PJ: Rapid upregulation of serum and glucocorticoid-regulated kinase (sgk) gene expression by corticosteroids in vivo. Mol.Cell Endocrinol. 166:129- 136, 2000

84. Alliston,TN, Maiyar,AC, Buse,P, Firestone,GL, Richards,JS: Follicle stimulating hormone-regulated expression of serum/glucocorticoid-inducible kinase in rat ovarian granulosa cells: a functional role for the Sp1 family in promoter activity. Mol.Endocrinol. 11:1934-1949, 1997

85. Lang,F, Klingel,K, Wagner,CA, Stegen,C, Warntges,S, Friedrich,B, Lanzendorfer,M, Melzig,J, Moschen,I, Steuer,S, Waldegger,S, Sauter,M, Paulmichl,M, Gerke,V, Risler,T, Gamba,G, Capasso,G, Kandolf,R, Hebert,SC, Massry,SG, Broer,S: Deranged transcriptional regulation of cell-volume-sensitive kinase hSGK in diabetic nephropathy. Proc.Natl.Acad.Sci.U.S.A 97:8157-8162, 2000

86. Waldegger,S, Klingel,K, Barth,P, Sauter,M, Rfer,ML, Kandolf,R, Lang,F: h-sgk serinethreonine protein kinase gene as transcriptional target of transforming growth factor beta in human intestine. Gastroenterology 116:1081-1088, 1999

87. Kumar,S, Harvey,KF, Kinoshita,M, Copeland,NG, Noda,M, Jenkins,NA: cDNA cloning, expression analysis, and mapping of the mouse Nedd4 gene. Genomics 40:435-443, 1997

88. Bell,LM, Leong,ML, Kim,B, Wang,E, Park,J, Hemmings,BA, Firestone,GL: Hyperosmotic stress stimulates promoter activity and regulates cellular utilization of the serum- and glucocorticoid-inducible protein kinase (Sgk) by a p38 MAPKdependent pathway. J.Biol.Chem. 275:25262-25272, 2000

89. Kobayashi,T, Deak,M, Morrice,N, Cohen,P: Characterization of the structure and regulation of two novel isoforms of serum- and glucocorticoid-induced protein kinase. Biochem.J. 344 Pt 1:189-197, 1999

90. Waldegger,S, Barth,P, Raber,G, Lang,F: Cloning and characterization of a putative human serine/threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume. Proc.Natl.Acad.Sci.U.S.A 94:4440-4445, 1997

91. Hollister,RD, Page,KJ, Hyman,BT: Distribution of the messenger RNA for the extracellularly regulated kinases 1, 2 and 3 in rat brain: effects of excitotoxic hippocampal lesions. Neuroscience 79:1111-1119, 1997

92. Imaizumi,K, Tsuda,M, Wanaka,A, Tohyama,M, Takagi,T: Differential expression of sgk mRNA, a member of the Ser/Thr protein kinase gene family, in rat brain after CNS injury. Brain Res.Mol.Brain Res. 26:189-196, 1994

93. Gonzalez-Robayna,IJ, Falender,AE, Ochsner,S, Firestone,GL, Richards,JS: Follicle- Stimulating hormone (FSH) stimulates phosphorylation and activation of protein kinase B (PKB/Akt) and serum and glucocorticoid-lnduced kinase (Sgk): evidence for A kinase-independent signaling by FSH in granulosa cells. Mol.Endocrinol. 14:1283- 1300, 2000

94. Waldegger,S, Erdel,M, Nagl,UO, Barth,P, Raber,G, Steuer,S, Utermann,G, Paulmichl,M, Lang,F: Genomic organization and chromosomal localization of the human SGK protein kinase gene. Genomics 51:299-302, 1998

95. Dai,F, Yu,L, He,H, Zhao,Y, Yang,J, Zhang,X, Zhao,S: Cloning and mapping of a novel human serum/glucocorticoid regulated kinase-like gene, SGKL, to chromosome 8q12.3-q13.1. Genomics 62:95-97, 1999

96. Klingel,K, Warntges,S, Bock,J, Wagner,CA, Sauter,M, Waldegger,S, Kandolf,R, Lang,F: Expression of cell volume-regulated kinase h-sgk in pancreatic tissue. Am.J.Physiol Gastrointest.Liver Physiol 279:G998-G1002, 2000

97. Liu,D, Yang,X, Songyang,Z: Identification of CISK, a new member of the SGK kinase family that promotes IL-3-dependent survival. Curr.Biol. 10:1233-1236, 2000

3.1 The Serum and Glucocorticoid inducible Kinase SGK1

The Serum and Glucocorticoid inducible protein Kinase 1 (SGK1) was identified in 1993 as an immediate early gene whose mRNA level increases noticeably within 30 minutes when mammary tumour or fibroblast cells are stimulated with serum or glucocorticoids1-3. SGK1 gene transcription was also shown to occur rapidly in response to many agonists like mineralocorticoids4-6, follicle stimulating hormone (FSH)7,8, transforming growth factor (TGF- β)9,10, thrombin11, hypertonicity12-14, high glucose9,11 and neuronal injury or excitotoxicity15,16.

SGK1 is a member of the ‘AGC’ subfamily of serine/threonine protein kinases, which include protein kinase A (PKA) or adenosine 3’, 5’ monophophate (cAMP)-dependent protein kinase, protein kinase G (PKG) or guanosine 3’, 5’ monophosphate (cGMP)-dependent protein kinase and isoforms of protein kinase C (PKC). SGK1 is present in the genomes of all eukaryotic organisms examined so far, including Caenorhabditis elegans, Drosphila, fish and mammals. Structure of SGK1 has been highly conserved through evolution like many other protein kinases8,13,17.

There are two other isoforms of SGK1 that have been identified in mammals and are named as SGK2 and SGK3. The catalytic domains of SGK2 and SGK3 isoforms share 80% amino acid sequence identity with one another and with SGK114. The human gene encoding SGK1 was found in chromosome 6q2317. The gene encoding SGK2 was identified in chromosome 20q12 and SGK-like gene which encodes a protein having predicted amino acid sequence identical to that of human SGK318 was found in chromosome 8q12.2.

SGK1 is expressed in all human tissues that have been studied including the pancreas, liver, heart, lung, skeletal muscle, placenta, kidney and brain13 but SGK1 is not expressed in all cell types within those tissues. For example, SGK1 transcript levels are found high in acinar cells in the pancreas19. High transcript levels of SGK1 are also found in the distal tubule and collecting duct of the kidney and in

[page 10]

thick ascending limb epithethial cells9. The expression of SGK2 mRNA is restricted in human tissues. It express most abundantly in liver, kidney and pancreas20. As like SGK1, SGK3 mRNA is present in all human and murine tissues examined but expression is particularly high in the mouse heart and spleen and in the embryo20,21.


1. Lang,F. & Cohen,P. Regulation and physiological roles of serum- and glucocorticoid-induced protein kinase isoforms. Sci. STKE. 2001, RE17 (2001).

2. Webster,M.K., Goya,L. & Firestone,G.L. Immediate-early transcriptional regulation and rapid mRNA turnover of a putative serine/threonine protein kinase. J. Biol. Chem. 268, 11482-11485 (1993).

3. Webster,M.K., Goya,L., Ge,Y., Maiyar,A.C. & Firestone,G.L. Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum. Mol. Cell Biol. 13, 2031-2040 (1993).

4. Brennan,F.E. & Fuller,P.J. Rapid upregulation of serum and glucocorticoidregulated kinase (sgk) gene expression by corticosteroids in vivo. Mol. Cell Endocrinol. 166, 129-136 (2000).

5. Chen,S.Y. et al. Epithelial sodium channel regulated by aldosterone-induced protein sgk. Proc. Natl. Acad. Sci. U. S. A 96, 2514-2519 (1999).

6. Shigaev,A., Asher,C., Latter,H., Garty,H. & Reuveny,E. Regulation of sgk by aldosterone and its effects on the epithelial Na(+) channel. Am. J. Physiol Renal Physiol 278, F613-F619 (2000).

7. Alliston,T.N., Maiyar,A.C., Buse,P., Firestone,G.L. & Richards,J.S. Follicle stimulating hormone-regulated expression of serum/glucocorticoid-inducible kinase in rat ovarian granulosa cells: a functional role for the Sp1 family in promoter activity. Mol. Endocrinol. 11, 1934-1949 (1997).

8. Gonzalez-Robayna,I.J., Falender,A.E., Ochsner,S., Firestone,G.L. & Richards,J.S. Follicle-Stimulating hormone (FSH) stimulates phosphorylation and activation of protein kinase B (PKB/Akt) and serum and glucocorticoid-lnduced kinase (Sgk): evidence for A kinase-independent signaling by FSH in granulosa cells. Mol. Endocrinol. 14, 1283-1300 (2000).

9. Lang,F. et al. Deranged transcriptional regulation of cell-volume-sensitive kinase hSGK in diabetic nephropathy. Proc. Natl. Acad. Sci. U. S. A 97, 8157- 8162 (2000).

10. Waldegger,S. et al. h-sgk serine-threonine protein kinase gene as transcriptional target of transforming growth factor beta in human intestine. Gastroenterology 116, 1081-1088 (1999).

11. Kumar,J.M., Brooks,D.P., Olson,B.A. & Laping,N.J. Sgk, a putative serine/threonine kinase, is differentially expressed in the kidney of diabetic mice and humans. J. Am. Soc. Nephrol. 10, 2488-2494 (1999).

12. Bell,L.M. et al. Hyperosmotic stress stimulates promoter activity and regulates cellular utilization of the serum- and glucocorticoid-inducible protein kinase (Sgk) by a p38 MAPK-dependent pathway. J. Biol. Chem. 275, 25262-25272 (2000).

13. Waldegger,S., Barth,P., Raber,G. & Lang,F. Cloning and characterization of a putative human serine/threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume. Proc. Natl. Acad. Sci. U. S. A 94, 4440-4445 (1997).

14. Kobayashi,T., Deak,M., Morrice,N. & Cohen,P. Characterization of the structure and regulation of two novel isoforms of serum- and glucocorticoidinduced protein kinase. Biochem. J. 344 Pt 1, 189-197 (1999).

15. Hollister,R.D., Page,K.J. & Hyman,B.T. Distribution of the messenger RNA for the extracellularly regulated kinases 1, 2 and 3 in rat brain: effects of excitotoxic hippocampal lesions. Neuroscience 79, 1111-1119 (1997).

16. Imaizumi,K., Tsuda,M., Wanaka,A., Tohyama,M. & Takagi,T. Differential expression of sgk mRNA, a member of the Ser/Thr protein kinase gene family, in rat brain after CNS injury. Brain Res. Mol. Brain Res. 26, 189-196 (1994).

17. Waldegger,S. et al. Genomic organization and chromosomal localization of the human SGK protein kinase gene. Genomics 51, 299-302 (1998).

18. Dai,F. et al. Cloning and mapping of a novel human serum/glucocorticoid regulated kinase-like gene, SGKL, to chromosome 8q12.3-q13.1. Genomics 62, 95-97 (1999).

19. Klingel,K. et al. Expression of cell volume-regulated kinase h-sgk in pancreatic tissue. Am. J. Physiol Gastrointest. Liver Physiol 279, G998-G1002 (2000).

20. Kobayashi,T. & Cohen,P. Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2. Biochem. J. 339 ( Pt 2), 319-328 (1999).

21. Liu,D., Yang,X. & Songyang,Z. Identification of CISK, a new member of the SGK kinase family that promotes IL-3-dependent survival. Curr. Biol. 10, 1233-1236 (2000).

Anmerkungen

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(Hindemith) LieschenMueller

[22.] Sj/Fragment 018 01 - Diskussion
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SGK1 has been observed as cytosolic in differentiated cells such as luteal cells (84;93) or in tumour cells arrested in the G1 phase of the cell division cycle by glucocorticoids (84;98). It has also been observed as nuclear in proliferating glomerulosa cells (84;93;98) or mammary tumour cells during the S and G2-M phases of the cell cycle{Gonzalez-Robayna, 1999 514 /id}. However, the localization of SGK1 in any given cell is regulated by extracellular signals. Thus, in serum-stimulated mammary epithelial cells, the endogenously expressed SGK1 is nuclear, but becomes cytosolic after the inhibition of phophatidylinositol (PI) 3- kinase. Translocation from the cytosol to the nucleus also occurs in response to serum stimulation of HEK293 or COS cells transfected with SGK1(99).

To become functional, SGK1 is activated by phosphorylation through a signaling cascade including phosphatidylinositol (PI) 3-kinase and phosphoinositide dependent kinase PDK1 and PDK2/H-motif kinase. While PDK1 phosphorylates SGK1 at 256Thr, PDK2/H-motif kinase phosphorylates the kinase at 422Ser. SGK2 and SGK3 may similarly be activated by PDK1 and PDK2/H-motif kinase. The equivalent phosphorylation sites for SGK2 and SGK3 are found at 193Thr/356Ser and 253Thr/419Ser, respectively (89)

Replacement of the serine at position 422 by aspartate in the human SGK1 leads to the constitutively active S422DSGK1 whereas replacement of lysine at position 127 with asparagine, within the ATP binding region required for the enzymatic activity, leads to the constitutively inactive K127NSGK1. Analogous mutations in SGK2 and SGK3 lead to the constitutively active S356DSGK2 and S419DSGK3, and the constitutively inactive K64NSGK2 and K191NSGK3 (89).

SGK isoforms resemble PKB in the substrate specificity, recognizing a serine or threonine residue lying in Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr sequence (where Xaa is a variable amino acid) and thereby phosphorylating it (89). SGK1 has a considerable physiological role through the regulation of transporters and ion channels. Sodium channel conductance stimulated by SGK1 may result in cell volume regulation (88;90;100). SGK1 mediated activation of sodium channels leads to Na+ entry which in turn depolarizes the cell membrane. The depolarized cell membrane allows the entry of chloride ions and the accumulation of NaCl that further increases the intracellular osmolarity. The osmotic gradient makes water to enter the cell by which the volume of cell increases (94). SGK1 was found to stimulate Na+, K+ and 2Cl- cotransporter activity in the thick ascending limb of the [kidney, a key nephron segment in urinary concentration, which is of importance in renal Na+ reabsorption (85).]


84. Alliston,TN, Maiyar,AC, Buse,P, Firestone,GL, Richards,JS: Follicle stimulating hormone-regulated expression of serum/glucocorticoid-inducible kinase in rat ovarian granulosa cells: a functional role for the Sp1 family in promoter activity. Mol.Endocrinol. 11:1934-1949, 1997

85. Lang,F, Klingel,K, Wagner,CA, Stegen,C, Warntges,S, Friedrich,B, Lanzendorfer,M, Melzig,J, Moschen,I, Steuer,S, Waldegger,S, Sauter,M, Paulmichl,M, Gerke,V, Risler,T, Gamba,G, Capasso,G, Kandolf,R, Hebert,SC, Massry,SG, Broer,S: Deranged transcriptional regulation of cell-volume-sensitive kinase hSGK in diabetic nephropathy. Proc.Natl.Acad.Sci.U.S.A 97:8157-8162, 2000

88. Bell,LM, Leong,ML, Kim,B, Wang,E, Park,J, Hemmings,BA, Firestone,GL: Hyperosmotic stress stimulates promoter activity and regulates cellular utilization of the serum- and glucocorticoid-inducible protein kinase (Sgk) by a p38 MAPKdependent pathway. J.Biol.Chem. 275:25262-25272, 2000

89. Kobayashi,T, Deak,M, Morrice,N, Cohen,P: Characterization of the structure and regulation of two novel isoforms of serum- and glucocorticoid-induced protein kinase. Biochem.J. 344 Pt 1:189-197, 1999

90. Waldegger,S, Barth,P, Raber,G, Lang,F: Cloning and characterization of a putative human serine/threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume. Proc.Natl.Acad.Sci.U.S.A 94:4440-4445, 1997

93. Gonzalez-Robayna,IJ, Falender,AE, Ochsner,S, Firestone,GL, Richards,JS: Follicle- Stimulating hormone (FSH) stimulates phosphorylation and activation of protein kinase B (PKB/Akt) and serum and glucocorticoid-lnduced kinase (Sgk): evidence for A kinase-independent signaling by FSH in granulosa cells. Mol.Endocrinol. 14:1283- 1300, 2000

94. Waldegger,S, Erdel,M, Nagl,UO, Barth,P, Raber,G, Steuer,S, Utermann,G, Paulmichl,M, Lang,F: Genomic organization and chromosomal localization of the human SGK protein kinase gene. Genomics 51:299-302, 1998

98. Buse,P, Tran,SH, Luther,E, Phu,PT, Aponte,GW, Firestone,GL: Cell cycle and hormonal control of nuclear-cytoplasmic localization of the serum- and glucocorticoid-inducible protein kinase, Sgk, in mammary tumor cells. A novel convergence point of anti-proliferative and proliferative cell signaling pathways. J.Biol.Chem. 274:7253-7263, 1999

99. Park,J, Leong,ML, Buse,P, Maiyar,AC, Firestone,GL, Hemmings,BA: Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI 3-kinase-stimulated signaling pathway. EMBO J. 18:3024-3033, 1999

100. Bohmer,C, Wagner,CA, Beck,S, Moschen,I, Melzig,J, Werner,A, Lin,JT, Lang,F, Wehner,F: The shrinkage-activated Na(+) conductance of rat hepatocytes and its possible correlation to rENaC. Cell Physiol Biochem. 10:187-194, 2000

SGK1 has been observed as cytosolic in differentiated cells such as luteal

cells22,23 or in tumour cells arrested in the G1 phase of the cell division cycle by glucocorticoids24. It has also been observed as nuclear in proliferating glomerulosa cells22,23 or mammary tumour cells during the S and G2-M phases of the cell cycle23. However, the localization of SGK1 in any given cell is regulated by extracellular signals. Thus, in serum-stimulated mammary epithelial cells, the endogenously expressed SGK1 is nuclear, but becomes cytosolic after the inhibition of phophatidylinositol (PI) 3-kinase. Translocation from the cytosol to the nucleus also occurs in response to serum stimulation of HEK293 or COS cells transfected with SGK125.

SGK1 is activated by phosphorylation through a signaling cascade including phosphatidylinositol (PI) 3-kinase and phosphoinositide dependent kinase PDK1 and PDK2/H-motif kinase. While PDK1 phosphorylates SGK1 at 256Thr, PDK2/Hmotif kinase phosphorylates the kinase at 422Ser. SGK2 and SGK3 may similarly be activated by PDK1 and PDK2/H-motif kinase. The equivalent phosphorylation sites for SGK2 and SGK3 are found at 193Thr/356Ser and 253Thr/419Ser, respectively14,20,25.

Replacement of the serine at position 422 by aspartate in the human SGK1 leads to the constitutively active S422DSGK1 whereas replacement of lysine at position 127 with asparagine leads to the constitutively inactive K127NSGK1. Analogous mutations in SGK2 and SGK3 lead to the constitutively active S356DSGK2 and S419DSGK3, and the constitutively inactive K64NSGK2 and K191NSGK314.

SGK isoforms resemble PKB in the substrate specificity, recognizing a serine or threonine residue lying in Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr sequence (where Xaa is a variable amino acid) and thereby phosphorylating it14,20,25.

SGK1 has a considerable physiological role through the regulation of transporters and ion channels. Sodium channel conductance stimulated by SGK1

[page 11]

may result in cell volume regulation12,13,26. SGK1 mediated activation of sodium channels leads to Na+ entry which in turn depolarizes the cell membrane. The depolarized cell membrane allows the entry of chloride ions and the accumulation of NaCl that further increases the intracellular osmolarity. The osmotic gradient makes water to enter the cell by which the volume of cell increases27,28. SGK1 was found to stimulate Na+, K+ and 2Cl- cotransporter activity in the thick ascending limb of the kidney, a key nephron segment in urinary concentration, which is of importance in renal Na+ reabsorption9.


9. Lang,F. et al. Deranged transcriptional regulation of cell-volume-sensitive kinase hSGK in diabetic nephropathy. Proc. Natl. Acad. Sci. U. S. A 97, 8157- 8162 (2000).

12. Bell,L.M. et al. Hyperosmotic stress stimulates promoter activity and regulates cellular utilization of the serum- and glucocorticoid-inducible protein kinase (Sgk) by a p38 MAPK-dependent pathway. J. Biol. Chem. 275, 25262-25272 (2000).

13. Waldegger,S., Barth,P., Raber,G. & Lang,F. Cloning and characterization of a putative human serine/threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume. Proc. Natl. Acad. Sci. U. S. A 94, 4440-4445 (1997).

14. Kobayashi,T., Deak,M., Morrice,N. & Cohen,P. Characterization of the structure and regulation of two novel isoforms of serum- and glucocorticoidinduced protein kinase. Biochem. J. 344 Pt 1, 189-197 (1999).

20. Kobayashi,T. & Cohen,P. Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2. Biochem. J. 339 ( Pt 2), 319-328 (1999).

22. Alliston,T.N., Gonzalez-Robayna,I.J., Buse,P., Firestone,G.L. & Richards,J.S. Expression and localization of serum/glucocorticoid-induced kinase in the rat ovary: relation to follicular growth and differentiation. Endocrinology 141, 385- 395 (2000).

23. Gonzalez-Robayna,I.J., Alliston,T.N., Buse,P., Firestone,G.L. & Richards,J.S. Functional and subcellular changes in the A-kinase-signaling pathway: relation to aromatase and Sgk expression during the transition of granulosa cells to luteal cells. Mol. Endocrinol. 13, 1318-1337 (1999).

24. Buse,P. et al. Cell cycle and hormonal control of nuclear-cytoplasmic localization of the serum- and glucocorticoid-inducible protein kinase, Sgk, in mammary tumor cells. A novel convergence point of anti-proliferative and proliferative cell signaling pathways. J. Biol. Chem. 274, 7253-7263 (1999).

25. Park,J. et al. Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI 3-kinase-stimulated signaling pathway. EMBO J. 18, 3024-3033 (1999).

26. Bohmer,C. et al. The shrinkage-activated Na(+) conductance of rat hepatocytes and its possible correlation to rENaC. Cell Physiol Biochem. 10, 187-194 (2000).

27. Lang,F. et al. Functional significance of cell volume regulatory mechanisms. Physiol Rev. 78, 247-306 (1998).

28. Lang,F., Busch,G.L. & Volkl,H. The diversity of volume regulatory mechanisms. Cell Physiol Biochem. 8, 1-45 (1998).

Anmerkungen

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[23.] Sj/Fragment 019 01 - Diskussion
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[SGK1 was found to stimulate Na+, K+ and 2Cl- cotransporter activity in the thick ascending limb of the] kidney, a key nephron segment in urinary concentration, which is of importance in renal Na+ reabsorption (85). Abundant SGK1 gene transcription has been observed in diabetic nephropathy (85-87), fibrosing pancreatitis (90) and inflammatory bowel disease (96) but SGK1 involvement in the formation of abnormal fibrosis tissues remains to be established.

Moreover SGK1 and its isoforms are well proved in stimulating the activity and the cell membrane abundance of several transporters and ion channels. For instance, SGK isoforms regulate the epithelial Na+ channel, ENaC (101) , the voltage-gated Na+ channel, SCN5A (101;102), the K+ channels ROMK1 (103), KCNE1/KCNQ1 (104) and Kv1.3 (105), the Na+/H+ exchanger NHE3 (103), the dicarboxylate transporter NaDCT (103), the glutamate transporters EAAT1 (106), EAAT3 (107), EAAT4 (108) and EAAT5 (109) and the Na+/K+- ATPase. The regulatory activity of SGK1 plays a diverse role in essential cell functions such as epithelial transport, excitability, cell proliferation and apoptosis.

[...]

To date, two modes of SGK1 action in regulating transporters and ion channels have been identified. It either regulates transporters by phosphorylating them at the putative consensus site (Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr) or by inhibiting the downregulating effect of protein ubiquitin ligase Nedd4-2. These two modes of regulation of SGK1 were observed in epithelial Na+ channel, ENaC (110) (Figure 5).


85. Lang,F, Klingel,K, Wagner,CA, Stegen,C, Warntges,S, Friedrich,B, Lanzendorfer,M, Melzig,J, Moschen,I, Steuer,S, Waldegger,S, Sauter,M, Paulmichl,M, Gerke,V, Risler,T, Gamba,G, Capasso,G, Kandolf,R, Hebert,SC, Massry,SG, Broer,S: Deranged transcriptional regulation of cell-volume-sensitive kinase hSGK in diabetic nephropathy. Proc.Natl.Acad.Sci.U.S.A 97:8157-8162, 2000

86. Waldegger,S, Klingel,K, Barth,P, Sauter,M, Rfer,ML, Kandolf,R, Lang,F: h-sgk serinethreonine protein kinase gene as transcriptional target of transforming growth factor beta in human intestine. Gastroenterology 116:1081-1088, 1999

87. Kumar,S, Harvey,KF, Kinoshita,M, Copeland,NG, Noda,M, Jenkins,NA: cDNA cloning, expression analysis, and mapping of the mouse Nedd4 gene. Genomics 40:435-443, 1997

90. Waldegger,S, Barth,P, Raber,G, Lang,F: Cloning and characterization of a putative human serine/threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume. Proc.Natl.Acad.Sci.U.S.A 94:4440-4445, 1997

96. Klingel,K, Warntges,S, Bock,J, Wagner,CA, Sauter,M, Waldegger,S, Kandolf,R, Lang,F: Expression of cell volume-regulated kinase h-sgk in pancreatic tissue. Am.J.Physiol Gastrointest.Liver Physiol 279:G998-G1002, 2000

101. Chen,SY, Bhargava,A, Mastroberardino,L, Meijer,OC, Wang,J, Buse,P, Firestone,GL, Verrey,F, Pearce,D: Epithelial sodium channel regulated by aldosterone-induced protein sgk. Proc.Natl.Acad.Sci.U.S.A 96:2514-2519, 1999

102. Boehmer,C, Wilhelm,V, Palmada,M, Wallisch,S, Henke,G, Brinkmeier,H, Cohen,P, Pieske,B, Lang,F: Serum and glucocorticoid inducible kinases in the regulation of the cardiac sodium channel SCN5A. Cardiovasc.Res. 57:1079-1084, 2003

103. Yun,CC, Palmada,M, Embark,HM, Fedorenko,O, Feng,Y, Henke,G, Setiawan,I, Boehmer,C, Weinman,EJ, Sandrasagra,S, Korbmacher,C, Cohen,P, Pearce,D, Lang,F: The serum and glucocorticoid-inducible kinase SGK1 and the Na+/H+ exchange regulating factor NHERF2 synergize to stimulate the renal outer medullary K+ channel ROMK1. J.Am.Soc.Nephrol. 13:2823-2830, 2002

104. Embark,HM, Bohmer,C, Vallon,V, Luft,F, Lang,F: Regulation of KCNE1-dependent K(+) current by the serum and glucocorticoid-inducible kinase (SGK) isoforms. Pflugers Arch. 445:601-606, 2003

105. Gamper,N, Fillon,S, Huber,SM, Feng,Y, Kobayashi,T, Cohen,P, Lang,F: IGF-1 upregulates K+ channels via PI3-kinase, PDK1 and SGK1. Pflugers Arch. 443:625-634, 2002

106. Boehmer,C, Henke,G, Schniepp,R, Palmada,M, Rothstein,JD, Broer,S, Lang,F: Regulation of the glutamate transporter EAAT1 by the ubiquitin ligase Nedd4-2 and the serum and glucocorticoid-inducible kinase isoforms SGK1/3 and protein kinase B. J.Neurochem. 86:1181-1188, 2003

107. Schniepp,R, Kohler,K, Ladewig,T, Guenther,E, Henke,G, Palmada,M, Boehmer,C, Rothstein,JD, Broer,S, Lang,F: Retinal colocalization and in vitro interaction of the glutamate transporter EAAT3 and the serum- and glucocorticoid-inducible kinase SGK1 [correction]. Invest Ophthalmol.Vis.Sci. 45:1442-1449, 2004

108. Rajamanickam,J, Palmada,M, Lang,F, Boehmer,C: EAAT4 phosphorylation at the SGK1 consensus site is required for transport modulation by the kinase. J.Neurochem. 102:858-866, 2007

109. Boehmer,C, Rajamanickam,J, Schniepp,R, Kohler,K, Wulff,P, Kuhl,D, Palmada,M, Lang,F: Regulation of the excitatory amino acid transporter EAAT5 by the serum and glucocorticoid dependent kinases SGK1 and SGK3. Biochem.Biophys.Res.Commun. 329:738-742, 2005

110. Debonneville,C, Flores,SY, Kamynina,E, Plant,PJ, Tauxe,C, Thomas,MA, Munster,C, Chraibi,A, Pratt,JH, Horisberger,JD, Pearce,D, Loffing,J, Staub,O: Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na(+) channel cell surface expression. EMBO J. 20:7052-7059, 2001

SGK1 was found to stimulate Na+, K+ and 2Cl- cotransporter activity in the thick ascending limb of the kidney, a key nephron segment in urinary concentration, which is of importance in renal Na+ reabsorption9. Abundant SGK1 gene transcription has been observed in diabetic nephropathy10,11,29, fibrosing pancreatitis13 and inflammatory bowel disease19 but SGK1 involvement in the formation of abnormal fibrosis tissues remains to be established.

Moreover SGK1 and its isoforms are well proved in stimulating the activity and the cell membrane abundance of several transporters and ion channels. For instance, SGK isoforms regulate the epithelial Na+ channel, ENaC5, the voltage-gated Na+ channel, SCN5A30, the K+ channels ROMK131, KCNE1/KCNQ132 and Kv1.333-35, the Na+/H+ exchanger NHE336, the dicarboxylate transporter NaDCT37, the glutamate transporters EAAT138, EAAT339, EAAT440 and EAAT541 and the Na+/K+-ATPase42. The regulatory activity of SGK1 plays a diverse role in essential cell functions such as epithelial transport, excitability, cell proliferation and apoptosis.

[page 12]

To date, two modes of SGK1 action in regulating transporters and ion channels have been identified. It either regulates transporters by phosphorylating them at the putative consensus site (Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr) or by inhibiting the downregulating effect of protein ubiquitin ligase Nedd4-2. These two modes of regulation of SGK1 were observed in epithelial Na+ channel, ENaC60,61 (Figure 1).


9. Lang,F. et al. Deranged transcriptional regulation of cell-volume-sensitive kinase hSGK in diabetic nephropathy. Proc. Natl. Acad. Sci. U. S. A 97, 8157-8162 (2000).

10. Waldegger,S. et al. h-sgk serine-threonine protein kinase gene as transcriptional target of transforming growth factor beta in human intestine. Gastroenterology 116, 1081-1088 (1999).

11. Kumar,J.M., Brooks,D.P., Olson,B.A. & Laping,N.J. Sgk, a putative serine/threonine kinase, is differentially expressed in the kidney of diabetic mice and humans. J. Am. Soc. Nephrol. 10, 2488-2494 (1999).

13. Waldegger,S., Barth,P., Raber,G. & Lang,F. Cloning and characterization of a putative human serine/threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume. Proc. Natl. Acad. Sci. U. S. A 94, 4440-4445 (1997).

19. Klingel,K. et al. Expression of cell volume-regulated kinase h-sgk in pancreatic tissue. Am. J. Physiol Gastrointest. Liver Physiol 279, G998-G1002 (2000).

29. Hoffman,B.B., Sharma,K., Zhu,Y. & Ziyadeh,F.N. Transcriptional activation of transforming growth factor-beta1 in mesangial cell culture by high glucose concentration. Kidney Int. 54, 1107-1116 (1998).

30. Boehmer,C. et al. Serum and glucocorticoid inducible kinases in the regulation of the cardiac sodium channel SCN5A. Cardiovasc. Res. 57, 1079-1084 (2003).

31. Yun,C.C. et al. The serum and glucocorticoid-inducible kinase SGK1 and the Na+/H+ exchange regulating factor NHERF2 synergize to stimulate the renal outer medullary K+ channel ROMK1. J. Am. Soc. Nephrol. 13, 2823-2830 (2002).

32. Embark,H.M., Bohmer,C., Vallon,V., Luft,F. & Lang,F. Regulation of KCNE1- dependent K(+) current by the serum and glucocorticoid-inducible kinase (SGK) isoforms. Pflugers Arch. 445, 601-606 (2003).

33. Gamper,N. et al. IGF-1 up-regulates K+ channels via PI3-kinase, PDK1 and SGK1. Pflugers Arch. 443, 625-634 (2002).

34. Gamper,N. et al. K+ channel activation by all three isoforms of serum- and glucocorticoid-dependent protein kinase SGK. Pflugers Arch. 445, 60-66 (2002).

35. Warntges,S. et al. Cerebral localization and regulation of the cell volumesensitive serum- and glucocorticoid-dependent kinase SGK1. Pflugers Arch. 443, 617-624 (2002).

36. Yun,C.C., Chen,Y. & Lang,F. Glucocorticoid activation of Na(+)/H(+) exchanger isoform 3 revisited. The roles of SGK1 and NHERF2. J. Biol. Chem. 277, 7676-7683 (2002).

37. Boehmer,C. et al. Stimulation of renal Na+ dicarboxylate cotransporter 1 by Na+/H+ exchanger regulating factor 2, serum and glucocorticoid inducible kinase isoforms, and protein kinase B. Biochem. Biophys. Res. Commun. 313, 998-1003 (2004).

38. Boehmer,C. et al. Regulation of the glutamate transporter EAAT1 by the ubiquitin ligase Nedd4-2 and the serum and glucocorticoid-inducible kinase isoforms SGK1/3 and protein kinase B. J. Neurochem. 86, 1181-1188 (2003).

39. Schniepp,R. et al. Retinal colocalization and in vitro interaction of the glutamate transporter EAAT3 and the serum- and glucocorticoid-inducible kinase SGK1 [correction]. Invest Ophthalmol. Vis. Sci. 45, 1442-1449 (2004).

40. Bohmer,C. et al. Stimulation of the EAAT4 glutamate transporter by SGK protein kinase isoforms and PKB. Biochem. Biophys. Res. Commun. 324, 1242-1248 (2004).

41. Boehmer,C. et al. Regulation of the excitatory amino acid transporter EAAT5 by the serum and glucocorticoid dependent kinases SGK1 and SGK3. Biochem. Biophys. Res. Commun. 329, 738-742 (2005).

60. Debonneville,C. et al. Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na(+) channel cell surface expression. EMBO J. 20, 7052-7059 (2001).

61. Friedrich,B. et al. The serine/threonine kinases SGK2 and SGK3 are potent stimulators of the epithelial Na+ channel alpha,beta,gamma-ENaC. Pflugers Arch. 445, 693-696 (2003).

Anmerkungen

The source is not mentioned.

By just copying the original text, Sj has eliminated the correct notation of ionic charges by superscripts

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Individuals carrying a certain variant of SGK1 [the combined presence of distinct polymorphisms in intron 6(I6CC) and in exon 8 (E866) have been shown to associated with increased blood pressure. It occurs due to enhanced stimulation of ENaC by SGK1. Moreover, the latter study revealed the role of SGK1 in the hypertension paralleling insulinemia. The outlined SGK1 gene variant may further accelerate intestinal glucose absorption by stimulation of SGLT1 and glucose accumulation in peripheral tissues including fat. Increased SGLT1 activity leads to accelerated intestinal glucose absorption, excessive insulin release, fat deposition, a subsequent decrease of plasma glucose concentration, and triggering of repeated glucose uptake and thus obesity. [page 1165]

As mentioned above, a certain variant of the SGK1 gene [the combined presence of distinct polymorphisms in intron 6 (I6CC) and in exon 8 (E8CC/CT)] has been shown to be associated with moderately enhanced blood pressure (56, 57). This correlation is presumably due to enhanced stimulation of ENaC by SGK1 in individuals carrying this variant.

[page 1166]

Moreover, the latter study revealed a relatively strong correlation between insulinemia and blood pressure in individuals carrying the SGK1 gene variant, suggesting a particular role of SGK1 in the hypertension paralleling hyperinsulinemia (339). [...]

The outlined SGK1 gene variant may further accelerate intestinal glucose absorption by stimulation of SGLT1 and glucose deposition in peripheral tissues including fat (see above). Enhanced SGLT1 activity, and subsequently accelerated intestinal glucose absorption, may lead to excessive insulin release, fat deposition, a subsequent decrease of plasma glucose concentration, and triggering of repeated glucose uptake and thus obesity (120).


[...]

Anmerkungen

The source is not given.

Note that in the source "the latter study" refers to a different study than in the dissertation of S. J.

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Sj 20 a diss.png

Figure 5. Schematic model showing molecular mechanisms of ENaC regulation by SGK1. Aldosterone binding to the mineralocorticoid receptor (MR) can stimulates the transcription of SGK1 as well as ENaC. Insulin or Insulin-like growth factor (IGF-1) phosphorylates SGK1 at Ser422 through PI3 kinase and PDK2/H-motify kinase signaling cascade. Activated (phosphorylated) SGK1 enhances ENaC plasma membrane abundance either directly by phosphorylating the channel or indirectly by inhibiting the downregulating effect of the ubiquitin ligase Nedd4-2.

Sj 20 a source.png

Figure 1 Schematic model showing molecular mechanisms of ENaC regulation by SGK1. Aldosterone binding to the mineralocorticoid receptor (MR) can stimulates the transcription of SGK1 as well as ENaC. Insulin or Insulin-like growth factor (IGF-1) phosphorylates SGK1 at Ser422 through PI3 kinase and PDK2/H-motify kinase signaling cascade. Activated (phosphorylated) SGK1 enhances ENaC plasma membrane abundance either directly by phosphorylating the channel or indirectly by inhibiting the downregulating effect of the ubiquitin ligase Nedd4-2.

Anmerkungen

No source is given.

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(Hindemith), LieschenMueller

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3.3 Xenopus laevis oocytes

One of the first and still most widely used assay system for quantifying an authentic protein biosynthetic process is the fully grown oocyte of the South African clawed frog, Xenopus laevis. The value of Xenopus laevis first became apparent in 1971, when Gurdon and co-workers discovered that the oocyte constitutes an efficient system for translating foreign messenger RNA.

The Xenopus oocyte is a cell specialized for the production and storage of proteins for later use during embryogenesis and developmentally divided into 6 stages (112). In addition, the complex architecture of the frog oocyte includes the subcellular systems involved in the export and import of proteins. Therefore, the mRNA-microinjected oocyte is an appropriate system to study the synthesis of specific polypeptides, as well as the storage of particular proteins in various subcellular organelles and the export of others into the extracellular space. Moreover, the subcellular compartmentalization, as well as the structure and biochemical, physiological, and biological properties of the synthesized protein, may be examined from exogenous proteins in the injected oocyte. For experimental studies oocytes of stages V-VI are used with a diameter of some 1.3 mm allowing easy preparation. The developmental stages V-VI are characterized by the occurence of 2 poles i.e. the vegetable (light) and the animal (dark) poles.

The main ion conductance in Xenopus oocytes is a Ca2+-dependent Cl- conductance governing the resting membrane potential close to the Cl- reversal potential of -40 mV. Despite their advantages, several precautions should be taken into consideration. First, the expression of endogenous carriers may interfere with the exogenously expressed proteins in various ways. For instance, it has been observed that injection of heterologous membrane proteins at high levels can induce endogenous channels (113). Second, due to the fact that Xenopus laevis is a poikilothermic animal, its oocytes are best kept at lower temperature and most experiments are carried out at room temperature. Hence, temperature sensitive processes i.e. protein trafficking or kinetics may be altered. Finally, since Xenopus oocytes may have different signaling pathways, precaution should be taken when studying the regulation of expressed proteins. It has been revealed that the PTH receptor regulates the internalization of the sodium-phosphate transporter NaPi, mediated by the PKA and PKC pathway.


112. Costa,PF, Emilio,MG, Fernandes,PL, Ferreira,HG, Ferreira,KG: Determination of ionic permeability coefficients of the plasma membrane of Xenopus laevis oocytes under voltage clamp. J.Physiol 413:199-211, 1989

113. Tzounopoulos,T, Maylie,J, Adelman,JP: Induction of endogenous channels by high levels of heterologous membrane proteins in Xenopus oocytes. Biophys.J. 69:904-908, 1995

1.8 Xenopus laevis oocytes and electrophysiological recording

One of the first and still most widely used assay system for quantifying an authentic protein biosynthetic process is the fully grown oocyte of the South African clawed frog, Xenopus laevis. The value of Xenopus laevis first became apparent in 1971, when Gurdon and co-workers discovered that the oocyte constitutes an efficient system for translating foreign messenger RNA (Gurdon et al., 1971).

The Xenopus oocyte is a cell specialized for the production and storage of proteins for later use during embryogenesis and developmentally divided into 6 stages (Dumont, 1972). In addition, the complex architecture of the frog oocyte includes the subcellular systems involved in the export and import of proteins. Therefore, the mRNA-microinjected oocyte is an appropriate system in which to study the synthesis of specific polypeptides, as well as the storage of particular proteins in various subcellular organelles and the export of others into the extracellular space. Moreover, the subcellular compartmentalization, as well as the structure and biochemical, physiological, and biological properties of the synthesized protein, may be examined from exogenous proteins in the injected oocyte (reviewed in Wagner et al., 2000).

For experimental studies oocytes of stages V-VI are used with a diameter of some 1.3 mm allowing easy preparation. The developmental stages V-VI are characterized by the occurence of 2 poles i.e. the vegetable (light) and the animal (dark) poles. [...] The main ion conductance in Xenopus oocytes is a Ca2+ -dependent Cl-conductance governing the resting membrane potential close to the Cl- reversal potential of -40 mV, (Dascal, 1987).

[page 31]

Despite their advantages, several precautions should be taken into consideration. First, the expression of endogenous carriers may interfere with the exogenously expressed proteins in various ways. For instance, it has been observed that injection of heterologous membrane proteins at high levels can induce endogenous channels (Tzounopoulos et al., 1995). Second, due to the fact that Xenopus laevis is a poikilothermic animal, its oocytes are best kept at lower temperature and most experiments are carried out at room temperature. Hence, temperature sensitive processes i.e. protein trafficking or kinetics may be altered (Wagner et al., 2000).

[page 32]

Finally, since Xenopus oocytes may have different signaling pathways, precaution should be taken when studying the regulation of expressed proteins. It has been revealed that the PTH receptor regulates the internalization of NaPi, mediated by the PKA and PKC pathway.

Anmerkungen

The source is not given.

Parts of this documented passage can also be found in an earlier source: Sj/Dublette/Fragment 024 01

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[27.] Sj/Fragment 025 01 - Diskussion
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[However, in NaPi-3 expressing Xenopus oocytes PKC-mediated PTH regulation] can not be observed. Instead, coupling to the PKA pathway leads to the alteration of PKA-regulated ion channels (114). Exposing the Xenopus oocytes to the regulators of intracellular signaling such as PKC activator phorbol esters may unspecifically lead to internalization of the plasma membrane and the expressed proteins (115;116). In summary, the Xenopus oocyte system has the advantage that channels, receptors and transporters can rapidly be expressed and identified by their electrophysiological properties. Once cDNA clones have been isolated, oocytes are an excellent system for correlating structure with function using a combination of molecular biological and electrophysiological techniques and analyzed both biochemically and electro physiologically in an in vivo situation.

114. Waldegger,S, Raber,G, Sussbrich,H, Ruppersberg,JP, Fakler,B, Murer,H, Lang,F, Busch,AE: Coexpression and stimulation of parathyroid hormone receptor positively regulates slowly activating IsK channels expressed in Xenopus oocytes. Kidney Int. 49:112-116, 1996

115. Vasilets,LA, Schwarz,W: Regulation of endogenous and expressed Na+/K+ pumps in Xenopus oocytes by membrane potential and stimulation of protein kinases. J.Membr.Biol. 125:119-132, 1992

116. Loo,DD, Hirsch,JR, Sarkar,HK, Wright,EM: Regulation of the mouse retinal taurine transporter (TAUT) by protein kinases in Xenopus oocytes. FEBS Lett. 392:250-254, 1996

However, in NaPi-3 expressing Xenopus oocytes PKC-mediated PTH regulation can not be observed (Wagner et al., 1996). Instead, coupling to the PKA pathway leads to the alteration of PKA-regulated ion channels (Waldegger et al., 1996). Exposing the Xenopus oocytes to the regulators of intracellular signaling such as PKC activator phorbol esters may unspecifically lead to internalization of the plasma membrane and the expressed proteins (Vasilets and Schwarz, 1992; Loo et al., 1996).

In summary, the Xenopus oocyte system has the advantage that channels, receptors and transporters can rapidly be expressed and analyzed both biochemically and electrophysiologically in an in vivo situation. The system can be used quite effectively as an assay for the functional cloning of channels that have only been identified by their electrophysiological properties. Once cDNA clones have been isolated, oocytes are an excellent system for correlating structure with function using a combination of molecular biological and electrophysiological techniques.

Anmerkungen

The source is not given.

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(Hindemith) LieschenMueller

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3.3.1 In vitro RNA transcription

In-vitro cRNA transcription involves 2 consecutive steps i.e. linearization of the plasmid DNA containing the inserted cDNA of interest by the corresponding restriction enzyme and the synthesis of RNA.

a. The inserted DNA should be cut at the 3’ end yielding a 5’ protruding or a blunt end by restriction enzyme. Plasmid DNA (10 μg) was incubated with 20 U restriction enzyme and an 10x buffer (5 μl) in a final volume of 50 μl at 37°C for 2 h or overnight.

b. To ascertain the linearization process, a 5 μl aliquot was taken out and analysed on a 1% agarose.

c. 1 volume isopropanol (50 μl) and 1/10 volume 3 M sodium acetate (5 μl) pH 5.2 was then added and incubated at room temperature for 10 min to precipitate the DNA.

d. The precipitated DNA was recovered by centrifugation at 17,000 rpm for 15 min at 4°C. The DNA pellet was washed by adding 100 μl of cold 70% ethanol to the pellet followed by centrifugation at 17,000 rpm for 5 min at 4°C. This washing stage was repeated. The DNA pellet was air dried and then resuspended in 10 μl of DNase free H2O. The concentration of DNA was determined spectrophotometrically by measuring the absorbance at 260 nm.

2.2.1 In vitro cRNA transcription

As illustrated in Fig. 12, in vitro cRNA transcription involves 2 consecutive steps i.e. linearisation of the plasmid DNA containing the inserted cDNA of interest by the corresponding restriction enzyme and the synthesis of RNA.

a. The inserted DNA should be cut at the 3’ end yielding a 5’ protruding or a blunt end by restriction enzyme. Plasmid DNA (10 μg) was incubated with 20 U restriction enzyme and an 10x buffer (5 μl) in a final volume of 50 μl at 37°C for 2 h or overnight.

b. To ascertain the linearization process, a 5 μl aliquot was taken out and analysed on a 1% agarose.

c. 1 volume isopropanol (50 μl) and 1/10 volume 3 M sodium acetate (5 μl) pH 5.2 was then added and incubated at room temperature for 10 min to precipitate the DNA.

d. The precipitated DNA was recovered by centrifugation at 17,000 rpm for 15 min at 4°C. The DNA pellet was washed by adding 100 μl of cold 70% ethanol to the pellet followed by centrifugation at 17,000 rpm for 5 min at 4°C. This washing stage was repeated. The DNA pellet was air dried and then resuspended in 10 μl of DNase free H2O. The concentration of DNA was determined spectrophotometrically by measuring the absorbance at 260 nm.

Anmerkungen

The source is not mentioned.

It is not surprising that identical procedures are used in different studies and it makes sense to describe those procedures with the same words, but it should be made transparent.

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(Hindemith) LieschenMueller

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e. 1 μg of linearised DNA was added to 1 μl rNTPS (20 nM), 2.5 μl Cap analogue (to prevent the degradation of the 5’ end of the synthesized RNA), 1 μl RNAase inhibitor (to protect the RNA from degradation by RNAase) and 2.4 μl 10 x transcription buffer(s). After mixing, 1 μl of T7 polymerase was added and the n incubated at 37°C for 1 hr. 1 μl DNase was added and the mixture was subsequently shaken for 15 min at 37°C. After addition of 100 μl DEPC-water and 125 μl phenol chloroform, the mixture was centrifuged at 13,000 rpm for 2 min. e. 1 μg of linearised DNA was added to 1 μl rNTPS (20 nM), 2.5 μl Cap analogue (to prevent the degradation of the 5’ end of the synthesized RNA), 1 μl RNAase inhibitor (to protect the RNA from degradation by RNAase) and 2.4 μl 10 x transcription buffer(s).

f. After mixing, 1 μl of T7 polymerase was added and the n incubated at 37°C for 1 h.

g. 1 μl DNase was added and the mixture was subsequently shaken for 15 min at 37°C.

h. After addition of 100 μl DEPC-water and 125 μl phenolchloroform, the mixture was centrifuged at 13,000 rpm for 2 min.

Anmerkungen

The source is not given.

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3.3.2 Preparation of Xenopus oocytes

An adult female Xenopus laevis frog was submersed in one liter of 3- aminobenzoic acid ethyl ester (0.1%) for about 15-30 min (Figure 6A). After the frog was fully anesthetized it was placed on ice for surgery.

A small abdominal incision (1 cm) was carried out and a segment of ovary was removed (Figure 6B,C). Subsequently the wound was closed with a reabsorbable suture (Figure 6D). The frog was then kept wet and warm by placing it in a cavity filled by a small amount of warm water to avoid drowning and hypothermia.

The ovarial sacs were manually separated into groups of 10-20 oocytes, put into a 15 ml tube and then enzymatically defolliculated by treatment with an OR- 2 (Oocytes-Ringer) (Table 1) solution containing 1-2 mg/ml collagenase A for 2-2.5 h at room temperature (Figure 6E) with gentle agitation. Defolliculation of the oocytes was stopped by washing several times with ND96 (Table 1).This step also removes all detritus permitting oocyte sorting. Oocytes were then sorted using a self-made apparatus (Figure 6F). Only large oocytes (stage V or VI) were selected and stored overnight in a ND96 storage solution at 16°C.

2.2.2 Preparation of oocytes

An adult female Xenopus laevis frog was submersed in one liter of 3-aminobenzoic acid ethyl ester (0.1%) for about 15-30 min (Fig. 13A). After the frog was fully anesthetized it was placed on ice for surgery. A small abdominal incision (1 cm) was carried out and a segment of ovary was removed (Fig. 13B, C). Subsequently the wound was closed with a reabsorbable suture (Fig. 13D). The frog was then kept wet and warm by placing it in a cavity filled by a small amount of warm water to avoid drowning and hypothermia.

The ovarial sacs were manually separated into groups of 10-20 oocytes, put into a 15 ml tube and then enzymatically defolliculated by treatment with an OR-2 (Oocytes-Ringer) solution containing 1-2 mg/ml collagenase A for 2-2.5 h at room temperature (Fig. 13E) with gentle agitation.

Defolliculation of the oocytes was stopped by washing several times with ND96. This step also removes all detritus permitting oocyte sorting. Oocytes were then sorted using a self-made apparatus (Fig. 13F). Only large oocytes (stage V or VI) were selected and stored overnight in a ND96 storage solution at 16°C.

Anmerkungen

Nothing has been marked as a citation.

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Sj 27 a diss.png

Figure 6. Oocytes preparation and injection. A The frog is anesthetized in 1 liter of 3-aminobenzoic acid ethyl ester (1%) in tap water at near room temperature. B The frog is placed on its back during operation. An incision about 1 cm long is made in the skin. C A small portion of the ovary is pulled out with forceps and removed with a pair of scissors. D The peritoneum and the muscle tissue are sewn up and then the skin closed off using cat gut. E The clump of oocytes is immediately transferred to a petri dish containing modified Barth medium with antibiotic. F Oocytes of stage V and VI are separated with a platinum wire loop. G For injection, the oocytes are aligned relative to the tip of the needle.

Sj 27 a source.png

Fig. 13. Steps of the oocytes preparation and injection. (A) The frog is anesthetized in 1 liter of 3-aminobenzoic acid ethyl ester (1%) in tap water at near room temperature. (B) The frog is placed on its back during operation. An incision about 1 cm long is made in the skin. (C) A small portion of the ovary is pulled out with forceps and removed with a pair of scissors. (D) The peritoneum and the muscle tissue are sewn up and then the skin closed off using cat gut. (E) The clump of oocytes is immediately transferred to a petri dish containing modified Barth medium with antibiotic. (F) Oocytes of stage V and VI are separated with a platinum wire loop. (G) For injection, the oocytes are aligned relative to the tip of the needle.

Anmerkungen

The source is not given.

The reader is given the impression that the figure shows photos of the lab work of the author.

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3.3.3 cRNA injection

After storing overnight, oocytes were injected using glass microcapillaries (filled with the required cRNA) mounted in a micromanipulator-controlled microinjector (Figure 7). Precaution was taken so that cRNA was not degraded by RNAases and that the injection capillary was not clogged with small particles. To avoid those problems several procedures were carried out such as using only sterile pipettes, gloves and DEPC treated water for dilution of cRNA. Glass capillaries were pulled using a normal puller. The tip was manually broken under the microscope (diameter of about 10-20 μm), backfilled with paraffin oil to seal the pipette from air and loaded with cRNA by suction (usually 1-2 μl).

2.2.3 cRNA injection

After storing overnight, oocytes were injected using glass microcapillaries (filled with the required cRNA) mounted in a micromanipulator-controlled microinjector (Fig. 13G). Precaution should be taken that cRNA was not contaminated with RNAases and that the injection capillary was not clogged with small particles. To avoid those problems several procedures were carried out such as using only sterile pipettes, gloves and DEPC treated water for dilution of cRNA.

Glass capillaries were pulled using a normal puller. The tip was manually broken under the microscope (diameter of about 10-20 μm), backfilled with paraffin oil to seal the pipette from air and loaded with cRNA by suction (usually 1-2 μl).

Anmerkungen

The source is not given.

Sichter
(Hindemith) LieschenMueller

[33.] Sj/Fragment 029 01 - Diskussion
Bearbeitet: 23. November 2016, 20:23 WiseWoman
Erstellt: 21. May 2015, 15:08 (Hindemith)
Embark 2004, Fragment, Gesichtet, SMWFragment, Schutzlevel sysop, Sj, Verschleierung

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Seite(n): 48, 49, Zeilen: 48: last paragraph; 49: 1ff
[Oocytes were] then placed into a 35 mm petri dish with a polypropylene mesh glued to the bottom to fix the oocytes and injected with a given volume of cRNA (usually 27.6 nl). The oocytes injected are listed in the Table 2.

After injection, oocytes were kept in storage solution at 15°C. To avoid sticking of oocytes to the petri dish or to other oocytes, the dish was gently shaken. At least every two days the storage solution was exchanged and damaged oocytes were removed to maximise the survival of the oocytes.

[page 48]

Ooytes were then placed into a 35 mm petri dish with a polypropylene mesh glued to the bottom to fix the oocytes and injected with a given volume of cRNA (usually 25 nl).

[page 49]

After injection, oocytes were kept in storage solution at 15°C. To avoid sticking of oocytes to the petri dish or to other oocytes, the dish was gently shaken. At least every two days the storage solution was exchanged and damaged oocytes were removed to maximise the survival of the oocytes.

Anmerkungen

The source is not given.

Note that after the documented text in the dissertation a figure follows (Figure 7) that is referenced on page 028. A source for that figure, datiert 15 Januar 2010, can also be found here: [2]. It has not been documented in this fragment, because it was not possible to prove that the image was on the site before the submission of the thesis.

Sichter
(Hindemith), WiseWoman

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