- Email: [email protected]
Role of protein tyrosine phosphatase-1B in diabetes and obesity
0 1999 editions
scientifiques
Biomed & Pharmacother 1999 ; 53 : 466-70 et mCdicales Elsevier SAS. All rights reserved
Dossier: Diabetes and obesity
Role of protein
tyrosine
phosphatase-1B
in diabetes and obesity
B .P. Kennedy Department
of Biochemistry and Molecular Biology, P.O. Box 1005, Pointe Claire-Dorval,
Merck Frosst Center for Therapeutic Quebec, Canada, H9R 4P8
Research,
Summary-Type
2 or non-insulin-dependent diabetes mellitus (NIDDM) is reaching epidemic proportions in industrialized countries. Obesity is a major factor in this disease, since about 75% of obese individuals will develop type 2 diabetes. There is an urgent need to develop new therapies for these diseases. Recently, the protein tyrosine phosphatase PTP-1B has been shown to be a negative regulator of the insulin signaling pathway, suggesting that inhibitors of this enzyme may be beneficial in the treatment of type 2 diabetes. Mice lacking PTP-IB are resistant to both diabetes and obesity. 0 1999 editions scientifiques et mtdicales Elsevier SAS
obesity / PTP-1B /type 2 diabetes
The insulin receptor consists of two ligand-binding a-subunits and two tyrosine kinase P-subunits [l]. When insulin binds to its receptor, the intrinsic tyrosine kinase activity of the P-subunit of the receptor is activated. This is followed by autophosphorylation of the receptor on a numberof crucial tyrosine residues,which is required for full activation of the tyrosine kinase activity [l]. The tyrosine kinase, once fully activated, phosphorylatesvarious insulin receptor substrateproteins including IRS-l, -2, -3 and -4 to propagate the insulin signalwhich leadseventually to glucoseuptake, glycogen synthesis,lipogenesisand various mitogenic events [2]. Although the events and proteins that are involved in transmitting the insulin signal are under intenseinvestigation [2,3], lesseffort hasbeendirected towards identifying the factors that turn the signal off. The counterpart of the tyrosine kinase, which adds phosphateto tyrosine residuesof the various signaling proteins, is the tyrosine phosphatase,which removes the phosphate. The equilibrium establishedbetween thesetwo enzymes controls the signalthrough the particular pathway. In the caseof the insulin receptor, the protein tyrosine phosphatase(PTP) that is responsible for the removal of phosphatefrom the tyrosine residues in the regulatory domain, hence inactivation of the receptor, is unknown. In this article, the role of the protein tyrosine phosphatasePTP- 1B in the negative regulation of insulin action is reviewed.
VANADATE AS AN INSULIN MIMETIC The involvement of PTPs in insulin action has been suggestedever sincethe discovery that vanadatecould mimic insulin activity [4]. Vanadateis a non-selective inhibitor of PTPs [5], and a number of studies have shown that treatment with vanadate can normalize blood glucoselevel in diabetes.In mouseand rat models of type 2 diabetes, chronic vanadate treatment resulted in normalization of glucose levels, lowering of insulin and triglyceride levels, and in somecases weight loss [4, 6, 71. The weight loss was not completely associatedwith the toxicity of vanadatebut due to someother mechanismaffected by vanadate treatment. For example, injection of low amountsof vanadate into a rat brain results in decreasedfood consumption and weight loss [S]. In clinical trials, vanadate improved insulin sensitivity in type 2 diabetic patients [9, lo]. The mechanismby which vanadate elicits these effects is unknown. The simplest explanation is that vanadate inhibits a negative regulatory PTP(s) that inactivates the insulin receptor. The effects of vanadate mimic the metabolic activities of insulin and do not appear to induce any of its mitogenic effects [ 111.The use of vanadate as a drug to treat type 2 diabetes is limited, due to its toxicity.
Role of protein
INVOLVEMENT
tyrosine
phosphatase-1B
OF PTPs IN INSULIN SIGNALING
A recent report suggested that the human genome encodes about 100 PTPs [ 121. This family of enzymes contains a common catalytic domain with the signature active site motif, HCXAGXXR(S/T)G, with cysteine as the active site nucleophile [ 131. The family can be classified into two groups, transmembrane receptor-like and cytosolic. PTP-1B falls into the latter category and was the first PTP to be identified [14]. PTP-1B consists of a single catalytic domain and a C-terminal tail that anchors it to the endoplasmic recticuium. The catalytic domain of PTP- 1B has been crystallized and the mechanism of phosphotyrosine catalysis established [ 131. The first suggestion that PTP-1B may be involved in the insulin signaling pathway came when the catalytic domain of PTP-IB was microinjected into frog oocytes and it prevented the insulin stimulation of both oocyte maturation and S6 peptide phosphorylation [ 151. Since then there have been many publications using a variety of techniques that have identified PTP-lB, as well as several other PTPs, most notably LAR, PTPa, PTPE and TC-PTP, as having an effect on insulin signaling [16, 171. Overexpression of PTP-1B in rat fibroblast cells significantly reduced insulin-stimulated insulin receptor autophosphorylation, whereas an inactive mutant had no effect. Similarly, rat primary adipocytes transfected with PTP- 1B cDNA inhibited translocation of the major insulin-sensitive glucose transporter, Glut 4. Osmotic loading of a PTP-1B inhibitory antibody into rat hepatoma cells increased insulin-stimulated activities in these cells, suggesting that inhibition of PTP-1B activity could augment insulin action. However, overexpression of LAR, a transmembrane receptor-like PTP, in cells resulted in reduced tyrosine phosphorylation of the insulin receptor. Conversely, antisense inhibition of LAR expression caused increased tyrosine phosphorylation of the insulin receptor. The caveat with these and many of the other in-vitro experiments is that in most cases any PTP when overexpressed can affect insulin action. This has been one of the criticisms against PTPs having specific functions since they display a lack of specificity when assayed in vitro. An important area of research that is only now being addressed is the in-vivo analysis of their activities. TARGETED
DISRUPTION OF THE MOUSE PTP-1B GENE
PTP activity has also been measured in various animal models of diabetes, and like the human studies
in diabetes
and obesity
467
described below, there are conflicting results. This is probably due to the complicated multifactorial nature of this disease, the differences in the animal models and the method for measurement of the PTP activity. For example, in the Zucker (fa/fa) rat, which has a mutation in the leptin receptor and has been used as a model of obesity and type 2 diabetes, PTP activity in skeletal muscle has been shown to be increased in one study [ 181 versus decreased in another [ 191. The reason for this is not clear but may be due to differences in methods of measurement of PTP activity. Conflicting results such as these and the lack of specificity when PTPs are overexpressed in cells suggest that alternative approaches are required to define the role of PTPs in the insulin signaling cascade. One way to determine the in-vivo function of a particular PTP is to develop selective inhibitors of that enzyme and investigate their effect in animal models. We have recently described some potent and selective peptide inhibitors of PTP- lB, but unfortunately they are not suitable for testing in vivo [20]. Non-peptide PTP1B inhibitors have also been reported but are about a loo-fold less active [21]. Alternatively, the targeted disruption of the PTP- 1B gene would be another method to examine the effect of the loss of PTP-1B enzyme activity on the insulin signaling cascade. Previous work has shown that disruption of PTP genes results in welldefined phenotypes, usually due to the impairment of specific signaling pathways (i.e., CD45 [22], PTPsigma [23], SHP-1 [24]), suggesting that the non-specificity of PTPs observed in vitro is not necessarily the case in vivo. Dismption of the PTP-1B gene in mice resulted in enhanced insulin sensitivity, in which slightly less than normal blood glucose concentrations were maintained with half the level of circulating insulin [25]. The enhanced insulin sensitivity was also observed in glucose and insulin tolerance tests and in insulinstimulated tyrosine phosphorylation of the insulin receptor, which was significantly increased in PTPlB-‘- mice compared to wild type littermate controls. One explanation of this phenotype is that PTP-1B is the insulin receptor protein tyrosine phosphatase. Its function is to dephosphotylate the activated insulin receptor, thus turning off the insulin signal. In mice, as in humans, obesity results in insulin resistance. To determine the effect of obesity on the enhanced insulin sensitivity of PTP-lB-deficient mice, they were put on a high fat diet to induce obesity. While the wild type littermates became fat and insulin-resistant, the PTP- 1B-jmice maintained insulin sensitivity and were obesityresistant. The obesity resistance observed in the PTPlB-I- mice is surprising given that these mice are
468
B.P. Kennedy
insulin-sensitive. Insulin is a potent anabolic agent promoting the storage of carbohydrates and fat, and why these mice are resistant to obesity is currently our major area of research. The targeted disruption of the mouse LAR gene, which has also been suggested to be a negative regulator of insulin signaling, has also been described. These mice have impaired mammary gland development but show no significant effects on glucose homeostasis [26]. LAR-deficient mice generated by insertional mutagenesis have also been reported, and these mice had a different phenotype [27]. They had stunted growth and were severely insulin-resistant, which would be contrary to the role of an insulin-receptor PTP. Therefore, the physiological role of LAR in insulin signaling is not clear and the results from these mice studies suggest that it is not an obvious target in diabetes. PTP ACTIVITY
IN DIABETES
There have been only a few studies examining the identities and level of PTP enzyme activities in diabetes and obesity. Measurement of the PTP activity in muscle tissue from obese insulin-resistant non-diabetic patients and lean insulin-sensitive controls was first reported by McGuire et al. [28]. They found that in insulin-resistant subjects, particulate PTP activity was 33% higher than in controls, suggesting that this increased PTP activity could be contributing to the insulin resistance observed in these patients. This was followed by a study by Kusari et al., who measured PTP activity in muscle from lean controls, obese insulin-resistant non-diabetics and obese NIDDM diabetics [29]. In contrast to the first report, obese insulin-resistant non-diabetic individuals were found to have a 21% decrease in PTP activity in the particulate muscle sample compared to lean controls. The reason for the discrepancy between these two studies is unclear and was suggested to be due to differences in the patient population used for each study. PTP activity in muscle of obese NIDDM subjects was also measured, and in this group particulate PTP activity was decreased 22%. In addition, there was a 38% decrease in the amount of PTP- 1B protein observed in the diabetic compared to non-diabetic subjects. In a more recent study, which was similar in scope to the previous one, Ahmad et al. measured PTP activity in muscle from three study groups: lean controls, insulinresistant non-diabetic individuals and obese diabetic individuals [30]. Consistent with the first study, PTP activity was elevated in soluble and particulate muscle fractions from obese insulin-resistant subjects compared to lean controls. In obese diabetic patient muscle,
PTP activity was decreased relative to lean controls. Furthermore, increased levels of PTP- 1B and LAR protein were observed in the particulate fraction of obese insulin-resistant subjects compared to lean controls. In the obese NIDDM subjects, PTP-1B and LAR protein levels were decreased in the particulate fraction compared to the controls. Finally, Worm et al. have also measured PTP activities in the particulate fraction from muscle of controls and type 2 diabetic patients and found no significant difference although soluble PTP activity was found to be significantly decreased [31]. Although there seem to be conflicting observations between the studies on PTP activities in diabetes and obesity (one has to keep in mind the limited number of subjects in these studies), it appears that PTP activities may be affected in obesity and diabetes. Combining the results from these four studies, one finds that the majority of obese non-diabetic insulin-resistant individuals have an increased particulate PTP activity in muscle. In one of the studies, this increased PTP activity coincides with increased levels of PTP-1B and LAR protein. In the obese NIDDM group, particulate and soluble PTP activity decreases, and in two studies this correlates with a decrease in PTP-1B levels, although LAR is also affected. It has also been reported that in adipose tissue from obese non-diabetic insulin-resistant subjects, PTP activity as well as PTP-1B and LAR protein levels are also increased [32]. Although this data is tenuous, the possibility exists that in the insulin-resistant state, PTP activity and PTP- 1B protein levels increase, whereas in the diabetic state both PTP activity and PTP-1B levels decrease. Whether this is the result of the normal progression for PTP- 1B expression in type 2 diabetes or just a consequence of the disease state of the tissue is not clear and requires further investigation. As discussed above, the role of PTP- 1B in human diabetes and insulin resistance is unclear. Specific PTP-1B inhibitors would certainly aid in clarifying this role; however, genetics may be another way to determine whether PTP-1B is involved in human diabetes and obesity. Recently, the mapping of a major human obesity quantitative trait locus (QTL) has been described [33, 341. QTL mapping involves a genome scan to determine if certain genetic markers can be statistically associated with a particular phenotype. The obesity QTL on chromosome 20q13.1 l-q13.2 is associated with susceptibility to obesity and hyperinsulinism. PTP-1B maps to chromosome 2Oq13. I-q13.2 [35] and could be a candidate gene for this QTL. Should this be the case, then one would predict that in these individuals there would be an overexpression or increased activity of PTP-lB, since a decrease in PTP-1B activity results in leanness.
Role of protein
tyrosine
phosphatase-1B
More work is needed to clarify this obesity QTL since other candidate genes are also located in this region of chromosome 20. CONCLUSION PTP- 1B was identified about ten years ago but its physiological role has remained elusive. The generation of the PTP-1B KO mouse has provided compelling evidence that PTP-1B functions in the insulin signaling pathway and has a major role in metabolism. Whether or not the results found in mice can be extrapolated to man is unknown and requires further investigation. Nevertheless, some of the human studies suggest that PTP activity increases in the insulin-resistant state and this correlates with increased levels of PTP-1B protein. Is this correct and is PTP-1B a factor in insulin resistance? The development of safe, specific PTP-1B inhibitors would help to answer this question. REFERENCES 1 White MF, Kahn CR. The insulin signaling system. J Biol Chem 1994 ; 269 : 1-4. 2 White ME The IRS-signaling system: a network of docking proteins that mediate insulin and cytokine action. Recent Prog Horm Res 1998 ; 53 : 119-38. 3 Virkamaki A, Ueki K, Kahn CR. Protein-protein interaction in insulin signaling and the molecular mechanisms of insulin resistance. Clin Invest 1999 ; 103 : 931-43. 4 Sekar N, Li J, ShechterY. Vanadium salts as insulin substitutes: mechanisms of action, a scientific and therapeutic tool in diabetes mellitus research. Crit Rev Biochem Mol Biol1996 : 31 : 339-59. 5 Huyer G, Liu S, Kelly J, Moffat J, Payette P, Kennedy B, et al. Mechanism of inhibition of protein-tyrosine phosphatases by vanadate and pervanadate. J Biol Chem 1997 ; 272 : 843-51. 6 Meyerovitch J, Rothenberg P, Shechter Y, Bonner-Weir S, Kahn CR. Vanadate normalizes hyperglycemia in two mouse models of non-insulin-dependent diabetes mellitus. J Clin Invest 1991 ; 87 : 1286-94. 7 Poucheret P, Verma S, Grynpas MD, McNeil1 JH. Vanadium and diabetes. Mol Cell Biochem 1998 : 188 : 73-80. 8 Meyerovitch J, ShechterY, Amir S. Vanadate stimulates in vivo glucose uptake in brain and arrests food intake and body weight gain in rats. Phvsiol Behav 1989 ; 45 : 1113-6. 9 Goldfine AB, Simonson DC, Folli F, Patti ME, Kahn CR. In vivo and in vitro studies of vanadate in human and rodent diabetes mellitus. Mol Cell Biochem 1995 ; 153 : 217-3 1. 10 Cohen N, Halberstam M, Shlimovich P, Chang CJ, Shamoon H, Rossetti L. Oral vanadyl sulfate improves hepatic and peripheral insulin sensitivity in patients with noninsulin-dependent diabetes mellitus. J Clin Invest 1995 ; 95 : 2501-9. 11 Fantus IG, Tsiani E. Multifunctional actions of vanadium compounds on insulin signaling pathways: evidence for preferential enhancement of metabolic versus mitogenic effects. Mol Cell Biochem 1998 ; 182 : 109-19. R. Protein tyrosine phosphatases: 12 Hooft van Huijsduijnen counting the trees in the forest. Gene 1998 ; 225 : t-8.
in diabetes
and obesity
469
13 Zhang ZY. Protein-tyrosine phosphatases: biological function, _ _ structural characteristics, and mechanism of catalysis. Crit Rev Biochem Mol Biol 1998 : 33 : l-52. 14 Tonks NK, Diltz CD, Fischer EH. Purification of the majorprotein-tyrosine-phosphatases of human placenta. J Biol Chem 1988 ; 263 : 6722-30. 15 Cicirelli MF, Tonks NK, Diltz CD, Weiel JE, Fischer EH, Krebs EG. Microinjection of a protein-tyrosine-phosphatase inhibits insulin action in Xenopus oocytes. Proc Nat1 Acad Sci USA 1990 ; 87 : 5514-8. 16 Byon JC, Kusari AB, Kusari J. Protein-tyrosine phosphatase1B acts as a negative regulator of insulin signal transduction. Mol Cell Biochem 1998 ; 182 : 101-8. their role in 17 Evans JL, Jallal B. Protein tyrosine phosphatases: insulin action and potential as drug targets. Exp Opin Invest Drugs 1999 ; 8 : 139-60. 18 Ahmad F, Goldstein BJ. Increased abundance of specific skeletal muscle protein-tyrosine phosphatases in a genetic model of insulin-resistant obesity and diabetes mellitus. Metabolism 1995 ; 44 : 1175-84. 19 Worm D, Handberg A, Hoppe E, Vinten J, Beck-Nielsen H. Decreased skeletal muscle phosphotyrosine phosphatase (PTPase) activity towards insulin receptors in insulin-resistant Zucker rats measured by delayed Europium fluorescence. Diabetologia 1996 ; 39 : 142-8. 20 Desmarais S. Friesen RW. Zamboni R, Ramachandran C. [Difluro(phosphono)methyl]phenylalanine-containing peptide inhibitors of protein tyrosine phosphatases. Biochem J 1999 ; 337 : 219-23. 21 Taing M, Keng YF, Shen K, Wu L, Lawrence DS, Zhang ZY. Potent and highly selective inhibitors of the protein tyrosine phosphatase-1B. Biochemistry 1999 ; 38 : 3793-803. 22 Kishihara K, Penninger J, Wallace VA, Kundig TM, Kawai K, Wakeham A, et al. Normal B lymphocyte development but impaired T cell maturation in dD45-exon6 protein tyrosine uhosuhatase-deficient mice. Cell 1993 ; 74 : 143-56. 23 Elchkbly M, Wagner J, Kennedy TE, Lanctot C, Michaliszyn E, Itie A, et al. Neuroendocrine dysplasia in mice lacking protein tyrosine phosphatase sigma. Nat Genet 1999 ; 21 : 330-3. 24 D’Ambrosio D, Hippen KL, Minskoff SA, Mellman I, Pani G, Siminovitch KA, et al. Recruitment and activation of PTPlC in negative regulation of antigen receptor signaling by Fc gamma RIIB 1. Science 1995 ; 268 : 293-7. 25 Elchebly M, Payette P, Michaliszyn E, Cromlish W, Collins S, Loy AL, et al. Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene. Science 1999 ; 283 : 1544-8. 26 Schaapveld RQ, Schepens JT, Robinson GW, Attema J, Oerlemans FT, Fransen JA, et al. Impaired mammary gland development and function in mice lacking LAR receptor-like tyrosine phosphatase activity. Dev Biol 1997 ; 188 : 134-46. 27 Ren JM, Li PM, Zhang WR, Sweet LJ, Cline G, Shulman GI, et al. Transgenic mice deficient in the LAR protein-tyrosine phosphatase exhibit profound defects in glucose homeostasis. Diabetes 1998 ; 47 : 493-7. MC, Fields RM, Nyomba BL, Raz I, Bogardus C, 28 McGuire Tonks NK, et al. Abnormal regulation of protein tyrosine phosphatase activities in skeletal muscle of insulin-resistant humans. Diabetes 1991 ; 40 : 939-42. 29 Kusari J, Kenner KA, Suh KI, Hill DE, Henry RR. Skeletal muscle protein tyrosine phosphatase activity and tyrosine phosphatase-1B protein content are associated with insulin action and resistance. J Clin Invest 1994 ; 93 : 1156-62. 30 Ahmad F, Azevedo JL, Cortright R, Dohm GL, Goldstein BJ. Alterations in skeletal muscle protein-tyrosine phosphatase
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activity and expression in insulin-resistant human besity and diabetes. J Clin Invest 1997 ; 100 : 449-58. 3 1 Worm D, Vinten J, Staehr P, Henriksen JE, Handberg A, BeckNielsen H. Altered basal and insulin-stimulated phosphotyrosine phosphatase (PTPase) activity in skeletal muscle from NIDDM patients compared with control subjects. Diabetologia 1996 ; 39 : 1208-14. 32 Ahmad F, Considine RV, Bauer TL, Ohannesian JP, Marco CC, Goldstein BJ. Improved sensitivity to insulin in obese subjects following weight loss is accompanied by reduced proteintyrosine phosphatases in adipose tissue. Metabolism 1997 ; 46 : 1140-5.
33 Lembertas AV, Perusse L, ChagnonYC, Fisler JS, Warden CH, Purcell-Huynh DA, et al. Identification of an obesity quantitative trait locus on mouse chromosome 2 and evidence of linkage to body fat and insulin on the human homologous region 20q. J Clin Invest 1997 ; 100 : 1240-7. 34 Lee JH, Reed DR. Li WD, Xu W, Joo EJ, Kilker RL, et al. Genome scan for human obesity and linkage to markers in 20a13. Am J Hum Genet 1999 : 64 : 196-209. 35 Brown-Shimer S, Johnson KA, Lawrence JB, Johnson C, Bruskin A, Green NR, et al. Molecular cloning and chromosome mapping of the human gene encoding protein phosphotyrosyl phosphatase-1B. Proc Nat1 Acad Sci USA 1990 ; 87 : 5148-52.
scientifiques
Biomed & Pharmacother 1999 ; 53 : 466-70 et mCdicales Elsevier SAS. All rights reserved
Dossier: Diabetes and obesity
Role of protein
tyrosine
phosphatase-1B
in diabetes and obesity
B .P. Kennedy Department
of Biochemistry and Molecular Biology, P.O. Box 1005, Pointe Claire-Dorval,
Merck Frosst Center for Therapeutic Quebec, Canada, H9R 4P8
Research,
Summary-Type
2 or non-insulin-dependent diabetes mellitus (NIDDM) is reaching epidemic proportions in industrialized countries. Obesity is a major factor in this disease, since about 75% of obese individuals will develop type 2 diabetes. There is an urgent need to develop new therapies for these diseases. Recently, the protein tyrosine phosphatase PTP-1B has been shown to be a negative regulator of the insulin signaling pathway, suggesting that inhibitors of this enzyme may be beneficial in the treatment of type 2 diabetes. Mice lacking PTP-IB are resistant to both diabetes and obesity. 0 1999 editions scientifiques et mtdicales Elsevier SAS
obesity / PTP-1B /type 2 diabetes
The insulin receptor consists of two ligand-binding a-subunits and two tyrosine kinase P-subunits [l]. When insulin binds to its receptor, the intrinsic tyrosine kinase activity of the P-subunit of the receptor is activated. This is followed by autophosphorylation of the receptor on a numberof crucial tyrosine residues,which is required for full activation of the tyrosine kinase activity [l]. The tyrosine kinase, once fully activated, phosphorylatesvarious insulin receptor substrateproteins including IRS-l, -2, -3 and -4 to propagate the insulin signalwhich leadseventually to glucoseuptake, glycogen synthesis,lipogenesisand various mitogenic events [2]. Although the events and proteins that are involved in transmitting the insulin signal are under intenseinvestigation [2,3], lesseffort hasbeendirected towards identifying the factors that turn the signal off. The counterpart of the tyrosine kinase, which adds phosphateto tyrosine residuesof the various signaling proteins, is the tyrosine phosphatase,which removes the phosphate. The equilibrium establishedbetween thesetwo enzymes controls the signalthrough the particular pathway. In the caseof the insulin receptor, the protein tyrosine phosphatase(PTP) that is responsible for the removal of phosphatefrom the tyrosine residues in the regulatory domain, hence inactivation of the receptor, is unknown. In this article, the role of the protein tyrosine phosphatasePTP- 1B in the negative regulation of insulin action is reviewed.
VANADATE AS AN INSULIN MIMETIC The involvement of PTPs in insulin action has been suggestedever sincethe discovery that vanadatecould mimic insulin activity [4]. Vanadateis a non-selective inhibitor of PTPs [5], and a number of studies have shown that treatment with vanadate can normalize blood glucoselevel in diabetes.In mouseand rat models of type 2 diabetes, chronic vanadate treatment resulted in normalization of glucose levels, lowering of insulin and triglyceride levels, and in somecases weight loss [4, 6, 71. The weight loss was not completely associatedwith the toxicity of vanadatebut due to someother mechanismaffected by vanadate treatment. For example, injection of low amountsof vanadate into a rat brain results in decreasedfood consumption and weight loss [S]. In clinical trials, vanadate improved insulin sensitivity in type 2 diabetic patients [9, lo]. The mechanismby which vanadate elicits these effects is unknown. The simplest explanation is that vanadate inhibits a negative regulatory PTP(s) that inactivates the insulin receptor. The effects of vanadate mimic the metabolic activities of insulin and do not appear to induce any of its mitogenic effects [ 111.The use of vanadate as a drug to treat type 2 diabetes is limited, due to its toxicity.
Role of protein
INVOLVEMENT
tyrosine
phosphatase-1B
OF PTPs IN INSULIN SIGNALING
A recent report suggested that the human genome encodes about 100 PTPs [ 121. This family of enzymes contains a common catalytic domain with the signature active site motif, HCXAGXXR(S/T)G, with cysteine as the active site nucleophile [ 131. The family can be classified into two groups, transmembrane receptor-like and cytosolic. PTP-1B falls into the latter category and was the first PTP to be identified [14]. PTP-1B consists of a single catalytic domain and a C-terminal tail that anchors it to the endoplasmic recticuium. The catalytic domain of PTP- 1B has been crystallized and the mechanism of phosphotyrosine catalysis established [ 131. The first suggestion that PTP-1B may be involved in the insulin signaling pathway came when the catalytic domain of PTP-IB was microinjected into frog oocytes and it prevented the insulin stimulation of both oocyte maturation and S6 peptide phosphorylation [ 151. Since then there have been many publications using a variety of techniques that have identified PTP-lB, as well as several other PTPs, most notably LAR, PTPa, PTPE and TC-PTP, as having an effect on insulin signaling [16, 171. Overexpression of PTP-1B in rat fibroblast cells significantly reduced insulin-stimulated insulin receptor autophosphorylation, whereas an inactive mutant had no effect. Similarly, rat primary adipocytes transfected with PTP- 1B cDNA inhibited translocation of the major insulin-sensitive glucose transporter, Glut 4. Osmotic loading of a PTP-1B inhibitory antibody into rat hepatoma cells increased insulin-stimulated activities in these cells, suggesting that inhibition of PTP-1B activity could augment insulin action. However, overexpression of LAR, a transmembrane receptor-like PTP, in cells resulted in reduced tyrosine phosphorylation of the insulin receptor. Conversely, antisense inhibition of LAR expression caused increased tyrosine phosphorylation of the insulin receptor. The caveat with these and many of the other in-vitro experiments is that in most cases any PTP when overexpressed can affect insulin action. This has been one of the criticisms against PTPs having specific functions since they display a lack of specificity when assayed in vitro. An important area of research that is only now being addressed is the in-vivo analysis of their activities. TARGETED
DISRUPTION OF THE MOUSE PTP-1B GENE
PTP activity has also been measured in various animal models of diabetes, and like the human studies
in diabetes
and obesity
467
described below, there are conflicting results. This is probably due to the complicated multifactorial nature of this disease, the differences in the animal models and the method for measurement of the PTP activity. For example, in the Zucker (fa/fa) rat, which has a mutation in the leptin receptor and has been used as a model of obesity and type 2 diabetes, PTP activity in skeletal muscle has been shown to be increased in one study [ 181 versus decreased in another [ 191. The reason for this is not clear but may be due to differences in methods of measurement of PTP activity. Conflicting results such as these and the lack of specificity when PTPs are overexpressed in cells suggest that alternative approaches are required to define the role of PTPs in the insulin signaling cascade. One way to determine the in-vivo function of a particular PTP is to develop selective inhibitors of that enzyme and investigate their effect in animal models. We have recently described some potent and selective peptide inhibitors of PTP- lB, but unfortunately they are not suitable for testing in vivo [20]. Non-peptide PTP1B inhibitors have also been reported but are about a loo-fold less active [21]. Alternatively, the targeted disruption of the PTP- 1B gene would be another method to examine the effect of the loss of PTP-1B enzyme activity on the insulin signaling cascade. Previous work has shown that disruption of PTP genes results in welldefined phenotypes, usually due to the impairment of specific signaling pathways (i.e., CD45 [22], PTPsigma [23], SHP-1 [24]), suggesting that the non-specificity of PTPs observed in vitro is not necessarily the case in vivo. Dismption of the PTP-1B gene in mice resulted in enhanced insulin sensitivity, in which slightly less than normal blood glucose concentrations were maintained with half the level of circulating insulin [25]. The enhanced insulin sensitivity was also observed in glucose and insulin tolerance tests and in insulinstimulated tyrosine phosphorylation of the insulin receptor, which was significantly increased in PTPlB-‘- mice compared to wild type littermate controls. One explanation of this phenotype is that PTP-1B is the insulin receptor protein tyrosine phosphatase. Its function is to dephosphotylate the activated insulin receptor, thus turning off the insulin signal. In mice, as in humans, obesity results in insulin resistance. To determine the effect of obesity on the enhanced insulin sensitivity of PTP-lB-deficient mice, they were put on a high fat diet to induce obesity. While the wild type littermates became fat and insulin-resistant, the PTP- 1B-jmice maintained insulin sensitivity and were obesityresistant. The obesity resistance observed in the PTPlB-I- mice is surprising given that these mice are
468
B.P. Kennedy
insulin-sensitive. Insulin is a potent anabolic agent promoting the storage of carbohydrates and fat, and why these mice are resistant to obesity is currently our major area of research. The targeted disruption of the mouse LAR gene, which has also been suggested to be a negative regulator of insulin signaling, has also been described. These mice have impaired mammary gland development but show no significant effects on glucose homeostasis [26]. LAR-deficient mice generated by insertional mutagenesis have also been reported, and these mice had a different phenotype [27]. They had stunted growth and were severely insulin-resistant, which would be contrary to the role of an insulin-receptor PTP. Therefore, the physiological role of LAR in insulin signaling is not clear and the results from these mice studies suggest that it is not an obvious target in diabetes. PTP ACTIVITY
IN DIABETES
There have been only a few studies examining the identities and level of PTP enzyme activities in diabetes and obesity. Measurement of the PTP activity in muscle tissue from obese insulin-resistant non-diabetic patients and lean insulin-sensitive controls was first reported by McGuire et al. [28]. They found that in insulin-resistant subjects, particulate PTP activity was 33% higher than in controls, suggesting that this increased PTP activity could be contributing to the insulin resistance observed in these patients. This was followed by a study by Kusari et al., who measured PTP activity in muscle from lean controls, obese insulin-resistant non-diabetics and obese NIDDM diabetics [29]. In contrast to the first report, obese insulin-resistant non-diabetic individuals were found to have a 21% decrease in PTP activity in the particulate muscle sample compared to lean controls. The reason for the discrepancy between these two studies is unclear and was suggested to be due to differences in the patient population used for each study. PTP activity in muscle of obese NIDDM subjects was also measured, and in this group particulate PTP activity was decreased 22%. In addition, there was a 38% decrease in the amount of PTP- 1B protein observed in the diabetic compared to non-diabetic subjects. In a more recent study, which was similar in scope to the previous one, Ahmad et al. measured PTP activity in muscle from three study groups: lean controls, insulinresistant non-diabetic individuals and obese diabetic individuals [30]. Consistent with the first study, PTP activity was elevated in soluble and particulate muscle fractions from obese insulin-resistant subjects compared to lean controls. In obese diabetic patient muscle,
PTP activity was decreased relative to lean controls. Furthermore, increased levels of PTP- 1B and LAR protein were observed in the particulate fraction of obese insulin-resistant subjects compared to lean controls. In the obese NIDDM subjects, PTP-1B and LAR protein levels were decreased in the particulate fraction compared to the controls. Finally, Worm et al. have also measured PTP activities in the particulate fraction from muscle of controls and type 2 diabetic patients and found no significant difference although soluble PTP activity was found to be significantly decreased [31]. Although there seem to be conflicting observations between the studies on PTP activities in diabetes and obesity (one has to keep in mind the limited number of subjects in these studies), it appears that PTP activities may be affected in obesity and diabetes. Combining the results from these four studies, one finds that the majority of obese non-diabetic insulin-resistant individuals have an increased particulate PTP activity in muscle. In one of the studies, this increased PTP activity coincides with increased levels of PTP-1B and LAR protein. In the obese NIDDM group, particulate and soluble PTP activity decreases, and in two studies this correlates with a decrease in PTP-1B levels, although LAR is also affected. It has also been reported that in adipose tissue from obese non-diabetic insulin-resistant subjects, PTP activity as well as PTP-1B and LAR protein levels are also increased [32]. Although this data is tenuous, the possibility exists that in the insulin-resistant state, PTP activity and PTP- 1B protein levels increase, whereas in the diabetic state both PTP activity and PTP-1B levels decrease. Whether this is the result of the normal progression for PTP- 1B expression in type 2 diabetes or just a consequence of the disease state of the tissue is not clear and requires further investigation. As discussed above, the role of PTP- 1B in human diabetes and insulin resistance is unclear. Specific PTP-1B inhibitors would certainly aid in clarifying this role; however, genetics may be another way to determine whether PTP-1B is involved in human diabetes and obesity. Recently, the mapping of a major human obesity quantitative trait locus (QTL) has been described [33, 341. QTL mapping involves a genome scan to determine if certain genetic markers can be statistically associated with a particular phenotype. The obesity QTL on chromosome 20q13.1 l-q13.2 is associated with susceptibility to obesity and hyperinsulinism. PTP-1B maps to chromosome 2Oq13. I-q13.2 [35] and could be a candidate gene for this QTL. Should this be the case, then one would predict that in these individuals there would be an overexpression or increased activity of PTP-lB, since a decrease in PTP-1B activity results in leanness.
Role of protein
tyrosine
phosphatase-1B
More work is needed to clarify this obesity QTL since other candidate genes are also located in this region of chromosome 20. CONCLUSION PTP- 1B was identified about ten years ago but its physiological role has remained elusive. The generation of the PTP-1B KO mouse has provided compelling evidence that PTP-1B functions in the insulin signaling pathway and has a major role in metabolism. Whether or not the results found in mice can be extrapolated to man is unknown and requires further investigation. Nevertheless, some of the human studies suggest that PTP activity increases in the insulin-resistant state and this correlates with increased levels of PTP-1B protein. Is this correct and is PTP-1B a factor in insulin resistance? The development of safe, specific PTP-1B inhibitors would help to answer this question. REFERENCES 1 White MF, Kahn CR. The insulin signaling system. J Biol Chem 1994 ; 269 : 1-4. 2 White ME The IRS-signaling system: a network of docking proteins that mediate insulin and cytokine action. Recent Prog Horm Res 1998 ; 53 : 119-38. 3 Virkamaki A, Ueki K, Kahn CR. Protein-protein interaction in insulin signaling and the molecular mechanisms of insulin resistance. Clin Invest 1999 ; 103 : 931-43. 4 Sekar N, Li J, ShechterY. Vanadium salts as insulin substitutes: mechanisms of action, a scientific and therapeutic tool in diabetes mellitus research. Crit Rev Biochem Mol Biol1996 : 31 : 339-59. 5 Huyer G, Liu S, Kelly J, Moffat J, Payette P, Kennedy B, et al. Mechanism of inhibition of protein-tyrosine phosphatases by vanadate and pervanadate. J Biol Chem 1997 ; 272 : 843-51. 6 Meyerovitch J, Rothenberg P, Shechter Y, Bonner-Weir S, Kahn CR. Vanadate normalizes hyperglycemia in two mouse models of non-insulin-dependent diabetes mellitus. J Clin Invest 1991 ; 87 : 1286-94. 7 Poucheret P, Verma S, Grynpas MD, McNeil1 JH. Vanadium and diabetes. Mol Cell Biochem 1998 : 188 : 73-80. 8 Meyerovitch J, ShechterY, Amir S. Vanadate stimulates in vivo glucose uptake in brain and arrests food intake and body weight gain in rats. Phvsiol Behav 1989 ; 45 : 1113-6. 9 Goldfine AB, Simonson DC, Folli F, Patti ME, Kahn CR. In vivo and in vitro studies of vanadate in human and rodent diabetes mellitus. Mol Cell Biochem 1995 ; 153 : 217-3 1. 10 Cohen N, Halberstam M, Shlimovich P, Chang CJ, Shamoon H, Rossetti L. Oral vanadyl sulfate improves hepatic and peripheral insulin sensitivity in patients with noninsulin-dependent diabetes mellitus. J Clin Invest 1995 ; 95 : 2501-9. 11 Fantus IG, Tsiani E. Multifunctional actions of vanadium compounds on insulin signaling pathways: evidence for preferential enhancement of metabolic versus mitogenic effects. Mol Cell Biochem 1998 ; 182 : 109-19. R. Protein tyrosine phosphatases: 12 Hooft van Huijsduijnen counting the trees in the forest. Gene 1998 ; 225 : t-8.
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