Lipid phosphatases as a possible therapeutic target in cases of type 2 diabetes and obesity

Pharmacology & Therapeutics 112 (2006) 799 – 809 www.elsevier.com/locate/pharmthera

Associate editor: I. Kimura

Lipid phosphatases as a possible therapeutic target in cases of type 2 diabetes and obesity Toshiyasu Sasaoka ⁎, Tsutomu Wada, Hiroshi Tsuneki Department of Clinical Pharmacology, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan

Abstract Phosphatidyl inositol 3-kinase (PI3-kinase) functions as a lipid kinase to produce PI(3,4,5)P3 from PI(4,5)P2 in vivo. PI(3,4,5)P3 is crucial as a lipid second messenger in various metabolic effects of insulin. Lipid phosphatases, src homology 2 domain containing inositol 5′-phosphatase 2 (SHIP2) and skeletal muscle and kidney-enriched inositol phosphatase (SKIP) hydrolyze PI(3,4,5)P3 to PI(3,4)P2 and phosphatase and tensin homolog deleted on chromosome ten (PTEN) hydrolyzes PI(3,4,5)P3 to PI(4,5)P2. SHIP2 negatively regulates insulin signaling relatively specifically via its 5′-phosphatase activity. Targeted disruption of the SHIP2 gene in mice resulted in increased insulin sensitivity and conferred protection from obesity induced by a high-fat diet. Polymorphisms in the human SHIP2 gene are associated, at least in part, with the insulin resistance of type 2 diabetes. Importantly, inhibition of endogenous SHIP2 through the liver-specific expression of a dominant-negative SHIP2 improves glucose metabolism and insulin resistance in diabetic db/db mice. Overexpression of PTEN and SKIP also inhibited insulin-induced phosphorylation of Akt and the uptake of glucose in cultured cells. Although a homozygous disruption of the PTEN gene in mice results in embryonic lethality, either skeletal muscle or adipose tissue-specific disruption of PTEN ameliorated glucose metabolism without formation of tumors in animal models of diabetes. The role of SKIP in glucose metabolism remains to be further clarified in vivo. Taken together, inhibition of endogenous SHIP2 in the whole body appears to be effective at improving the insulin resistance associated with type 2 diabetes and/or obesity. Inhibition of PTEN in the tissues specifically targeted, including skeletal muscle and fat, may result in an amelioration of insulin resistance in type 2 diabetes, although caution against the formation of tumors is needed. © 2006 Elsevier Inc. All rights reserved. Keywords: Lipid phosphatase; SHIP2; PTEN; SKIP; Type 2 diabetes; Obesity Abbreviations: GSK, glycogen synthase kinase; IRS, insulin receptor substrate; PI3-kinase, phosphatidyl inositol 3-kinase; PKC, protein kinase C; PTEN, phosphatase and tensin homolog deleted on chromosome ten; SHIP2, src homology 2 domain containing inositol 5′-phosphatase 2; SKIP, skeletal muscle and kidney-enriched inositol phosphatase.

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Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . SHIP2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Role of SHIP2 in cultured cells . . . . . . . . . . . . 2.2. Role of SHIP2 in mice . . . . . . . . . . . . . . . . 2.3. SHIP2 gene polymorphisms in human subjects . . . . 2.4. Impact of inhibition of endogenous SHIP2 on glucose PTEN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Role of PTEN in cultured cells . . . . . . . . . . . . 3.2. Role of PTEN in mice . . . . . . . . . . . . . . . . 3.3. PTEN gene polymorphisms in human subjects . . . .

⁎ Corresponding author. Tel.: +81 76 434 7550; fax: +81 76 434 5067. E-mail address: [email protected] (T. Sasaoka). 0163-7258/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2006.06.001

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Role of lipid phosphatases in brain function . . . . . . . . . . . . . 4.1. Expression of lipid phosphatases in brain . . . . . . . . . . . 4.2. Role of lipid phosphatases in neurodegenerative disorders . . 4.3. Role of lipid phosphatases in feeding and energy expenditure. 5. Role of lipid phosphatases in atherosclerosis . . . . . . . . . . . . . 6. SKIP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Type 2 diabetes mellitus is characterized by insulin resistance caused by a defect in glucose uptake in skeletal muscle and fat, and impaired glucose production in the liver (White, 2002; Taniguchi et al., 2006). The biological actions of insulin are initiated by binding to the insulin receptor, resulting in phosphorylation of insulin receptor substrates (IRS) at tyrosine residues (Saltiel & Kahn, 2001; White, 2002). The tyrosine-phosphorylated IRS binds to the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase, resulting in the activation of the p110 catalytic subunit (White, 2002; Taniguchi et al., 2006). Phosphatidyl inositol 3-kinase (PI3-kinase) acts as a lipid kinase producing PI(3,4,5)P3 from PI(4,5)P2 in vivo (Shepherd et al., 1998). PI(3,4,5)P3 is a key lipid second messenger in various metabolic effects of insulin (Saltiel & Kahn, 2001; Khan & Pessin, 2002). PI(3,4,5)P3 mediates the

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transfer of signals to downstream molecules including Akt and atypical protein kinase C (PKC) (Sasaoka et al., 2001; Wada et al., 2001). These are the key signaling molecules in the activation of glucose uptake in skeletal muscle and fat tissue. The PI3-kinase-dependent pathway is also important in the regulation of mRNA expression for gluconeogenesis, glycolysis, and lipid synthesis in the liver (Miyake et al., 2002; Taniguchi et al., 2006). In addition to the crucial role that PI3kinase plays in controlling the glucose homeostasis in peripheral target tissues of insulin, increasing evidence indicates a key role for PI3-kinase pathway in the control of feeding and energy expenditure in the central nervous system (CNS) (Morrison et al., 2005; Morton et al., 2005). Lipid phosphatases hydrolyze the important PI3-kinase product PI(3,4,5)P3 (Fig. 1). Src homology 2 domain containing inositol 5′-phosphatase 2 (SHIP2) and skeletal muscle and kidney-enriched inositol phosphatase (SKIP) were identified as 5′-lipid phosphatases that hydrolyze PI(3,4,5)P3 to PI(3,4)P2 (Pesesse et al., 1997; Ishihara et al., 1999; Ijuin et al., 2000). Phosphatase and tensin homolog deleted on chromosome ten (PTEN) acts as a 3′-phosphatase that hydrolyzes PI(3,4,5)P3 to PI(4,5)P2 (Maehama & Dixon, 1998). In this article, we discuss the impact of negative regulation of the PI3-kinase pathway by these lipid phosphatases on glucose homeostasis and energy expenditure in a state of insulin resistance as well as in physiological states. We aim to clarify the effect of inhibition of the lipid phosphatase on the amelioration of insulin resistance in type 2 diabetes and prevention of obesity. 2. SHIP2 2.1. Role of SHIP2 in cultured cells

Fig. 1. Regulation of insulin signaling by lipid phosphatases in skeletal muscle and fat. The 5′-lipid phosphatase SHIP2 and SKIP hydrolyze the PI3-kinase product PI(3,4,5)P3 to PI(3,4)P2, and the 3′-lipid phosphatase PTEN hydrolyzes PI(3,4,5)P3 to PI(4,5)P2. The regulatory activity of these lipid phosphatases attenuates the activity of downstream targets of PI3-kinase including Akt and atypical PKC, resulting in decreased glucose uptake and glycogen synthesis.

We and Pesesse et al. identified SHIP2 as a 5′-phosphatase that hydrolyzes the PI3-kinase product PI(3,4,5)P3 to PI(3,4)P2 (Pesesse et al., 1997; Ishihara et al., 1999). Although the expression of SHIP1 is mainly limited to hematopoietic tissue, SHIP2 is more ubiquitously expressed including in skeletal muscle, heart, and brain, and to a lesser extent in liver and kidney (Pesesse et al., 1997; Vollenweider et al., 1999). We showed that adenovirus-mediated expression of wild-type SHIP2 inhibited insulin-induced activation of Akt and atypical PKC leading to decreased glucose uptake and glycogen synthesis, whereas these actions of insulin were enhanced by expression of a dominantnegative SHIP2 which lacks catalytic activity in 3T3-L1 adipocytes (Wada et al., 2001; Murakami et al., 2004). Insulin-

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(3,4,5)P3 production, although only insulin is able to stimulate glucose uptake via the PI3-kinase (Sasaoka et al., 2004). In this context, SHIP2 is specifically translocated from the cytosol to plasma membrane in response to insulin, and SHIP2 mainly regulates insulin-induced phosphorylation of Akt2 at the plasma membrane (Ishihara et al., 2002; Sasaoka et al., 2004). Furthermore, SHIP2 appears to regulate both IRS-1 and IRS-2mediated phosphorylation of Akt (Sasaoka et al., 2004). By these possible molecular mechanisms, SHIP2 appears to function as a relatively specific negative regulator of insulin signaling in target tissues. 2.2. Role of SHIP2 in mice

Fig. 2. Molecular mechanisms by which insulin activates SHIP2. Most SHIP2 localizes to the cytosol in the basal state. Insulin induces the redistribution of a small amount of SHIP2 from the cytosol to the plasma membrane. The association of SHIP2 with Shc appears to be required for the translocation at least in specific cell types. SHIP2 dehydrolyzes PI(3,4,5)P3 at the plasma membrane, a process which is generated by both IRS-1 and IRS-2-mediated activation of PI3-kinase. Interestingly, SHIP2 negatively regulates insulininduced phosphorylation of Akt2, but not Akt1, at the plasma membrane.

induced glycogen synthesis via glycogen synthase kinase (GSK) 3β and protein phosphatase 1 (PP1) was also inhibited by expression of wild-type SHIP2, whereas it was enhanced by expression of the dominant-negative SHIP2 in 3T3-L1 adipocytes and L6 myocytes (Sasaoka et al., 2001; Wada et al., 2001) (Fig. 1). These results indicate that SHIP2, via its 5′-phosphatase activity, negatively regulates insulin signaling leading to glucose uptake and glycogen synthesis in cultured fat and skeletal muscle cells. Since SHIP2 appears to be a physiologically important negative regulator of insulin signaling, clarification of the molecular mechanism by which SHIP2 is activated in response to insulin is important (Fig. 2). In this regard, we focused on the regulation of the phosphorylation of Akt, which is one of the key targets of PI3-kinase in the mediation of insulin's metabolic action (Cho et al., 2001a, 2001b; Easton et al., 2005). Akt has 3 isoforms; Akt1, Akt2, and Akt3. Akt1 and Akt2 are broadly expressed and play crucial roles in insulin's action, whereas the expression of Akt3 is limited to the brain and testis (Cho et al., 2001a, 2001b; Easton et al., 2005). Mice with a targeted disruption of Akt1 demonstrate a defect in growth. However, Akt1 knockout mice are normal with respect to glucose homeostasis (Cho et al., 2001a). In contrast, mice lacking Akt2 are normal in size but show insulin resistance in the target tissues of insulin (Cho et al., 2001b). These results indicate that the PI3-kinase/Akt2 pathway, and not the PI3-kinase/Akt1 pathway, links insulin signaling to the metabolic actions of insulin. We showed that SHIP2 regulates insulin-induced phosphorylation of Akt2 but not Akt1 in 3T3-L1 adipocytes (Sasaoka et al., 2004). In addition, both insulin and platelet-derived growth factor (PDGF) activate PI3-kinase and PI

Clément et al. (2001) reported an analysis of mice with a targeted disruption of SHIP2 (Table 1). The mice exhibited postnatal hypoglycemia and died within 3 days of birth. The expression of genes implicated in hepatic glucose production including the genes for PEPCK and G6Pase was markedly decreased in the mice. Notably, mice with a heterozygous knockout of SHIP2 showed enhanced insulin sensitivity. In addition, other biological systems were considered to be not impaired based on a histological analysis in these mice (Clément et al., 2001). These results indicated that SHIP2 is a key negative regulator of insulin signaling in vivo. However, the targeting construct used in the knockout mice was made to Table 1 Features of glucose metabolism in lipid phosphatase knockout mice Phenotype SHIIP2 Knockout 1. First SHIP2 KO (SHIP2−/− and Phox2a−/−) (Clément et al., 2001) 2. Second SHIP2 KO (Sleeman et al., 2005)

PTEN Knockout 1. General PTEN KO (Di Cristofano et al., 1998; Stambolic et al., 1998) 2. Liver-specific PTEN KO (Stiles et al., 2004; Horie et al., 2004) 3. Muscle-specific PTEN KO (Wijesekara et al., 2005)

• Postneonatal death due to severe hypoglycemia in SHIP2−/− mice. • Increased sensitivity to insulin in SHIP2+/− mice. • Blood glucose and insulin levels were not significantly changed, whereas phosphorylation of Akt was enhanced in liver and skeletal muscle under normal chow diet. • Body weight gain is mild, and blood glucose and insulin levels were ameliorated under high-fat diet.

• Embryonic lethal due to tumor formation.

• Enhanced insulin action in liver with systemically improved glucose metabolism. Hepatomegaly and hepatosteatosis are accompanied. • No apparent change in glucose metabolism and body weight under normal chow. Skeletal muscle insulin action was enhanced with amelioration of insulin resistance by high-fat diet. 4. Fat-specific PTEN KO • Improved systemic glucose tolerance and insulin (Kuralwalla-Martinez sensitivity, and protective from developing diabetes et al., 2005) by STZ injection. 5. Pancreas-specific • Increased pancreatic β cell mass, and protective PTEN KO (Stiles from developing diabetes by STZ injection. et al., 2006) SKIP Knockout; unestablished.

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delete the C-terminal exons 19–28 of the SHIP2 gene, and the 18 N-terminal exons including the SH2 domain and the 5′phosphatase domain might have remained in the mice. In addition, the targeting construct also resulted in deletion of the neighboring Phox2a gene (Clément et al., 2004). The Phox2a gene product is known to function as a transcription factor involved in neuronal development and differentiation, and the relation of Phox2a with glucose homeostasis is so far unclear (Clément et al., 2004). Therefore, it is uncertain whether the phenotype seen in the first knockout mice is derived from the deletion of either the SHIP2 gene, the Phox2a gene, or both. Recently, Sleeman et al. generated a second SHIP2 knockout mouse that lacks the entire SHIP2 gene but possesses an intact Phox2a gene (Sleeman et al., 2005). Thus, a targeting construct for the second SHIP2 knockout mice was made to delete the first 18 exons encoding the SHIP2 gene, whereby the remaining C-terminus was also ablated. The phenotype of the second SHIP2 knockout mice was different from that of the first. The second mice had truncated facial features caused by an abnormality in skeletal growth, and reached adulthood, in contrast to the first. Insulin-stimulated phosphorylation of Akt was enhanced in the liver and skeletal muscle of the second SHIP2 null mice. Regardless of the enhancement, glucose and insulin levels were not significantly different between the SHIP2 knockout and control mice, although the trend of glucose and insulin levels was lower in the second SHIP2 knockout mice. Interestingly, the second SHIP2 knockout mice did not become obese when fed a 45% high-fat diet for 6 weeks. In this regard, these knockout mice were not insulin resistant, whereas the control mice were, when fed the high-fat diet (Sleeman et al., 2005). It remains to be elucidated why enhanced phosphorylation of Akt in the skeletal muscle and liver does not apparently affect the glucose homeostasis in the second SHIP2 knockout mice fed a normal chow diet. Although there is a difference in phenotype between the 2 SHIP2 knockout mice, it is apparent that SHIP2 is key to the control of glucose homeostasis and energy expenditure especially in diabetes with insulin resistance. 2.3. SHIP2 gene polymorphisms in human subjects Concerning the impact of SHIP2 in pathological states with insulin resistance, the amount of SHIP2 protein is increased in adipose tissue and skeletal muscle of diabetic db/db mice (Hori et al., 2002). The SHIP2 gene in humans is located at chromosome 11q13–14, and is assumed to be related to type 2 diabetes with insulin resistance and hypertension (Kaisaki et al., 2004). Along these lines, a SHIP2 gene with a 16-bp deletion in the 3′untranslated regulatory region was found more frequently in diabetic patients than in control subjects in UK and Belgian populations (Marion et al., 2002). Transfection of the deletion mutant into human embryonic kidney cells resulted in enhanced promoter activity of SHIP2, indicating a possible elevation of SHIP2 levels in diabetic patients (Marion et al., 2002). More recently, some polymorphisms of the SHIP2 gene identified in British and French populations were found to be associated with metabolic syndromes including type 2 diabetes and hypertension,

although the molecular mechanism by which the SHIP2 mutants are involved in a state of insulin resistance was not examined (Kaisaki et al., 2004). We also studied the possible association of SHIP2 gene polymorphisms with type 2 diabetes in a Japanese cohort (Kagawa et al., 2005) (Fig. 3). A polymorphism identified in the 5′-phosphatase catalytic region of SHIP2 was more common in control subjects than in type 2 diabetic patients. Since transfection studies with the mutant SHIP2 revealed that the 5′phosphatase activity is reduced for the negative regulation of insulin signaling, the polymorphism might confer protection from insulin resistance in a Japanese population (Kagawa et al., 2005). Taken together, some polymorphisms in SHIP2 may, at least in part, be implicated in the pathogenesis of human type 2 diabetes with insulin resistance. 2.4. Impact of inhibition of endogenous SHIP2 on glucose metabolism Since SHIP2 is implicated in insulin resistance as a cause of type 2 diabetes in addition to the control of glucose homeostasis and energy expenditure, inhibiting the function of endogenous SHIP2 may be a valuable approach to ameliorating insulin's actions in a state of insulin resistance. Hyperinsulinemia is a hallmark of insulin resistance, and chronic hyperinsulinemia is known to cause a desensitization to subsequent insulin responses, leading to the vicious cycle involved in the pathogenesis of type 2 diabetes (Sasaoka et al., 2005). In this regard, chronic insulin treatment impaired subsequent acute insulin stimulation of Akt phosphorylation and glucose uptake in 3T3-L1 adipocytes. Interestingly, inhibition of the endogenous SHIP2's function through the expression of a dominantnegative SHIP2 effectively ameliorated the decreased phosphorylation of Akt to almost control levels, and partly (∼ 50%) restored the reduced glucose uptake. Partial amelioration of glucose uptake appears to originate from a direct impairment of the Glut4 translocation system by chronic insulin treatment (Sasaoka et al., 2005). Based on experiments involving the tissue-specific knockout of the insulin receptor in mice, the liver appears to be the most critical target of insulin's action in the control of glucose homeostasis (Michael et al., 2000). Therefore, we examined the impact of the liver-specific expression of the dominant-negative SHIP2 by conducting adenovirus-mediated gene transfer in diabetic db/db mice. Inhibition of the function of SHIP2 in the liver ameliorated hepatic insulin signaling with decreased mRNA expression for the gluconeogenic enzymes G6Pase and PEPCK. As a result, the degree of hyperglycemia and hyperinsulinemia was apparently improved in db/db mice (Fukui et al., 2005). In addition, muscle denervation is known to cause insulin resistance characterized by a decrease in the ability of insulin to stimulate glucose uptake and glycogen synthesis in rats (Bertelli et al., 2003). Although the extent of SHIP2 expression is not altered by muscle denervation, treatment with an antisense oligonucleotide against SHIP2 mRNA ameliorated insulin resistance in rats. A reduction of SHIP2 expression by 50% achieved using this approach led to a reversal of the denervation-induced decrease in insulin-induced

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Fig. 3. SHIP2 gene polymorphisms in a Japanese cohort. SHIP2 is composed of 28 exons. Ten polymorphisms with four missense mutations were found in the cohort. Among them, SNP3 (L632I) was located in the 5′phosphatase catalytic region, and SNP5 (N982S) was adjacent to the phosphotyrosine-binding domain consensus motif in the C-terminus. SNP3 and SNP5 may affect metabolic and mitogenic insulin signaling, respectively.

glucose uptake (Bertelli et al., 2003). Based on these results, it is interesting to speculate that inhibition of the expression and/or function of SHIP2 would be effective in the treatment of glucose metabolism in cases of type 2 diabetes with insulin resistance. 3. PTEN 3.1. Role of PTEN in cultured cells PTEN was originally identified as a candidate tumor suppressor and shares homology with protein tyrosine phosphatases (Li et al., 1997; Steck et al., 1997). Subsequent analysis demonstrated that PTEN functions as a lipid phosphatase to dephosphorylate PI(3,4,5)P3 at position 3 on the inositol ring (Maehama & Dixon, 1998). PTEN is a critical regulator for cell growth and survival, and its gene is frequently mutated or deleted in patients with cancer (Li et al., 1997; Steck et al., 1997). PTEN has been shown to dephosphorylate PI(3,4,5)P3 to PI(4,5)P2, resulting in decreased levels of PI(3,4,5)P3 and a reduction of Akt activity (Di Cristofano et al., 1998; Stambolic et al., 1998). In addition to the key role of PI3-kinase in cell growth and survival, the activation of PI3-kinase and Akt is a crucial step in insulin's control of glucose homeostasis by facilitating glucose uptake in skeletal muscle and fat tissue and by inhibiting hepatic glucose output (Saltiel & Kahn, 2001; Khan & Pessin, 2002; White, 2002). Therefore, one can speculate that PTEN modulates insulin signaling via its 3′phosphatase activity to hydrolyze the PI3-kinase product PI (3,4,5)P3 to PI(3,4)P2. Overexpression of wild-type PTEN

inhibited insulin-induced activation of Akt and glucose uptake in 3T3-L1 adipocytes (Nakashima et al., 2000). On the other hand, expression of a dominant-negative mutant PTEN did not affect the metabolic action of insulin, whereas the amount of PI (3,4,5)P3 was increased (Ono et al., 2001). In contrast, depletion of PTEN protein by siRNA-mediated gene silencing enhanced insulin-induced phosphorylation of Akt and glucose uptake in 3T3-L1 adipocytes (Tang et al., 2005). Although some discrepancies exist between experiments, endogenous PTEN appears to play an important role in the negative regulation of insulin signaling in the peripheral target tissues. 3.2. Role of PTEN in mice Mice with a homozygous deletion of the PTEN gene died in the embryonic stage from tumors (Table 1). Mice with a heterozygous ablation of the gene were viable but had tumors in various organs (Di Cristofano et al., 1998; Stambolic et al., 1998). It is well recognized that mice lacking IRS-2 develop diabetes within the first 3 months of life (Withers et al., 1999). Concerning the impact of PTEN on glucose homeostasis, a heterozygous deletion of the PTEN gene in IRS-2 knockout mice conferred protection from insulin resistance and enhanced islet growth (Kushner et al., 2005). As regards with the tissue-specific role of PTEN, antisense oligonucleotide-mediated inhibition of endogenous PTEN expression enhanced insulin-induced phosphorylation of Akt in the liver of mice (Butler et al., 2002). As a result, the reduction in PTEN levels improved elevated glucose levels and ameliorated insulin sensitivity with decreased insulin

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concentrations in diabetic ob/ob and db/db mice (Butler et al., 2002). Liver-specific PTEN knockout mice were generated and studied until 6 months of age (Stiles et al., 2004). Deletion of PTEN specifically in liver resulted in an enhanced hepatic action of insulin with improved systemic glucose tolerance, although it led to increased fatty acid synthesis accompanied by hepatomegaly and fatty liver. Interestingly, hepatosteatosis was reported to be accompanied by improved glucose tolerance, relative hypoinsulinemia, and lean body weight in the mice. It is postulated that PTEN knockout mice differ in fatty liver phenotype from hyperinsulinemic and insulinresistant models (Stiles et al., 2004). Given this report, inhibition of PTEN in liver may be a therapeutic target in the treatment of type 2 diabetes. However, another study reported that hepatocyte-specific deletion of PTEN results in hepatocellular carcinomas in addition to insulin hypersensitivity and steatohepatitis in mice (Horie et al., 2004). About half of liverspecific PTEN knockout mice develop adenoma by 44 weeks of age. All of the mice had adenomas in the liver and 66% had hepatocellular carcinomas by 74–78 weeks of age (Horie et al., 2004). Therefore, caution is needed when inhibiting the function of PTEN in the liver because of the formation of tumors. Adipose tissue-specific PTEN knockout mice demonstrated improved systemic glucose tolerance and insulin sensitivity, associated with decreased fasting insulin levels, enhanced Glut4 translocation, and decreased serum resistin levels (KuralwallaMartinez et al., 2005). Insulin signaling and AMP kinase activity in the liver were also enhanced in these mice. Adipose tissue-specific knockout of PTEN protected mice from streptozotocin-induced diabetes without altering adiposity or plasma fatty acid levels. Tumors were not observed in the adipose tissue-specific PTEN knockout mice (KuralwallaMartinez et al., 2005). These results suggest that the inhibition of PTEN in adipose tissue may be beneficial for the amelioration of insulin resistance and type 2 diabetes. Muscle-specific deletion of PTEN in mice did not affect body weight, muscle weight, or fat pad weight (Wijesekara et al., 2005). Glucose tolerance and muscle insulin signaling were unchanged in muscle-specific PTEN knockout mice fed a normal chow diet. However, the muscle-specific knockout of PTEN resulted in increased insulin-induced Akt phosphorylation and glucose uptake in the soleus, but not in the extensor digitorum longus muscle compared to the control after high-fat feeding. These mice are protected from the hyperinsulinemia and islet hyperplasia caused by a high-fat diet, and free from tumors (Wijesekara et al., 2005). Therefore, muscle PTEN may also be a therapeutic target in the treatment of insulin resistance and type 2 diabetes. Deletion of the PTEN gene specifically in pancreatic ß cells also resulted in increased cell proliferation and decreased cell death (Stiles et al., 2006). Pancreatic islets were increased in number and size with a significant increase in ß cell mass in pancreatic ß cell-specific PTEN knockout mice. The PTEN knockout mice are reported to protect from developing diabetes after the injection of streptozotocin, and not to have tumors (Stiles et al., 2006).

3.3. PTEN gene polymorphisms in human subjects Glucose tolerance and insulin sensitivity are reported to be enhanced in patients with Cowden syndrome who possess germline mutations of the PTEN gene (Iida et al., 2000). A possible association of the PTEN gene with type 2 diabetes was examined in Danish Caucasian patients and Japanese patients with type 2 diabetes (Hansen et al., 2001; Ishihara et al., 2003). Although 4 intronic polymorphisms were identified in the Danish patients, they were not associated with type 2 diabetes. None of these 4 polymorphisms were found in Japanese type 2 diabetics. On the other hand, 3 variants of the PTEN gene were identified in the Japanese diabetic patients (Ishihara et al., 2003). These differences may arise from a different genetic background between the 2 ethnic groups. Among the 3 variants seen in the Japanese population, the frequency of the substitution of C with G at position − 9 (− 9C→G) (SNP1), located in the untranslated region of exon 1, was significantly higher in type 2 diabetic patients than in control subjects. Transfection of the PTEN gene with SNP1 resulted in a significantly higher expression level of PTEN protein, leading to decreased phosphorylation of Akt compared to that of the wild-type PTEN gene in Cos1 cells and Rat1 cells (Ishihara et al., 2003). These results indicate that the SNP1 polymorphism of the PTEN gene is associated with the insulin resistance of type 2 diabetes due possibly to a potentiated hydrolysis of the PI3-kinase product, at least in part, in the Japanese population. 4. Role of lipid phosphatases in brain function 4.1. Expression of lipid phosphatases in brain SHIP2 is abundantly expressed in the brain including the cerebral cortex, cerebellum, hypothalamus, thalamus, hippocampus, striatum, and midbrain in mice (Muraille et al., 2001; Sleeman et al., 2005; Sasaoka et al., unpublished data). PTEN is also expressed in most of the neurons in the brain (Li et al., 2003; Bossy-Wetzel et al., 2004). Therefore, one can speculate that SHIP2 and PTEN control neuronal functions regarding food intake and energy expenditure and/or central insulin/IGF-1 signaling to protect against apoptosis in neuronal cells. 4.2. Role of lipid phosphatases in neurodegenerative disorders In addition to the importance of PTEN as a tumor suppressor, mutations in PTEN are known to be a cause of Cowden disease and Bannayan-Zonana syndrome, in which hamartomas develop in multiple organs (Liaw et al., 1997; Marsh et al., 1997). Notably, some patients with these disorders show defects in neuronal development (Liaw et al., 1997; Marsh et al., 1997). In mice, deletion of PTEN in neuronal precursor cells had a profound effect on the development of the brain. The mice had an enlarged brain due to increased cell proliferation, decreased cell death, and enlarged cell size (Groszer et al., 2001). More recently, mutations in PTEN-induced kinase 1 (PINK1) were

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identified as a cause of hereditary early-onset Parkinson's disease (Valente et al., 2004). These reports implicate PTEN signaling in human neurodegenerative diseases. In addition, since Akt activity is known to be impaired in those with Alzheimer's disease, possible change in the expression of PTEN in the brain could be a cause of this disease (BossyWetzel et al., 2004; Griffin et al., 2005; Rickle et al., 2006). However, the expression of PTEN has also been reported to be decreased or unaltered (Griffin et al., 2005; Rickle et al., 2006). It is still unknown whether the expression and/ or function of PTEN is altered in the brain of patients with type 2 diabetes and insulin resistance. Epidemiological studies indicate that insulin resistance and type 2 diabetes increase the risk for age-related cognitive decline (Schubert et al., 2003, 2004). Although long-term hyperglycemia causes vascular complications, defects in the insulin/IGF-1 signaling system may contribute directly to memory loss and dementia. Thus, insulin resistance and diabetes are known to be associated with an increased risk of Alzheimer's disease (Schubert et al., 2003, 2004). It is known that at least 50% of brain and body growth is mediated by insulin/IGF-1 signaling (Schubert et al., 2003). The receptors for insulin and IGF-1 are tyrosine kinases that mediate phosphorylation of the IRS (Araki et al., 1994; Withers et al., 1998). IRS-1 mediates the effect of IGF-1 on somatic growth, because IRS-1 null mice are ~ 50% smaller than control mice (Araki et al., 1994; Withers et al., 1998). Although body size in IRS-2 null mice is normal, the IRS-2 pathway is responsible for the effect of the insulin/IGF-1 system on brain size (Schubert et al., 2003). Alzheimer's disease is characterized by an accumulation of extracellular deposits of amyloid ß-peptide that promotes inflammation, and the intracellular accumulation of neurofibrillary tangles composed of paired helical filaments assembled from hyperphosphorylated forms of the microtubuleassociated protein Tau (Schubert et al., 2003, 2004). Active GSK3ß is known to phosphorylate Tau. Insulin and IGF-1, via an IRS/PI3-kinase/Akt-dependent pathway, induce phosphorylation of GSK3ß for its inactivation. In this context, Tau is hyperphosphorylated in both neuron-specific insulin receptor knockout mice (NIRKO) and IRS-2 knockout mice, although the sites of its phosphorylation were different (Schubert et al., 2003, 2004). Therefore, it is speculated that lipid phosphatases hydrolyzing the PI3-kinase product PI(3,4,5)P3 are involved in the neurodegenerative signaling. In this regard, IGF-1-induced inhibition of the phosphorylation of Tau was attenuated, resulting in enhanced apoptosis in cerebellar granule cells derived from wild-type SHIP2 transgenic mice (Tsuneki, H., Soeda, Y., and Sasaoka, T., unpublished data). Further study will be needed to clarify the impact of the lipid phosphatases SHIP2 and PTEN on the neurodegeneration associated with type 2 diabetes (Fig. 4). 4.3. Role of lipid phosphatases in feeding and energy expenditure Leptin is a key regulator of feeding and energy homeostasis, which reduces food intake and body fat content (Bates & Myers,

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Fig. 4. Lipid phosphatases regulate insulin and IGF-1 signaling for protection from apoptosis in brain. Insulin and IGF-1, via an IRS/PI3-kinase/Akt dependent pathway, induce phosphorylation of GSK3ß for its inactivation. Thus, insulin and IGF-1 inhibit GSK3ß-induced phosphorylation of Tau. Assembled hyperphosphorylated forms of the microtubule-associated protein Tau consist of paired helical filaments for neurofibrillary tangles seen in Alzheimer's disease. Thus, attenuation of the insulin and IGF-1 signaling by lipid phosphatases SHIP2 and PTEN might lead to neurodegeneration.

2003; Frühbeck, 2006). Thus, obesity is frequently associated with leptin resistance. Leptin affects POMC/CART neurons positively and AgRP/NPY neurons negatively to control the energy balance mainly in the hypothalamic arcuate nucleus (Bates & Myers, 2003; Frühbeck, 2006). Studies first indicated that leptin's function is mediated by a Janus-activated kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway (Bates & Myers, 2003; Frühbeck, 2006). Recently, however, there is increasing evidence to suggest the importance of PI3-kinase in the neuronal function of leptin (Morrison et al., 2005; Morton et al., 2005; Xu et al., 2005). In this context, feeding and energy homeostasis are also known to be regulated by insulin in the CNS (Brüning et al., 2000; Burks et al., 2000). Along these lines, female NIRKO and IRS-2 knockout mice exhibited an increase in food intake and obesity (Brüning et al., 2000; Burks et al., 2000). Both mice showed leptin resistance with elevated leptin levels. Apparently, PI3kinase is a crucial mediator for the actions of both insulin and leptin in the regulation of feeding and energy homeostasis in the hypothalamus (Morrison et al., 2005; Morton et al., 2005; Xu et al., 2005). Based on these findings, one can speculate that enhancement of the PI3-kinase pathway by inhibition of lipid phosphatase leads to a decrease in food intake and negative energy balance. In this regard, the second SHIP2 knockout mouse was conferred protection from obesity caused by a highfat diet (Sleeman et al., 2005). In addition, serum leptin levels were decreased in the SHIP2 knockout mice compared to

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Fig. 5. Lipid phosphatases regulate insulin and leptin signaling for control of feeding and energy expenditure in hypothalamus. POMC/CART neurons positively and AgRP/NPY neurons negatively control the energy balance mainly in the hypothalamic arcuate nucleus. PI3-kinase plays a crucial role in both insulin- and leptininduced control of feeding and energy expenditure. Attenuation of the insulin and leptin signaling by lipid phosphatases SHIP2 and PTEN might result in excess feeding leading to obesity.

control mice (Sleeman et al., 2005). These results indicate that ablation of SHIP2 may lead to enhanced sensitivity of both insulin and leptin in the hypothalamus. Thus, an increase in energy expenditure on a high-fat diet is evident in the SHIP2 knockout mice from measurements of the basal metabolic rate (BMR) and increased expression of UCP3 mRNA in the skeletal muscle (Sleeman et al., 2005). Leptin sensitivityinduced enhancement of sympathetic nerve activity may lead to increased energy expenditure in peripheral tissue. Inconsistent with a role for the lipid phosphatase SHIP2 in the brain, overall food intake was reported to be unchanged in SHIP2 knockout mice (Sleeman et al., 2005). Ablation of lipid phosphatases in peripheral tissues also appears to have an impact on enhanced leptin sensitivity. In this context, liver-specific knockout of PTEN in mice resulted in reduced body fat content and plasma leptin levels (Stiles et al., 2004). Studies with neuron-specific SHIP2 knockout mice may shed light on whether or not the effect of SHIP2 to regulate food intake and energy expenditure is mediated by SHIP2 in the CNS (Fig. 5). 5. Role of lipid phosphatases in atherosclerosis SHIP2 is known to be a relatively specific negative regulator of insulin signaling (Clément et al., 2001; Sasaoka et al., 2004; Sleeman et al., 2005). However, SHIP2 is tyrosine-phosphorylated in response to platelet-derived growth factor (PDGF), epidermal growth factor (EGF), nerve growth factor (NGF), and IGF-1, as well as insulin (Habib et al., 1998). PDGF- and EGFinduced generation of PI(3,4,5)P3 was decreased by overexpression of wild-type SHIP2 in Cos-7 cells and aortic

vascular smooth muscle cells (VSMC) (Pesesse et al., 2001; Sasaoka et al., 2003). SHIP2 is known to interact with p130Cas, filamin, vinexin, Cbl, and Shc, which are related to cell adhesion and migration, and cytoskeletal reorganization (Prasad et al., 2001; Paternotte et al., 2005; Prasad & Decker, 2005). We therefore examined the effect of the expression of SHIP2 on the PDGF- and IGF-1-induced signaling leading to cell growth and apoptosis in VSMC. Interestingly, both the PDGF- and IGF-1induced phosphorylation of Akt and protection from apoptosis were inhibited by SHIP2 via its 5′-phosphatase activity. In contrast, the PDGF- and IGF-1-induced activation of MAP kinase and DNA synthesis were decreased by expression of SHIP2 independent of the 5′-phosphatase activity (Sasaoka et al., 2003; Gao et al., 2005). These results indicate that SHIP2 also regulates PDGF- and IGF-1 signaling via 5′-phosphatasedependent and -independent mechanisms in VSMC. Based on this, one can speculate that SHIP2 is involved in the development and progression of atherosclerosis. In this regard, some polymorphisms in the SHIP2 gene are associated with metabolic syndrome and hypertension in French and British populations (Kaisaki et al., 2004). A mutation in the SHIP2 gene was found adjacent to the C-terminus tyrosine phosphorylation site in a Japanese diabetic patient. Insulin-induced phosphorylation of MAP kinase was increased in cells expressing this mutant SHIP2 as compared to those expressing wild-type SHIP2 (Kagawa et al., 2005). These results indicate a possible involvement in the progression of atherosclerosis. However, the significant alterations observed in the pathways after the knockout of SHIP2 were not unforeseen (Clément et al., 2001; Sleeman et al., 2005). Mice with a deletion of SHIP2 did not

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demonstrate overt changes in cell growth and cell survival except for the facial cytoskeletal change (Sleeman et al., 2005). No apparent change in vessels is seen in transgenic mice overexpressing SHIP2 either (Tsuneki and Sasaoka, unpublished data). To clarify the impact of SHIP2 on atherosclerosis, further studies will be required to examine possible subtle changes of cell growth and cell survival in aged mice with knockout and/or overexpression of SHIP2. PTEN appears to be involved in the proliferation, migration, and survival of VSMC. Overexpression of PTEN inhibited PDGF-induced phosphorylation of p70-S6 kinase, Akt, GSK3, but not MAP kinase, resulting in decreased cell proliferation and migration in VSMC (Huang & Kontos, 2002). In addition, the adenovirus-mediated intraarterial delivery of PTEN inhibited balloon-injured neonatal hyperplasia via induction of apoptosis and inhibition of medial cell proliferation (Huang et al., 2004). Furthermore, propylthiouracil (PTU)-induced inhibition of proliferation and migration in VSMC to prevent the development of atherosclerosis was reported to be mediated via upregulation of the production of PTEN protein (Chen et al., 2004). These results indicate that overexpression of PTEN in VSMC is a therapeutic approach to inhibiting the progression of atherosclerosis. 6. SKIP SKIP was identified as a 5′-inositol phosphatase predominantly expressed in skeletal and cardiac muscle, and kidney, and categorized as a type 2 lipid phosphatase (Ijuin et al., 2000). Originally, SKIP was reported to be the 5′-lipid phosphatase hydrolyzing PI(4,5)P2 to PI(4)P, thereby involved in regulation of the cytoskeletal reorganization including actin rearrangement (Ijuin et al., 2000). Thereafter, it was found that SKIP functions to hydrolyze PI(3,4,5)P3 to PI(3,4)P2 (Ijuin & Takenawa, 2003). Insulin-induced intracellular production of PI(3,4,5)P3 and phosphorylation of Akt decreased when SKIP was overexpressed, but increased when endogenous levels of SKIP were reduced by means of antisense oligonucleotide in Chinese hamster ovary cells overexpressing insulin receptors. On the other hand, the insulin-induced phosphorylation of MAP kinase was unaffected. Interestingly, overexpression of SKIP inhibited insulininduced glucose uptake and glycogen synthesis in L6myoblasts (Ijuin & Takenawa, 2003). Based on these results, it is possible that SKIP regulates insulin-induced metabolic signaling especially in skeletal muscles. Further study will be needed to clarify the role of SKIP in differentiated cells of the target tissues of insulin. Analyses of knockout mice and/or transgenic mice will also be required to determine the role of SKIP in vivo. Elucidation of possible changes in the expression and/or enzymatic function in cases of type 2 diabetes with insulin resistance will also be required. 7. Conclusions and perspectives Lipid phosphatases SHIP2, PTEN, and SKIP function to regulate the metabolic action of insulin by hydrolyzing the PI3-

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kinase product PI(3,4,5)P3. Although PTEN negatively regulates insulin signaling in the liver, skeletal muscle, fat, and pancreas, whole-body inhibition of PTEN is not an adequate approach to the treatment of type 2 diabetes. In fact, homologous targeted disruption of PTEN in mice is lethal due to the formation of malignant tumors. However, if tissuespecific inhibition of PTEN in skeletal muscle and fat tissue is possible, it might be a therapeutic approach for type 2 diabetes, since the formation of tumors is not observed in these tissuespecific knockout mice. On the other hand, if the VSMCspecific expression of PTEN is possible, it would be of therapeutic value in inhibiting the progression of atherosclerosis. More studies will be required to determine whether or not the inhibition of SKIP is an appropriate target in the treatment of type 2 diabetes. Importantly, SHIP2 appears to be an important negative regulator of insulin signaling with some pathological impact on insulin resistance in type 2 diabetes. In addition, SHIP2 is a key factor in the control of energy expenditure, although it remains to be elucidated whether its function is derived from the CNS or peripheral tissue. In any case, inhibition of SHIP2 appears to be of therapeutic value for the insulin resistance with type 2 diabetes and/or obesity. Although an efficient inhibitor of endogenous SHIP2 has yet to be identified, the development of such an agent would ameliorate insulin resistance without gain in body weight. Acknowledgments This work was supported in part by a Grant-in-aid for Scientific Research from the Japan Society for the Promotion of Science. We are grateful to Professor Dr. I. Kimura and Professor Dr. M. Kobayashi (University of Toyama, Toyama, Japan) for their continuous encouragement, and Drs. H. Ishihara, M. Ishiki, H. Hori, S. Murakami, K. Fukui, M. Ikubo, S. Kagawa, S. Yaguchi, and Y. Soeda (University of Toyama, Toyama, Japan) for their expertise. References Araki, E., Lipes, M. A., Patti, M. E., Bruning, J. C., Haag, B., III, Johnson, R. S., et al. (1994). Alternative pathway of insulin signaling in targeted disruption of the IRS-1 gene. Nature 372, 186−190. Bates, S. H., & Myers, M. G. (2003). The role of leptin receptor signaling in feeding and neuroendocrine function. TRENDS Endocrinol Metab 14, 447−452. Bertelli, D. F., Ueno, M., Amaral, M. E. C., Toyama, M. H., Carneiro, E. M., Marangoni, S., et al. (2003). Reversal of denervation-induced insulin resistance by SHIP2 protein synthesis blockade. Am J Physiol Endocrinol Metab 284, E679−E687. Bossy-Wetzel, E., Schwarzenbacher, R., & Lipton, S. A. (2004). Molecular pathways to neurodegeneration. Nat Med 10, S2−S9. Brüning, J. C., Gautam, D., Burks, D. J., Gillette, J., Schubert, M., Orban, P. C., et al. (2000). Role of brain insulin receptor in control of body weight and reproduction. Science 289, 2122−2125. Burks, D. J., deMora, J. F., Schubert, M., Withers, D. J., Myers, M. G., Towery, H. H., et al. (2000). IRS-2 pathways integrate female reproduction and energy homeostasis. Nature 407, 377−382. Butler, M., McKay, R. A., Popoff, I. J., Gaarde, W. A., Witchell, D., Murray, S. F., et al. (2002). Specific inhibition of PTEN expression reverses hyperglycemia in diabetic mice. Diabetes 51, 1028−1034.

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