Insulin receptor substrate (IRS)-2؊/؊ mice develop diabetes because of insulin resistance in the liver and failure to undergo ␤-cell hyperplasia. Here we show by DNA chip microarray analysis that expression of the sterol regulatory element-binding protein (SREBP)-1 gene, a downstream target of insulin, was paradoxically increased in 16-week-old IRS-2 ؊/؊ mouse liver, where insulin-mediated intracellular signaling events were substantially attenuated. The expression of SREBP-1 downstream genes, such as the spot 14, ATP citratelyase, and fatty acid synthase genes, was also increased. Increased liver triglyceride content in IRS-2 ؊/؊ mice assures the physiological importance of SREBP-1 gene induction. IRS-2 ؊/؊ mice showed leptin resistance; low dose leptin administration, enough to reduce food intake and body weight in wild-type mice, failed to do so in IRS-2 ؊/؊ mice. Interestingly, high dose leptin administration reduced SREBP-1 expression in IRS-2 ؊/؊ mouse liver. Thus, IRS-2 gene disruption results in leptin resistance, causing an SREBP-1 gene induction, obesity, fatty liver, and diabetes.The pathogenesis of type 2 diabetes involves complex interactions among multiple physiological defects. Transgenic and knockout technology to create animal models of type 2 diabetes have had a major impact on assessment of the function of newly identified molecules implicated in the regulation of glucose homeostasis in vivo (1). The insulin receptor substrate (IRS) 1 proteins play a key role in signal transduction from the insulin receptor (reviewed in Refs. 2-4). These molecules are major intracellular phosphorylation targets of activated insulin receptor tyrosine kinase. The mammalian IRS protein family contains at least four members, ubiquitous IRS-1 (5) and IRS-2 (6), adipose tissue-predominant IRS-3 (7), and IRS-4, which are expressed in thymus, brain, and kidney (8). The physiological roles of each protein have been evaluated by gene targeting strategies. IRS-1 Ϫ/Ϫ mice are growth-retarded and insulin-resistant (9, 10) but do not develop diabetes, because an alternate substrate IRS-2 (10) or pp190 (11) compensates for the lack of IRS-1 in liver (11) and, at least in part, in skeletal muscle (12). In addition, hyperinsulinemia associated with ␤-cell hyperplasia effectively countervailed the insulin-resistant states (13). IRS-2 Ϫ/Ϫ mice, however, developed diabetes because of inadequate ␤-cell proliferation combined with liver-insulin resistance (14 -16). Mice lacking IRS-3 or IRS-4 had milder phenotypes (17, 18).Liver is a major target organ for insulin action, contributing to energy storage in the fed state by regulating catabolic and anabolic pathways. Liver-specific insulin receptor knockout mice exhibit dramatic insulin resistance (19). Insulin decreases gluconeogenic enzyme mRNAs (20) and increases lipogenic enzyme mRNAs. A transcription factor of sterol regulatory element-binding protein 1c (SREBP-1c) (21-23) or adipocyte differentiation and determination factor (24) plays a central role in insulin-mediated lipogen...

We previously reported that insulin receptor substrate-2 (IRS-2)-deficient mice develop diabetes as a result of insulin resistance in the liver and failure of ␤-cell hyperplasia. In this study we introduced the IRS-2 gene specifically into the liver of Irs2 ؊/؊ mice with adenovirus vectors. Glucose tolerance tests revealed that the IRS-2 restoration in the liver ameliorated the hyperglycemia, but the improvement in hyperinsulinemia was only partial. Endogenous glucose production (EGP) and the rate of glucose disappearance (Rd) were measured during hyperinsulinemic-euglycemic clamp studies: EGP was increased 2-fold in the Irs2 ؊/؊ mice, while Rd decreased by 50%. Restoration of IRS-2 in the liver suppressed EGP to a level similar to that in wild-type mice, but Rd remained decreased in the Adeno-IRS-2-infected Irs2 ؊/؊ mice. Irs2 ؊/؊ mice also exhibit obesity and hyperleptinemia associated with impairment of hypothalamic phosphatidylinositol 3-kinase activation. Continuous intracerebroventricular leptin infusion or caloric restriction yielded Irs2 ؊/؊ mice whose adiposity was comparable to that of Irs2 ؉/؉ mice, and both the hyperglycemia and the hyperinsulinemia of these mice improved with increased Rd albeit partially. Finally combination treatment consisting of adenovirus-mediated gene transfer of IRS-2 and continuous intracerebroventricular leptin infusion completely reversed the hyperglycemia and hyperinsulinemia in Irs2 ؊/؊ mice. EGP and Rd also became normal in these mice as well as in mice treated by caloric restriction plus adenoviral gene transfer. We therefore concluded that a combination of increased EGP due to insulin signaling defects in the liver and reduced Rd due to obesity accounts for the systemic insulin resistance in Irs2

Acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes catalyze the final step in mammalian triglyceride synthesis, and their functions are considered to be involved in the mechanisms of obesity, insulin resistance, and leptin resistance. Insulin receptor substrate-2 (IRS-2)-deficient mice exhibit obesity-associated with hypertrophic adipocytes and leptin resistance. Screening for transcripts of genes involved in fatty acid and triglyceride synthesis to investigate the mechanism of the hypertrophic change in the adipocytes showed that expression of DGAT2 mRNA was up-regulated in the white adipose tissue (WAT) of Irs2 ؊/؊ mice, whereas that of DGAT1 was down-regulated. This reciprocal expression of DGAT1 and DGAT2 was also observed in WAT of leptin-deficient ob/ob mice. A high fat diet also resulted in increased DGAT2 and reduced DGAT1 in the WAT of C57BL/6 mice. Induction of adipocyte hypertrophy in vitro up-regulated both DGAT1 and DGAT2 expression in 3T3-L1 cells, suggesting that adipocyte non-autonomous mechanism in vivo is required for the reciprocal changes in expression of DGAT1 and DGAT2. In fact, intracerebroventricular infusion of leptin reduced DGAT2 expression in WAT of Irs2 ؊/؊ mice and ob/ob mice, independently of DGAT1 expression. We propose the hypothesis that leptin regulates adipocyte size by altering expression patterns of DGAT via central nervous system to determine the levels of triglyceride synthesis.We have hypothesized that the size of adipocytes is inversely correlated with insulin sensitivity; namely, that larger adipocytes are associated with insulin resistance and smaller adipocytes are associated with insulin sensitivity (1). Acyl-CoA:diacylglycerol acyltransferase (DGAT) 1 is a key enzyme that catalyzes the final step in mammalian triglyceride synthesis (2), and two DGAT enzymes have been identified (3-5). Although the genes encoding DGAT1 and DGAT2 belong to different gene families, both genes are ubiquitously expressed and the enzymes they encode have similar substrate specificity (5). Dgat1 Ϫ/Ϫ mice have been reported to exhibit normal growth on a chow diet, and to be resistant to diet-induced obesity (6). Interestingly, Dgat1Ϫ/Ϫ mice exhibit increased insulin sensitivity and a leptin-sensitive phenotype associated with decreased tissue triglyceride content, suggesting that DGAT1 is somehow involved in insulin and leptin action throughout the body (7, 8). Recently, Dgat2Ϫ/Ϫ mice have been reported to exhibit marked reduction of triglyceride and fatty acids in the body, suggesting a critical role of DGAT2 in lipogenesis; however, the physiological roles of DGAT2 in adult mice are still unknown because Dgat2 Ϫ/Ϫ mice die soon after birth (9). The insulin receptor substrate (IRS) proteins play a key role in signal transduction from the insulin receptor (10, 11) and are major intracellular phosphorylation targets of activated insulin receptor tyrosine kinase. Irs2 Ϫ/Ϫ mice develop diabetes because of inadequate ␤-cell proliferation combined with insulin resistance (12-14). Another n...

Inducing mitochondrial uncoupling (mUncoupling) is an attractive therapeutic strategy for treating metabolic diseases because it leads to calorie-wasting by reducing the efficiency of oxidative phosphorylation (OXPHOS) in mitochondria. Here we report a safe mUncoupler, OPC-163493, which has unique pharmacokinetic characteristics. OPC-163493 shows a good bioavailability upon oral administration and primarily distributed to specific organs: the liver and kidneys, avoiding systemic toxicities. It exhibits insulin-independent antidiabetic effects in multiple animal models of type I and type II diabetes and antisteatotic effects in fatty liver models. These beneficial effects can be explained by the improvement of glucose metabolism and enhancement of energy expenditure by OPC-163493 in the liver. Moreover, OPC-163493 treatment lowered blood pressure, extended survival, and improved renal function in the rat model of stroke/hypertension, possibly by enhancing NO bioavailability in blood vessels and reducing mitochondrial ROS production. OPC-163493 is a liver-localized/targeted mUncoupler that ameliorates various complications of diabetes.

1 We discovered the ®rst nonpeptide arginine-vasopressin (AVP) V 2 -receptor agonist, OPC-51803. Pharmacological properties of OPC-51803 were elucidated using HeLa cells expressing human AVP receptor subtypes (V 2 , V 1a and V 1b ) and compared with those of 1-desamino-8-D-arginine vasopressin (dDAVP), a peptide V 2 -receptor agonist. 2 OPC-51803 and dDAVP displaced [3 H]-AVP binding to human V 2 -and V 1a -receptors with K i values of 91.9+10.8 nM (n=6) and 3.12+0.38 nM (n=6) for V 2 -receptors, and 819+39 nM (n=6) and 41.5+9.9 nM (n=6) for V 1a -receptors, indicating that OPC-51803 was about nine times more selective for V 2 -receptors, similar to the selectivity of dDAVP. OPC-51803 scarcely displaced [3 H]-AVP binding to human V 1b -receptors even at 10 74 M, while dDAVP showed potent anity to human V 1b -receptors with the K i value of 13.7+3.2 nM (n=4). 3 OPC-51803 concentration-dependently increased cyclic adenosine 3', 5'-monophosphate (cyclic AMP) production in HeLa cells expressing human V 2 -receptors with an EC 50 value of 189+14 nM (n=6). The concentration-response curve for cyclic AMP production induced by OPC-51803 was shifted to the right in the presence of a V 2 -antagonist, OPC-31260. ] i in HeLa cells expressing human V 1a -and V 1b -receptors in a concentration-dependent fashion. 5 From these results, OPC-51803 has been con®rmed to be the ®rst nonpeptide agonist for human AVP V 2 -receptors without agonistic activities for V 1a -and V 1b -receptors. OPC-51803 may be useful for the treatment of AVP-de®cient pathophysiological states and as a tool for AVP researches.

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Aim/hypothesis Lowering the body weight set point is a prerequisite for the maintenance of reduced body weight. In this context, obesity is known to be strongly linked to leptin resistance, and it remains to be clarified whether recovery from leptin resistance might lower the body weight set point to allow sustained body weight loss. Methods Obese IRS-2 knockout (IRS-2 -/-) mice were subjected to calorie restriction (CR) or b3-adrenergic receptor (AR) agonist treatment. The physiological effects of leptin, hypothalamic leptin signaling, and alterations of the body weight set point were evaluated. Results In the CR mice, recovery from acquired leptin resistance was observed, as shown by the restoration of the suppressive effects of leptin on food intake and weight gain, as well as the recovery of signal transducer and activator of transcription 3 (STAT3) phosphorylation. Nevertheless, the body weight quickly rebounded to the original body weight after cessation of the CR, suggesting that CR failed to overcome the primary defect in IRS-2/ phosphatidylinositol 3-kinase (PI3K) signaling. On the other hand, after 2 weeks b3-AR agonist treatment, the mice began to lose body weight, indicating that the treatment was able to overcome the primary defect in IRS-2/ PI3K signaling and lower the body weight set point. Conclusions/interpretation Recovery of acquired leptin resistance does not lead to a resetting of the body weight set point in obese IRS-2/PI3K-defective mice. b3-AR agonist treatment may act on some pathways distal to or independent of PI3K and/or STAT3, inducing resetting of the body weight set point. Keywords b3-adrenergic receptor agonist Á Body weight set point Á Calorie restriction Á IRS-2 -/mice Á Leptin resistance Á Signal transducer and activator of transcription 3 (STAT3)

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