Obesity is an epidemic in Western society, and causes rapidly accelerating rates of type 2 diabetes and cardiovascular disease. The evolutionarily conserved serine/threonine kinase, AMP-activated protein kinase (AMPK), functions as a 'fuel gauge' to monitor cellular energy status. We investigated the potential role of AMPK in the hypothalamus in the regulation of food intake. Here we report that AMPK activity is inhibited in arcuate and paraventricular hypothalamus (PVH) by the anorexigenic hormone leptin, and in multiple hypothalamic regions by insulin, high glucose and refeeding. A melanocortin receptor agonist, a potent anorexigen, decreases AMPK activity in PVH, whereas agouti-related protein, an orexigen, increases AMPK activity. Melanocortin receptor signalling is required for leptin and refeeding effects on AMPK in PVH. Dominant negative AMPK expression in the hypothalamus is sufficient to reduce food intake and body weight, whereas constitutively active AMPK increases both. Alterations of hypothalamic AMPK activity augment changes in arcuate neuropeptide expression induced by fasting and feeding. Furthermore, inhibition of hypothalamic AMPK is necessary for leptin's effects on food intake and body weight, as constitutively active AMPK blocks these effects. Thus, hypothalamic AMPK plays a critical role in hormonal and nutrient-derived anorexigenic and orexigenic signals and in energy balance.

AMP-activated protein kinase (AMPK) is a key regulator of cellular energy balance and of the effects of leptin on food intake and fatty acid oxidation. Obesity is usually associated with resistance to the effects of leptin on food intake and body weight. To determine whether diet-induced obesity (DIO) impairs the AMPK response to leptin in muscle and/or hypothalamus, we fed FVB mice a high fat (55%) diet for 10 -12 weeks. Leptin acutely decreased food intake by ϳ30% in chow-fed mice. DIO mice tended to eat less, and leptin had no effect on food intake. Leptin decreased respiratory exchange ratio in chow-fed mice indicating increased fatty acid oxidation. Respiratory exchange ratio was low basally in high fat-fed mice, and leptin had no further effect. Leptin (3 mg/kg intraperitoneally) increased ␣2-AMPK activity 2-fold in muscle in chow-fed mice but not in DIO mice. Leptin decreased acetyl-CoA carboxylase activity 40% in muscle from chow-fed mice. In muscle from DIO mice, acetyl-CoA carboxylase activity was basally low, and leptin had no further effect. In paraventricular, arcuate, and medial hypothalamus of chow-fed mice, leptin inhibited ␣2-AMPK activity but not in DIO mice. In addition, leptin increased STAT3 phosphorylation 2-fold in arcuate of chow-fed mice, but this effect was attenuated because of elevated basal STAT3 phosphorylation in DIO mice. Thus, DIO in FVB mice alters ␣2-AMPK in muscle and hypothalamus and STAT3 in hypothalamus and impairs further effects of leptin on these signaling pathways. Defective responses of AMPK to leptin may contribute to resistance to leptin action on food intake and energy expenditure in obese states.

Accumulating evidence indicates an important role for serine phosphorylation of IRS-1 in the regulation of insulin action. Recent studies suggest that Rho-kinase (ROK) is a mediator of insulin signaling, via interaction with IRS-1. Here we show that insulin stimulation of glucose transport is impaired when ROK is chemically or biologically inhibited in cultured adipocytes and myotubes and in isolated soleus muscle ex vivo. Inactivation of ROK also reduces insulin-stimulated IRS-1 tyrosine phosphorylation and PI3K activity. Moreover, inhibition of ROK activity in mice causes insulin resistance by reducing insulin-stimulated glucose uptake in skeletal muscle in vivo. Mass spectrometry analysis identifies IRS-1 Ser632/635 as substrates of ROK in vitro, and mutation of these sites inhibits insulin signaling. These results strongly suggest that ROK regulates insulin-stimulated glucose transport in vitro and in vivo. Thus, ROK is an important regulator of insulin signaling and glucose metabolism.

The nuclear hormone receptor peroxisome proliferator-activated receptor ␥ (PPAR␥) plays central roles in adipogenesis and glucose homeostasis and is the molecular target for the thiazolidinedione (TZD) class of antidiabetic drugs. Activation of PPAR␥ by TZDs improves insulin sensitivity; however, this is accompanied by the induction of several undesirable side effects. We have identified a novel synthetic PPAR␥ ligand, T2384, to explore the biological activities associated with occupying different regions of the receptor ligand-binding pocket. X-ray crystallography studies revealed that T2384 can adopt two distinct binding modes, which we have termed "U" and "S", interacting with the ligand-binding pocket of PPAR␥ primarily via hydrophobic contacts that are distinct from full agonists. The different binding modes occupied by T2384 induced distinct patterns of coregulatory protein interaction with PPAR␥ in vitro and displayed unique receptor function in cell-based activity assays. We speculate that these unique biochemical and cellular activities may be responsible for the novel in vivo profile observed in animals treated systemically with T2384. When administered to diabetic KKAy mice, T2384 rapidly improved insulin sensitivity in the absence of weight gain, hemodilution, and anemia characteristics of treatment with rosiglitazone (a TZD). Moreover, upon coadministration with rosiglitazone, T2384 was able to antagonize the side effects induced by rosiglitazone treatment alone while retaining robust effects on glucose disposal. These results are consistent with the hypothesis that interactions between ligands and specific regions of the receptor ligand-binding pocket might selectively trigger a subset of receptor-mediated biological responses leading to the improvement of insulin sensitivity, without eliciting less desirable responses associated with full activation of the receptor. We suggest that T2384 may represent a prototype for a novel class of PPAR␥ ligand and, furthermore, that molecules sharing some of these properties would be useful for treatment of type 2 diabetes.

PTP1B؊/؊ mice are resistant to diet-induced obesity due to leptin hypersensitivity and consequent increased energy expenditure. We aimed to determine the cellular mechanisms underlying this metabolic state. AMPK is an important mediator of leptin's metabolic effects. We find that ␣1 and ␣2 AMPK activity are elevated and acetylcoenzyme A carboxylase activity is decreased in the muscle and brown adipose tissue (BAT) of PTP1B ؊/؊ mice. The effects of PTP1B deficiency on ␣2, but not ␣1, AMPK activity in BAT and muscle are neuronally mediated, as they are present in neuron-but not muscle-specific PTP1B ؊/؊ mice. In addition, AMPK activity is decreased in the hypothalamic nuclei of neuronal and whole-body PTP1B ؊/؊ mice, accompanied by alterations in neuropeptide expression that are indicative of enhanced leptin sensitivity. Furthermore, AMPK target genes regulating mitochondrial biogenesis, fatty acid oxidation, and energy expenditure are induced with PTP1B inhibition, resulting in increased mitochondrial content in BAT and conversion to a more oxidative muscle fiber type. Thus, neuronal PTP1B inhibition results in decreased hypothalamic AMPK activity, isoform-specific AMPK activation in peripheral tissues, and downstream gene expression changes that promote leanness and increased energy expenditure. Therefore, the mechanism by which PTP1B regulates adiposity and leptin sensitivity likely involves the coordinated regulation of AMPK in hypothalamus and peripheral tissues.Protein tyrosine phosphatase 1B (PTP1B) belongs to a family of tyrosine phosphatases with diverse roles in eukaryotes (2, 4). PTP1B attenuates insulin signaling by dephosphorylating the insulin receptor (19,22,61) and possibly IRS-1 (9, 23) and leptin signaling by dephosphorylating JAK2, which phosphorylates the leptin receptor and associated substrates (10, 45, 67). PTP1B-deficient mice are insulin hypersensitive, lean, and resistant to diet-induced obesity (20, 36) due, at least in part, to increased energy expenditure (36). The leanness can be explained by the absence of PTP1B in neurons, because neuronspecific PTP1B Ϫ/Ϫ mice also have reduced body weight and adiposity and increased energy expenditure (6). In contrast, muscle-and liver-specific PTP1B-deficient mice have normal body weight with improved insulin sensitivity, whereas adipose-PTP1B-deficient mice have increased body weight (6,15,16). These data suggest that PTP1B in peripheral tissues such as muscle and liver is an important mediator of peripheral insulin sensitivity, whereas PTP1B in the nervous system plays a critical role in regulating energy expenditure and adiposity (6).The adipocyte-derived hormone leptin plays an essential

A tyrosine kinase adaptor protein containing pleckstrin homology and SH2 domains (APS) is rapidly and strongly tyrosine phosphorylated by insulin receptor kinase upon insulin stimulation. The function of APS in insulin signaling has heretofore remained unknown. APS-deficient (APS ؊/؊ ) mice were used to investigate its function in vivo. The blood glucose-lowering effect of insulin, as assessed by the intraperitoneal insulin tolerance test, was increased in APS ؊/؊ mice. Plasma insulin levels during fasting and in the intraperitoneal glucose tolerance test were lower in APS ؊/؊ mice. APS ؊/؊ mice showed an increase in the whole-body glucose infusion rate as assessed by the hyperinsulinemic-euglycemic clamp test. These findings indicated that APS ؊/؊ mice exhibited increased sensitivity to insulin. However, overexpression of wild-type or dominant-negative APS in 3T3L1 adipocytes did not affect insulin receptor numbers, phosphorylations of insulin receptor, insulin receptor substrate-1, or Akt and mitogen-activated protein kinase. The glucose uptake and GLUT4 translocation were not affected by insulin stimulation in these cells. Nevertheless, the insulin-stimulated glucose transport in isolated adipocytes of APS ؊/؊ mice was increased over that of APS ؉/؉ mice. APS ؊/؊ mice also showed increased serum levels of leptin and adiponectin, which might explain the increased insulin sensitivity of adipocytes. Diabetes 52: [2657][2658][2659][2660][2661][2662][2663][2664][2665] 2003 I nsulin signaling begins with the binding of insulin to its receptor present on the cell surface, and activation of the insulin receptor tyrosine kinase results in tyrosine phosphorylation of a number of intracellular substrates. These molecules, including the insulin receptor substrate (IRS) family (1), src homology and collagen (2), Gab1 (3), and Grb10 (4), act as adaptor molecules that link between the insulin receptor and downstream signaling effectors. Adaptor protein containing a pleckstrin homology and SH2 domain (APS) is also one of the substrates that tyrosine phosphorylated by insulin receptor kinase (5,6).APS was first described to interact with an oncogenic mutant of the tyrosine kinase receptor c-Kit (7), and APS was isolated by the two-hybrid system using the cytoplasmic domain of the human insulin receptor as bait (5,6). APS (66.5 kDa) forms an adaptor protein family together with Lnk (8,9) and SH2-B (SH2-B␣, SH2-B␤, SH2-B␥, and SH2-B␦) (10 -13), whose members share a homologous NH 2 -terminal region with proline-rich stretches, pleckstrin homology and SH2 domains, and a conserved COOHterminal tyrosine phosphorylation site. It has been demonstrated that some members of this adaptor protein family act as modulators of signaling through various tyrosine kinase receptors. Lnk plays a role in regulating production of B-cell precursors and hematopoietic progenitor cells (8,14). SH2-B is an important signaling molecule in the insulin-like growth factor I (IGF-1) mediated reproductive pathway (13).APS is highly expressed in insulin-r...

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It has been proposed that mitochondrial oxidative phosphorylation in pancreatic beta-cells plays an important role in insulin secretion. To examine the impact of mitochondrial dysfunction on insulin secretion, we created a MIN6 cell line that depleted mitochondrial DNA (mtDNA) by treatment with ethidium bromide (EtBr), and studied the response of the cell line to various secretagogues. MIN6 cells cultured with 0.5 microg/ml EtBr for over 2 months (termed MIN6 deltamt cells) revealed a marked (>90%) decrease in mtDNA content and a lack of mRNAs encoded by mtDNA. MIN6 deltamt cells showed the defects of cytochrome c oxidase activity, glucose- and leucine-induced increase in cellular ATP content, and respiratory chain-driven ATP synthesis, suggesting that MIN6 deltamt cells lost oxidative phosphorylation activity due to the selective disruption of the subunits of respiratory chain enzymes encoded by mtDNA. MIN6 deltamt cells also showed a decrease in glucose utilization, suggesting the impairment of the glycolytic pathway as well. After stimulation with glucose and leucine, MIN6 deltamt cells showed no response in insulin secretion or intracellular free Ca2+ concentration ([Ca2+]i). On the other hand, arginine stimulated insulin secretion and an increase in [Ca2+]i in MIN6 deltamt cells as in MIN6 cells. Glibenclamide also stimulated insulin secretion and an increase in [Ca2+]i in both types of cells, but the responses of MIN6 deltamt cells were significantly lower than those of MIN6 cells. These results suggest the importance of ATP production in insulin secretion and an increase in [Ca2+]i, both induced by glucose and leucine. Moreover, mitochondrial function turns out to be not essential but important for the activation of sulfonylurea-induced insulin secretion.