In obesity and type 2 diabetes, expression of the GLUT4 glucose transporter is decreased selectively in adipocytes. Adipose-specific Glut4 (also known as Slc2a4) knockout (adipose-Glut4(-/-)) mice show insulin resistance secondarily in muscle and liver. Here we show, using DNA arrays, that expression of retinol binding protein-4 (RBP4) is elevated in adipose tissue of adipose-Glut4(-/-) mice. We show that serum RBP4 levels are elevated in insulin-resistant mice and humans with obesity and type 2 diabetes. RBP4 levels are normalized by rosiglitazone, an insulin-sensitizing drug. Transgenic overexpression of human RBP4 or injection of recombinant RBP4 in normal mice causes insulin resistance. Conversely, genetic deletion of Rbp4 enhances insulin sensitivity. Fenretinide, a synthetic retinoid that increases urinary excretion of RBP4, normalizes serum RBP4 levels and improves insulin resistance and glucose intolerance in mice with obesity induced by a high-fat diet. Increasing serum RBP4 induces hepatic expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) and impairs insulin signalling in muscle. Thus, RBP4 is an adipocyte-derived 'signal' that may contribute to the pathogenesis of type 2 diabetes. Lowering RBP4 could be a new strategy for treating type 2 diabetes.

Protein-tyrosine phosphatase 1B (PTP-1B) is a major protein-tyrosine phosphatase that has been implicated in the regulation of insulin action, as well as in other signal transduction pathways. To investigate the role of PTP-1B in vivo, we generated homozygotic PTP-1B-null mice by targeted gene disruption. PTP-1B-deficient mice have remarkably low adiposity and are protected from diet-induced obesity. Decreased adiposity is due to a marked reduction in fat cell mass without a decrease in adipocyte number. Leanness in PTP-1B-deficient mice is accompanied by increased basal metabolic rate and total energy expenditure, without marked alteration of uncoupling protein mRNA expression. In addition, insulin-stimulated whole-body glucose disposal is enhanced significantly in PTP-1B-deficient animals, as shown by hyperinsulinemic-euglycemic clamp studies. Remarkably, increased insulin sensitivity in PTP-1B-deficient mice is tissue specific, as insulin-stimulated glucose uptake is elevated in skeletal muscle, whereas adipose tissue is unaffected. Our results identify PTP-1B as a major regulator of energy balance, insulin sensitivity, and body fat stores in vivo.Obesity and diabetes mellitus represent major public health problems. Type 2 diabetes is a polygenic disease affecting over 100 million people worldwide. The risk of developing type 2 diabetes is increased in populations that lead a sedentary lifestyle and consume a typical western diet, in which more than 50% of the calories are derived from fat (34, 37). A high-fat diet and low energy expenditure predispose to obesity, a condition characterized by increased insulin resistance in insulinresponsive tissues, such as skeletal muscle, liver, and white adipose tissue (9, 42). Body weight also is subject to polygenic regulation (18). Many of the key genes that regulate body mass and glucose homeostasis remain to be identified (27).Insulin plays a critical role in regulating glucose homeostasis, lipid metabolism, and energy balance. Insulin signaling is initiated by binding of insulin to the insulin receptor (IR), a receptor tyrosine kinase. Insulin binding evokes a cascade of phosphorylation events, beginning with the autophosphorylation of the IR on multiple tyrosyl residues. Autophosphorylation enhances IR kinase activity and triggers downstream signaling events. These include tyrosyl phosphorylation of IR substrate (IRS) proteins (IRS-1 to -4) and other adapter molecules (e.g., Grb2 and Shc), whose combined actions mediate the biological effects of insulin (reviewed in references 24, 43, 54, and 69).

Mice lacking the protein-tyrosine phosphatase PTP1B are hypersensitive to insulin and resistant to obesity. However, the molecular basis for resistance to obesity has been unclear. Here we show that PTP1B regulates leptin signaling. In transfection studies, PTP1B dephosphorylates the leptin receptor-associated kinase, Jak2. PTP1B is expressed in hypothalamic regions harboring leptin-responsive neurons. Compared to wild-type littermates, PTP1B(-/-) mice have decreased leptin/body fat ratios, leptin hypersensitivity, and enhanced leptin-induced hypothalamic Stat3 tyrosyl phosphorylation. Gold thioglucose treatment, which ablates leptin-responsive hypothalamic neurons, partially overcomes resistance to obesity in PTP1B(-/-) mice. Our data indicate that PTP1B regulates leptin signaling in vivo, likely by targeting Jak2. PTP1B may be a novel target to treat leptin resistance in obesity.

Protein-tyrosine phosphatase 1B (PTP1B) is a major negative regulator of insulin and leptin sensitivity. PTP1B overexpression in adipose tissue and skeletal muscle of humans and rodents may contribute to insulin resistance and obesity. The mechanisms mediating PTP1B overexpression in obese and diabetic states have been unclear. We find that adipose tissue inflammation and the pro-inflammatory cytokine tumor necrosis factor ␣ (TNF␣) regulate PTP1B expression in vivo. High fat feeding of mice increased PTP1B expression 1.5-to 7-fold in adipose tissue, liver, skeletal muscle, and arcuate nucleus of hypothalamus. PTP1B overexpression in high fat-fed mice coincided with increased adipose tissue expression of the macrophage marker CD68 and TNF␣, which is implicated in causing obesity-induced insulin resistance. TNF␣ increased PTP1B mRNA and protein levels by 2-to 5-fold in a dose-and time-dependent manner in adipocyte and hepatocyte cell lines. TNF␣ administration in mice increased PTP1B mRNA 1.4-to 4-fold in adipose tissue, liver, skeletal muscle, and hypothalamic arcuate nucleus and PTP1B protein 2-fold in liver. Actinomycin D treatment blocked, and high dose salicylate treatment inhibited by 80%, TNF␣-induced PTP1B expression in adipocyte cell lines, suggesting TNF␣ may induce PTP1B transcription via nuclear factor B (NFB) activation. Chromatin immunoprecipitation from adipocyte cell lines and liver of mice demonstrated TNF␣-induced recruitment of NFB subunit p65 to the PTP1B promoter in vitro and in vivo. In mice with diet-induced obesity, TNF␣ deficiency also partly blocked PTP1B overexpression in adipose tissue. Our data suggest that PTP1B overexpression in multiple tissues in obesity is regulated by inflammation and that PTP1B may be a target of anti-inflammatory therapies.According to current World Health Organization estimates, twice as many people worldwide suffer ill health effects from the accumulation of excess adipose mass (1.6 billion) than from malnutrition (800 million). Obesity contributes to the pathogenesis of many important human diseases, including type 2 diabetes and cancer (1). Obesity is accompanied by resistance to insulin and leptin, key hormones regulating glucose homeostasis and body weight (2). The molecular mechanisms underlying leptin and insulin resistance in obesity are not completely understood. PTP1B 2 is a major negative regulator of insulin and leptin sensitivity, acting to dephosphorylate the insulin receptor and the leptin receptor-associated Janus kinase 2 (3, 4). PTP1B may also dephosphorylate more distal components of these signaling pathways, such as insulin receptor substrate 1 (5, 6). In vivo, PTP1B is widely expressed in multiple cell types and tissues, including skeletal muscle, liver, adipose tissue, and brain (3, 4). PTP1B deficiency enhances insulin signaling and sensitivity in skeletal muscle and liver (7-10). PTP1B Ϫ/Ϫ mice also have reduced adiposity and are protected from diet-induced obesity (7, 8) due to enhanced leptin action (11, 12) in neurons (9). Conversely...

Simian virus 40 (SV40) large T antigen is a potent transcriptional activator of both viral and cellular promoters. Within the SV40 late promoter, a specific upstream element necessary for T-antigen transcriptional activation is the binding site for transcription-enhancing factor 1 (TEF-1). The promoter structure necessary for T-antigen-mediated transcriptional activation appears to be simple. For example, a promoter consisting of upstream TEF-1 binding sites (or other factor-binding sites) and a downstream TATA or initiator element is efficiently activated. It has been demonstrated that transcriptional activation by T antigen does not require direct binding to the DNA; thus, the most direct effect that T antigen could have on these simple promoters would be through protein-protein interactions with either upstream-bound transcription factors, the basal transcription complex, or both. To determine whether such interactions occur, full-length T antigen or segments of it was fused to the glutathione-binding site (GST fusions) or to the Gal4 DNA-binding domain (amino acids 1 to 147) (Gal4 fusions). With the GST fusions, it was found that TEF-1 and the TATA-binding protein (TBP) bound different regions of T antigen. A GST fusion containing amino acids 5 to 172 (region Ti) efficiently bound TBP. TEF-1 bound neither region Ti nor a region between amino acids 168 and 373 (region T2); however, it bound efficiently to the combined region (T5) containing amino acids 5 to 383. The Gal4 fusions demonstrated that no region of T antigen could activate a promoter containing Gal4-binding sites, suggesting that T antigen does not contain an activation domain of the type defined by this assay. However, the Gal4 fusion proteins maintained their ability to activate promoters known to be activated by wild-type T antigen. The fusion with region Ti, which binds only TBP, modestly activated the SV40 late promoter and the simple TEF-1/TATA promoter. Region T5, which binds both TBP and TEF-1, activated each of these promoters to levels equivalent to that of wild-type T antigen. The correlation between the binding of both TEF-1 and TBP and the ability to mediate wild-type levels of transcriptional activation suggests that T antigen causes activation through direct interactions with multiple factors in the transcription complex.The simian virus 40 (SV40) early-gene product large T antigen is a potent viral oncoprotein that interacts with a number of cellular proteins known to affect cell growth and gene expression. These interactions may account for the wide variety of functions attributed to T antigen (reviewed in references 14, 28, and 36). One of these functions is the transcriptional activation of both viral and cellular promoters, which was first detected through studies of activation of the SV40 late promoter (Fig. 1A) (7, 24). Subsequently, large T antigen was shown to be a promiscuous transcriptional activator because of its effect on many viral and cellular promoters (1,17,31,42, 52). This activation of cellular promoters may be as...

Insulin signaling is essential for normal glucose homeostasis. Rho-kinase (ROCK) isoforms have been shown to participate in insulin signaling and glucose metabolism in cultured cell lines. To investigate the physiological role of ROCK1 in the regulation of whole body glucose homeostasis and insulin sensitivity in vivo, we studied mice with global disruption of ROCK1. Here we show that, at 16 -18 weeks of age, ROCK1-deficient mice exhibited insulin resistance, as revealed by the failure of blood glucose levels to decrease after insulin injection. However, glucose tolerance was normal in the absence of ROCK1. These effects were independent of changes in adiposity. Interestingly, ROCK1 gene ablation caused a significant increase in glucose-induced insulin secretion, leading to hyperinsulinemia. To determine the mechanism(s) by which deletion of ROCK1 causes insulin resistance, we measured the ability of insulin to activate phosphatidylinositol 3-kinase and multiple distal pathways in skeletal muscle. Insulin-stimulated phosphatidylinositol 3-kinase activity associated with IRS-1 or phospho-tyrosine was also reduced ϳ40% without any alteration in tyrosine phosphorylation of insulin receptor in skeletal muscle. Concurrently, serine phosphorylation of IRS-1 at serine 632/635, which is phosphorylated by ROCK in vitro, was also impaired in these mice. Insulin-induced phosphorylation of Akt, AS160, S6K, and S6 was also decreased in skeletal muscle. These data suggest that ROCK1 deficiency causes systemic insulin resistance by impairing insulin signaling in skeletal muscle. Thus, our results identify ROCK1 as a novel regulator of glucose homeostasis and insulin sensitivity in vivo, which could lead to new treatment approaches for obesity and type 2 diabetes.The ability of insulin to acutely stimulate glucose uptake and metabolism in peripheral tissues such as skeletal muscle and adipose tissue is critical for the regulation of normal glucose homeostasis (1). Impairments in insulin secretion and in the response of peripheral tissues to insulin (i.e. insulin resistance) are major pathogenic features of type 2 diabetes and contribute to the morbidity of obesity (1, 2). Insulin action involves a series of signaling cascades initiated by insulin binding to its receptor, eliciting receptor autophosphorylation and activation of the receptor tyrosine kinase, resulting in tyrosine phosphorylation of insulin receptor substrates (IRSs) 4 (3). Phosphorylation of IRSs leads to activation of phosphatidylinositol 3-kinase (PI3K) and subsequently to activation of Akt and its downstream mediator AS160, all of which are important steps for the stimulation of glucose transport induced by insulin (4 -6). Although the mechanism(s) underlying insulin resistance are not completely understood in peripheral tissues such as skeletal muscle, they are thought to result, at least in part, from impaired insulin-stimulated signal transduction (7).Rho-kinase (ROCK) is a Ser/Thr protein kinase identified as a GTP-Rho-binding protein (8). There are two ...

Previous studies implicate protein-tyrosine phosphatase 1B (PTP1B) and leukocyte antigen-related phosphatase (LAR) as negative regulators of insulin signaling. The expression and/or activity of PTP1B and LAR are increased in muscle of insulin-resistant rodents and humans. Overexpression of LAR selectively in muscle of transgenic mice causes whole body insulin resistance. To determine whether overexpression of PTP1B also causes insulin resistance, we generated transgenic mice overexpressing human PTP1B selectively in muscle at levels similar to those observed in insulin-resistant humans. Insulin-stimulated insulin receptor (IR) tyrosyl phosphorylation and phosphatidylinositol 3-kinase activity were impaired by 35% and 40 -60% in muscle of PTP1B-overexpressing mice compared with controls. Insulin stimulation of protein kinase C (PKC)/ activity, which is required for glucose transport, was impaired in muscle of PTP1B-overexpressing mice compared with controls, showing that PTP1B overexpression impairs activation of these PKC isoforms. Furthermore, hyperinsulinemic-euglycemic clamp studies revealed that whole body glucose disposal and muscle glucose uptake were decreased by 40 -50% in PTP1B-overexpressing mice. Overexpression of PTP1B or LAR alone in muscle caused similar impairments in insulin action; however, compound overexpression achieved by crossing PTP1B-and LAR-overexpressing mice was not additive. Antibodies against specific IR phosphotyrosines indicated overlapping sites of action of PTP1B and LAR. Thus, overexpression of PTP1B in vivo impairs insulin sensitivity, suggesting that overexpression of PTP1B in muscle of obese humans and rodents may contribute to their insulin resistance. Lack of additive impairment of insulin signaling by PTP1B and LAR suggests that these PTPs have overlapping actions in causing insulin resistance in vivo.

Diabetic foot ulceration is a severe complication of diabetes that lacks effective treatment. Mast cells (MCs) contribute to wound healing, but their role in diabetes skin complications is poorly understood. Here we show that the number of degranulated MCs is increased in unwounded forearm and foot skin of patients with diabetes and in unwounded dorsal skin of diabetic mice (P < 0.05). Conversely, postwounding MC degranulation increases in nondiabetic mice, but not in diabetic mice. Pretreatment with the MC degranulation inhibitor disodium cromoglycate rescues diabetes-associated wound-healing impairment in mice and shifts macrophages to the regenerative M2 phenotype (P < 0.05). Nevertheless, nondiabetic and diabetic mice deficient in MCs have delayed wound healing compared with their wild-type (WT) controls, implying that some MC mediator is needed for proper healing. MCs are a major source of vascular endothelial growth factor (VEGF) in mouse skin, but the level of VEGF is reduced in diabetic mouse skin, and its release from human MCs is reduced in hyperglycemic conditions. Topical treatment with the MC trigger substance P does not affect wound healing in MC-deficient mice, but improves it in WT mice. In conclusion, the presence of nondegranulated MCs in unwounded skin is required for proper wound healing, and therapies inhibiting MC degranulation could improve wound healing in diabetes.