Hypoinsulinaemia, glucose intolerance and diminished beta-cell size in S6K1-deficient mice

Insulin controls glucose homeostasis by regulating glucose use in peripheral tissues, and its own production and secretion in pancreatic beta cells. These responses are largely mediated downstream of the insulin receptor substrates, IRS-1 and IRS-2 (refs 4-8), through distinct signalling pathways. Although a number of effectors of these pathways have been identified, their roles in mediating glucose homeostasis are poorly defined. Here we show that mice deficient for S6 kinase 1, an effector of the phosphatidylinositide-3-OH kinase signalling pathway, are hypoinsulinaemic and glucose intolerant. Whereas insulin resistance is not observed in isolated muscle, such mice exhibit a sharp reduction in glucose-induced insulin secretion and in pancreatic insulin content. This is not due to a lesion in glucose sensing or insulin production, but to a reduction in pancreatic endocrine mass, which is accounted for by a selective decrease in beta-cell size. The observed phenotype closely parallels those of preclinical type 2 diabetes mellitus, in which malnutrition-induced hypoinsulinaemia predisposes individuals to glucose intolerance.     

MicroRNAs 103 and 107 regulate insulin sensitivity

Defects in insulin signalling are among the most common and earliest defects that predispose an individual to the development of type 2 diabetes. MicroRNAs have been identified as a new class of regulatory molecules that influence many biological functions, including metabolism. However, the direct regulation of insulin sensitivity by microRNAs in vivo has not been demonstrated. Here we show that the expression of microRNAs 103 and 107 (miR-103/107) is upregulated in obese mice. Silencing of miR-103/107 leads to improved glucose homeostasis and insulin sensitivity. In contrast, gain of miR-103/107 function in either liver or fat is sufficient to induce impaired glucose homeostasis. We identify caveolin-1, a critical regulator of the insulin receptor, as a direct target gene of miR-103/107. We demonstrate that caveolin-1 is upregulated upon miR-103/107 inactivation in adipocytes and that this is concomitant with stabilization of the insulin receptor, enhanced insulin signalling, decreased adipocyte size and enhanced insulin-stimulated glucose uptake. These findings demonstrate the central importance of miR-103/107 to insulin sensitivity and identify a new target for the treatment of type 2 diabetes and obesity.     

InsP4 facilitates store-operated calcium influx by inhibition of InsP3 5-phosphatase

Receptor-mediated generation of inositol 1,4,5-trisphosphate (InsP3) initiates Ca2+ release from intracellular stores and the subsequent activation of store-operated calcium influx. InsP3 is metabolized within seconds by 5-phosphatase and 3-kinase, yielding Ins(1,4)P2 and inositol 1,3,4,5-tetrakisphosphate (InsP4), respectively. Some studies have suggested that InsP4 controls Ca2+ influx in combination with InsP3 (refs 3 and 4), but another study did not find the same result. Some of the apparent conflicts between these previous studies have been resolved; however, the physiological function of InsP4 remains elusive. Here we have investigated the function of InsP4 in Ca2+ influx in the mast cell line RBL-2H3, and we show that InsP4 inhibits InsP3 metabolism through InsP3 5-phosphatase, thereby facilitating the activation of the store-operated Ca2+ current I(CRAC) (ref. 9). Physiologically, this mechanism opens a discriminatory time window for coincidence detection that enables selective facilitation of Ca2+ influx by appropriately timed low-level receptor stimulation. At higher concentrations, InsP4 acts as an inhibitor of InsP3 receptors, enabling InsP4 to act as a potent bi-modal regulator of cellular sensitivity to InsP3, which provides both facilitatory and inhibitory feedback on Ca2+ signalling.     

The hormone resistin links obesity to diabetes

Diabetes mellitus is a chronic disease that leads to complications including heart disease, stroke, kidney failure, blindness and nerve damage. Type 2 diabetes, characterized by target-tissue resistance to insulin, is epidemic in industrialized societies and is strongly associated with obesity; however, the mechanism by which increased adiposity causes insulin resistance is unclear. Here we show that adipocytes secrete a unique signalling molecule, which we have named resistin (for resistance to insulin). Circulating resistin levels are decreased by the anti-diabetic drug rosiglitazone, and increased in diet-induced and genetic forms of obesity. Administration of anti-resistin antibody improves blood sugar and insulin action in mice with diet-induced obesity. Moreover, treatment of normal mice with recombinant resistin impairs glucose tolerance and insulin action. Insulin-stimulated glucose uptake by adipocytes is enhanced by neutralization of resistin and is reduced by resistin treatment. Resistin is thus a hormone that potentially links obesity to diabetes.     

Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity

Elucidating the signalling mechanisms by which obesity leads to impaired insulin action is critical in the development of therapeutic strategies for the treatment of diabetes. Recently, mice deficient for S6 Kinase 1 (S6K1), an effector of the mammalian target of rapamycin (mTOR) that acts to integrate nutrient and insulin signals, were shown to be hypoinsulinaemic, glucose intolerant and have reduced beta-cell mass. However, S6K1-deficient mice maintain normal glucose levels during fasting, suggesting hypersensitivity to insulin, raising the question of their metabolic fate as a function of age and diet. Here, we report that S6K1-deficient mice are protected against obesity owing to enhanced beta-oxidation. However on a high fat diet, levels of glucose and free fatty acids still rise in S6K1-deficient mice, resulting in insulin receptor desensitization. Nevertheless, S6K1-deficient mice remain sensitive to insulin owing to the apparent loss of a negative feedback loop from S6K1 to insulin receptor substrate 1 (IRS1), which blunts S307 and S636/S639 phosphorylation; sites involved in insulin resistance. Moreover, wild-type mice on a high fat diet as well as K/K A(y) and ob/ob (also known as Lep/Lep) mice-two genetic models of obesity-have markedly elevated S6K1 activity and, unlike S6K1-deficient mice, increased phosphorylation of IRS1 S307 and S636/S639. Thus under conditions of nutrient satiation S6K1 negatively regulates insulin signalling.     

Attenuation of FGF signalling in mouse beta-cells leads to diabetes

Fibroblast growth factor (FGF) signalling has been implicated in patterning, proliferation and cell differentiation in many organs, including the developing pancreas. Here we show that the FGF receptors (FGFRs) 1 and 2, together with the ligands FGF1, FGF2, FGF4, FGF5, FGF7 and FGF10, are expressed in adult mouse beta-cells, indicating that FGF signalling may have a role in differentiated beta-cells. When we perturbed signalling by expressing dominant-negative forms of the receptors, FGFR1c and FGFR2b, in the pancreas, we found that that mice with attenuated FGFR1c signalling, but not those with reduced FGFR2b signalling, develop diabetes with age and exhibit a decreased number of beta-cells, impaired expression of glucose transporter 2 and increased proinsulin content in beta-cells owing to impaired expression of prohormone convertases 1/3 and 2. These defects are all characteristic of patients with type-2 diabetes. Mutations in the homeobox gene Ipf1/Pdx1 are linked to diabetes in both mouse and human. We also show that Ipf1/Pdx1 is required for the expression of FGFR1 signalling components in beta-cells, indicating that Ipf1/Pdx1 acts upstream of FGFR1 signalling in beta-cells to maintain proper glucose sensing, insulin processing and glucose homeostasis.     

Critical role for the p110alpha phosphoinositide-3-OH kinase in growth and metabolic regulation

The eight catalytic subunits of the mammalian phosphoinositide-3-OH kinase (PI(3)K) family form the backbone of an evolutionarily conserved signalling pathway; however, the roles of most PI(3)K isoforms in organismal physiology and disease are unknown. To delineate the role of p110alpha, a ubiquitously expressed PI(3)K involved in tyrosine kinase and Ras signalling, here we generated mice carrying a knockin mutation (D933A) that abrogates p110alpha kinase activity. Homozygosity for this kinase-dead p110alpha led to embryonic lethality. Mice heterozygous for this mutation were viable and fertile, but displayed severely blunted signalling via insulin-receptor substrate (IRS) proteins, key mediators of insulin, insulin-like growth factor-1 and leptin action. Defective responsiveness to these hormones led to reduced somatic growth, hyperinsulinaemia, glucose intolerance, hyperphagia and increased adiposity in mice heterozygous for the D933A mutation. This signalling function of p110alpha derives from its highly selective recruitment and activation to IRS signalling complexes compared to p110beta, the other broadly expressed PI(3)K isoform, which did not contribute to IRS-associated PI(3)K activity. p110alpha was the principal IRS-associated PI(3)K in cancer cell lines. These findings demonstrate a critical role for p110alpha in growth factor and metabolic signalling and also suggest an explanation for selective mutation or overexpression of p110alpha in a variety of cancers.     

Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes

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.     

Essential roles of PI(3)K-p110beta in cell growth, metabolism and tumorigenesis

On activation by receptors, the ubiquitously expressed class IA isoforms (p110alpha and p110beta) of phosphatidylinositol-3-OH kinase (PI(3)K) generate lipid second messengers, which initiate multiple signal transduction cascades. Recent studies have demonstrated specific functions for p110alpha in growth factor and insulin signalling. To probe for distinct functions of p110beta, we constructed conditional knockout mice. Here we show that ablation of p110beta in the livers of the resulting mice leads to impaired insulin sensitivity and glucose homeostasis, while having little effect on phosphorylation of Akt, suggesting the involvement of a kinase-independent role of p110beta in insulin metabolic action. Using established mouse embryonic fibroblasts, we found that removal of p110beta also had little effect on Akt phosphorylation in response to stimulation by insulin and epidermal growth factor, but resulted in retarded cell proliferation. Reconstitution of p110beta-null cells with a wild-type or kinase-dead allele of p110beta demonstrated that p110beta possesses kinase-independent functions in regulating cell proliferation and trafficking. However, the kinase activity of p110beta was required for G-protein-coupled receptor signalling triggered by lysophosphatidic acid and had a function in oncogenic transformation. Most strikingly, in an animal model of prostate tumour formation induced by Pten loss, ablation of p110beta (also known as Pik3cb), but not that of p110alpha (also known as Pik3ca), impeded tumorigenesis with a concomitant diminution of Akt phosphorylation. Taken together, our findings demonstrate both kinase-dependent and kinase-independent functions for p110beta, and strongly indicate the kinase-dependent functions of p110beta as a promising target in cancer therapy.     

CAP defines a second signalling pathway required for insulin-stimulated glucose transport

Insulin stimulates the transport of glucose into fat and muscle cells. Although the precise molecular mechanisms involved in this process remain uncertain, insulin initiates its actions by binding to its tyrosine kinase receptor, leading to the phosphorylation of intracellular substrates. One such substrate is the Cbl proto-oncogene product. Cbl is recruited to the insulin receptor by interaction with the adapter protein CAP, through one of three adjacent SH3 domains in the carboxy terminus of CAP. Upon phosphorylation of Cbl, the CAP-Cbl complex dissociates from the insulin receptor and moves to a caveolin-enriched, triton-insoluble membrane fraction. Here, to identify a molecular mechanism underlying this subcellular redistribution, we screened a yeast two-hybrid library using the amino-terminal region of CAP and identified the caveolar protein flotillin. Flotillin forms a ternary complex with CAP and Cbl, directing the localization of the CAP-Cbl complex to a lipid raft subdomain of the plasma membrane. Expression of the N-terminal domain of CAP in 3T3-L1 adipocytes blocks the stimulation of glucose transport by insulin, without affecting signalling events that depend on phosphatidylinositol-3-OH kinase. Thus, localization of the Cbl-CAP complex to lipid rafts generates a pathway that is crucial in the regulation of glucose uptake.     

Inositol-1,4,5-trisphosphate receptor regulates hepatic gluconeogenesis in fasting and diabetes

In the fasted state, increases in circulating glucagon promote hepatic glucose production through induction of the gluconeogenic program. Triggering of the cyclic AMP pathway increases gluconeogenic gene expression via the de-phosphorylation of the CREB co-activator CRTC2 (ref. 1). Glucagon promotes CRTC2 dephosphorylation in part through the protein kinase A (PKA)-mediated inhibition of the CRTC2 kinase SIK2. A number of Ser/Thr phosphatases seem to be capable of dephosphorylating CRTC2 (refs 2, 3), but the mechanisms by which hormonal cues regulate these enzymes remain unclear. Here we show in mice that glucagon stimulates CRTC2 dephosphorylation in hepatocytes by mobilizing intracellular calcium stores and activating the calcium/calmodulin-dependent Ser/Thr-phosphatase calcineurin (also known as PP3CA). Glucagon increased cytosolic calcium concentration through the PKA-mediated phosphorylation of inositol-1,4,5-trisphosphate receptors (InsP(3)Rs), which associate with CRTC2. After their activation, InsP(3)Rs enhanced gluconeogenic gene expression by promoting the calcineurin-mediated dephosphorylation of CRTC2. During feeding, increases in insulin signalling reduced CRTC2 activity via the AKT-mediated inactivation of InsP(3)Rs. InsP(3)R activity was increased in diabetes, leading to upregulation of the gluconeogenic program. As hepatic downregulation of InsP(3)Rs and calcineurin improved circulating glucose levels in insulin resistance, these results demonstrate how interactions between cAMP and calcium pathways at the level of the InsP(3)R modulate hepatic glucose production under fasting conditions and in diabetes.     

Insulin receptor tyrosine kinase is defective in skeletal muscle of insulin-resistant obese mice

Obese syndromes of genetic origin or experimentally induced are characterized by resistance to insulin both in vivo (association of hyperglycaemia and hyperinsulinaemia) and in vitro. Thus, skeletal muscle of obese mice, which is the most important target organ for the action of insulin, displays a reduced response to insulin. This hormonal resistance cannot be explained by the moderate decrease in the number of insulin receptors found in obese animals. In fact, it is generally believed that a biochemical event occurring very early after binding of insulin to its receptor, which is the first step in insulin action, is defective in obesity. One of the earliest post-binding events so far recognized, and which is thought to have a key role in cellular signalling by the insulin receptor, is the insulin-stimulated phosphorylation of its receptor. In an effort to localize the defect responsible for the insulin resistance in obesity, we have studied the insulin receptor protein kinase activity and we show here that insulin receptors from skeletal muscles of insulin-resistant obese mice have an altered kinase activity for phosphorylation of both the receptor itself and of exogeneous substrates.     

Reactive oxygen species have a causal role in multiple forms of insulin resistance

Insulin resistance is a cardinal feature of type 2 diabetes and is characteristic of a wide range of other clinical and experimental settings. Little is known about why insulin resistance occurs in so many contexts. Do the various insults that trigger insulin resistance act through a common mechanism? Or, as has been suggested, do they use distinct cellular pathways? Here we report a genomic analysis of two cellular models of insulin resistance, one induced by treatment with the cytokine tumour-necrosis factor-alpha and the other with the glucocorticoid dexamethasone. Gene expression analysis suggests that reactive oxygen species (ROS) levels are increased in both models, and we confirmed this through measures of cellular redox state. ROS have previously been proposed to be involved in insulin resistance, although evidence for a causal role has been scant. We tested this hypothesis in cell culture using six treatments designed to alter ROS levels, including two small molecules and four transgenes; all ameliorated insulin resistance to varying degrees. One of these treatments was tested in obese, insulin-resistant mice and was shown to improve insulin sensitivity and glucose homeostasis. Together, our findings suggest that increased ROS levels are an important trigger for insulin resistance in numerous settings.     

The inositol tris/tetrakisphosphate pathway--demonstration of Ins(1,4,5)P3 3-kinase activity in animal tissues

Recent advances in our understanding of the role of inositides in cell signalling have led to the central hypothesis that a receptor-stimulated phosphodiesteratic hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) results in the formation of two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). The existence of another pathway of inositide metabolism was first suggested by the discovery that a novel inositol trisphosphate, Ins(1,3,4)P3, is formed in stimulated tissues; the metabolic kinetics of Ins(1,3,4)P3 are entirely different from those of Ins(1,4,5)P3 (refs 6, 7). The probable route of formation of Ins(1,3,4)P3 was recently shown to be via a 5-dephosphorylation of inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), a compound which is rapidly formed on muscarinic stimulation of brain slices, and which can be readily converted to Ins(1,3,4)P3 by a 5-phosphatase in red blood cell membranes. However, the source of Ins(1,3,4,5)P4 is unclear, and an attempt to detect a possible parent lipid, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), was unsuccessful. The recent discovery that the higher phosphorylated forms of inositol (InsP5 and InsP6) also exist in animal cells suggested that inositol phosphate kinases might not be confined to plant and avian tissues, and here we show that a variety of animal tissues contain an active and specific Ins(1,4,5)P3 3-kinase. We therefore suggest that an inositol tris/tetrakisphosphate pathway exists as an alternative route to the dephosphorylation of Ins(1,4,5)P3. The function of this novel pathway is unknown.     

Adipose-selective targeting of the GLUT4 gene impairs insulin action in muscle and liver

The earliest defect in developing type 2 diabetes is insulin resistance, characterized by decreased glucose transport and metabolism in muscle and adipocytes. The glucose transporter GLUT4 mediates insulin-stimulated glucose uptake in adipocytes and muscle by rapidly moving from intracellular storage sites to the plasma membrane. In insulin-resistant states such as obesity and type 2 diabetes, GLUT4 expression is decreased in adipose tissue but preserved in muscle. Because skeletal muscle is the main site of insulin-stimulated glucose uptake, the role of adipose tissue GLUT4 downregulation in the pathogenesis of insulin resistance and diabetes is unclear. To determine the role of adipose GLUT4 in glucose homeostasis, we used Cre/loxP DNA recombination to generate mice with adipose-selective reduction of GLUT4 (G4A-/-). Here we show that these mice have normal growth and adipose mass despite markedly impaired insulin-stimulated glucose uptake in adipocytes. Although GLUT4 expression is preserved in muscle, these mice develop insulin resistance in muscle and liver, manifested by decreased biological responses and impaired activation of phosphoinositide-3-OH kinase. G4A-/- mice develop glucose intolerance and hyperinsulinaemia. Thus, downregulation of GLUT4 and glucose transport selectively in adipose tissue can cause insulin resistance and thereby increase the risk of developing diabetes.     

Platelet-derived growth factor stimulates synthesis of PtdIns(3,4,5)P3 by activating a PtdIns(4,5)P2 3-OH kinase.

Although the hormone-stimulated synthesis of 3-phosphorylated inositol lipids is known to form an intracellular signalling system, there is no consensus on the crucial receptor-regulated event in this pathway and it is still not clear which of the intermediates represent potential output signals. We show here that the key step in the synthesis of 3-phosphorylated inositol lipids in 3T3 cells stimulated by platelet-derived growth factor is the activation of a phosphatidylinositol(4,5)-bisphosphate (3)-hydroxy (PtdIns(4,5)P2 3-OH) kinase. A similar conclusion has been applied to explain the actions of formyl-Met-Leu-Phe on neutrophils, and it may be that receptors that couple through intrinsic tyrosine kinases or through G proteins stimulate the same step in 3-phosphorylated inositol lipid metabolism. The close parallel between these two mechanisms for the activation of PtdIns(4,5)P2 3-OH kinase and those described for the activation of another key signalling enzyme, phospholipase C (ref. 7), focuses attention on the product of the PtdIns(4,5)P2 3-OH kinase, PtdIns(3,4,5)P3, as a possible new second messenger.     

Insulin stimulation of glucose uptake can be mediated by diacylglycerol in adipocytes

An early effect of insulin in adipocytes is to stimulate glucose uptake. The increased uptake appears to be due to mobilization of glucose transporters from an intracellular location to the plasma membrane and to enhanced intrinsic activity of the transporters. Little is known about the insulin-generated signals causing these changes. Phorbol esters have been shown to mimic the insulin effect, but phosphorylation of the transporter does not seem to be involved. A phospho-oligosaccharide was recently shown to mimic the effects of insulin on protein phosphorylation, suggesting that it could be a mediator for some intracellular metabolic effects of the hormone, but it did not affect glucose uptake. A diacyglycerol is produced in the plasma membrane in conjunction with the generation of the phospho-oligosaccharide. Here I show that added 1,2-diacylglycerols potently increase glucose transporter-mediated uptake of glucose in rat adipocytes, but without activation of protein kinase C.     

Pyruvate kinase M2 is a phosphotyrosine-binding protein

Growth factors stimulate cells to take up excess nutrients and to use them for anabolic processes. The biochemical mechanism by which this is accomplished is not fully understood but it is initiated by phosphorylation of signalling proteins on tyrosine residues. Using a novel proteomic screen for phosphotyrosine-binding proteins, we have made the observation that an enzyme involved in glycolysis, the human M2 (fetal) isoform of pyruvate kinase (PKM2), binds directly and selectively to tyrosine-phosphorylated peptides. We show that binding of phosphotyrosine peptides to PKM2 results in release of the allosteric activator fructose-1,6-bisphosphate, leading to inhibition of PKM2 enzymatic activity. We also provide evidence that this regulation of PKM2 by phosphotyrosine signalling diverts glucose metabolites from energy production to anabolic processes when cells are stimulated by certain growth factors. Collectively, our results indicate that expression of this phosphotyrosine-binding form of pyruvate kinase is critical for rapid growth in cancer cells.     

胰岛素抵抗及高血糖对GFAT活性的影响

分别观察了胰岛素抵抗和高血糖水平对己糖胺途径限速酶GFAT活性的影响.在alloxan高血糖小鼠模型中,与正常对照组比较,血清果糖胺水平升高了15.9%,肾组织GFAT活性升高了32.8%;经胰岛素治疗后,血清果糖胺水平降低了9.7%,其肾组织GFAT活性也降低了l9.4%.在高糖高脂饲料诱导的胰岛素抵抗的IR小鼠中,与同批正常对照组比较,正糖钳实验中稳态时葡萄糖输注率G IR值降低了69.3%,胰岛素耐量实验中的AUC值升高了38.1%,其肾脏组织GFAT活性也增加了26.6%.在胰岛素诱导的具有胰岛素抵抗的IR-H IR c细胞模型中,与正常H IR c细胞比较,其10、25 nmol/L胰岛素诱导的葡萄糖摄取能力分别降低了25.3%、21.1%,而GFAT活性分别增加了29.7%、46.5%.可见,GFAT活性与一段时间的平均血糖水平和胰岛素抵抗状态密切正相关.