The lipid phosphatase SHIP2 controls insulin sensitivity

Insulin is the primary hormone involved in glucose homeostasis, and impairment of insulin action and/or secretion has a critical role in the pathogenesis of diabetes mellitus. Type-II SH2-domain-containing inositol 5-phosphatase, or 'SHIP2', is a member of the inositol polyphosphate 5-phosphatase family. In vitro studies have shown that SHIP2, in response to stimulation by numerous growth factors and insulin, is closely linked to signalling events mediated by both phosphoinositide-3-OH kinase and Ras/mitogen-activated protein kinase. Here we report the generation of mice lacking the SHIP2 gene. Loss of SHIP2 leads to increased sensitivity to insulin, which is characterized by severe neonatal hypoglycaemia, deregulated expression of the genes involved in gluconeogenesis, and perinatal death. Adult mice that are heterozygous for the SHIP2 mutation have increased glucose tolerance and insulin sensitivity associated with an increased recruitment of the GLUT4 glucose transporter and increased glycogen synthesis in skeletal muscles. Our results show that SHIP2 is a potent negative regulator of insulin signalling and insulin sensitivity in vivo.     

Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein.

Since the discovery of insulin nearly 70 years ago, there has been no problem more fundamental to diabetes research than understanding how insulin works at the cellular level. Insulin binds to the alpha subunit of the insulin receptor which activates the tyrosine kinase in the beta subunit, but the molecular events linking the receptor kinase to insulin-sensitive enzymes and transport processes are unknown. Our discovery that insulin stimulates tyrosine phosphorylation of a protein of relative molecular mass between 165,000 and 185,000, collectively called pp185, showed that the insulin receptor kinase has specific cellular substrates. The pp185 is a minor cytoplasmic phosphoprotein found in most cells and tissues; its phosphorylation is decreased in cells expressing mutant receptors defective in signalling. We have now cloned IRS-1, which encodes a component of the pp185 band. IRS-1 contains over ten potential tyrosine phosphorylation sites, six of which are in Tyr-Met-X-Met motifs. During insulin stimulation, the IRS-1 protein undergoes tyrosine phosphorylation and binds phosphatidylinositol 3-kinase, suggesting that IRS-1 acts as a multisite 'docking' protein to bind signal-transducing molecules containing Src-homology 2 and Src-homology-3 domains. Thus IRS-1 may link the insulin receptor kinase and enzymes regulating cellular growth and metabolism.     

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.     

IRS-2 pathways integrate female reproduction and energy homeostasis

Severe dietary restriction, catabolic states and even short-term caloric deprivation impair fertility in mammals. Likewise, obesity is associated with infertile conditions such as polycystic ovary syndrome. The reproductive status of lower organisms such as Caenorhabditis elegans is also modulated by availability of nutrients. Thus, fertility requires the integration of reproductive and metabolic signals. Here we show that deletion of insulin receptor substrate-2 (IRS-2), a component of the insulin/insulin-like growth factor-1 signalling cascade, causes female infertility. Mice lacking IRS-2 have small, anovulatory ovaries with reduced numbers of follicles. Plasma concentrations of luteinizing hormone, prolactin and sex steroids are low in these animals. Pituitaries are decreased in size and contain reduced numbers of gonadotrophs. Females lacking IRS-2 have increased food intake and obesity, despite elevated levels of leptin. Our findings indicate that insulin, together with leptin and other neuropeptides, may modulate hypothalamic control of appetite and reproductive endocrinology. Coupled with findings on the role of insulin-signalling pathways in the regulation of fertility, metabolism and longevity in C. elegans and Drosophila, we have identified an evolutionarily conserved mechanism in mammals that regulates both reproduction and energy homeostasis.     

Free fatty acids regulate insulin secretion from pancreatic beta cells through GPR40

Diabetes, a disease in which carbohydrate and lipid metabolism are regulated improperly by insulin, is a serious worldwide health issue. Insulin is secreted from pancreatic beta cells in response to elevated plasma glucose, with various factors modifying its secretion. Free fatty acids (FFAs) provide an important energy source as nutrients, and they also act as signalling molecules in various cellular processes, including insulin secretion. Although FFAs are thought to promote insulin secretion in an acute phase, this mechanism is not clearly understood. Here we show that a G-protein-coupled receptor, GPR40, which is abundantly expressed in the pancreas, functions as a receptor for long-chain FFAs. Furthermore, we show that long-chain FFAs amplify glucose-stimulated insulin secretion from pancreatic beta cells by activating GPR40. Our results indicate that GPR40 agonists and/or antagonists show potential for the development of new anti-diabetic drugs.     

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.     

Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation

While bile acids (BAs) have long been known to be essential in dietary lipid absorption and cholesterol catabolism, in recent years an important role for BAs as signalling molecules has emerged. BAs activate mitogen-activated protein kinase pathways, are ligands for the G-protein-coupled receptor (GPCR) TGR5 and activate nuclear hormone receptors such as farnesoid X receptor alpha (FXR-alpha; NR1H4). FXR-alpha regulates the enterohepatic recycling and biosynthesis of BAs by controlling the expression of genes such as the short heterodimer partner (SHP; NR0B2) that inhibits the activity of other nuclear receptors. The FXR-alpha-mediated SHP induction also underlies the downregulation of the hepatic fatty acid and triglyceride biosynthesis and very-low-density lipoprotein production mediated by sterol-regulatory-element-binding protein 1c. This indicates that BAs might be able to function beyond the control of BA homeostasis as general metabolic integrators. Here we show that the administration of BAs to mice increases energy expenditure in brown adipose tissue, preventing obesity and resistance to insulin. This novel metabolic effect of BAs is critically dependent on induction of the cyclic-AMP-dependent thyroid hormone activating enzyme type 2 iodothyronine deiodinase (D2) because it is lost in D2-/- mice. Treatment of brown adipocytes and human skeletal myocytes with BA increases D2 activity and oxygen consumption. These effects are independent of FXR-alpha, and instead are mediated by increased cAMP production that stems from the binding of BAs with the G-protein-coupled receptor TGR5. In both rodents and humans, the most thermogenically important tissues are specifically targeted by this mechanism because they coexpress D2 and TGR5. The BA-TGR5-cAMP-D2 signalling pathway is therefore a crucial mechanism for fine-tuning energy homeostasis that can be targeted to improve metabolic control.     

Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes

Calorie restriction extends lifespan and produces a metabolic profile desirable for treating diseases of ageing such as type 2 diabetes. SIRT1, an NAD+-dependent deacetylase, is a principal modulator of pathways downstream of calorie restriction that produce beneficial effects on glucose homeostasis and insulin sensitivity. Resveratrol, a polyphenolic SIRT1 activator, mimics the anti-ageing effects of calorie restriction in lower organisms and in mice fed a high-fat diet ameliorates insulin resistance, increases mitochondrial content, and prolongs survival. Here we describe the identification and characterization of small molecule activators of SIRT1 that are structurally unrelated to, and 1,000-fold more potent than, resveratrol. These compounds bind to the SIRT1 enzyme-peptide substrate complex at an allosteric site amino-terminal to the catalytic domain and lower the Michaelis constant for acetylated substrates. In diet-induced obese and genetically obese mice, these compounds improve insulin sensitivity, lower plasma glucose, and increase mitochondrial capacity. In Zucker fa/fa rats, hyperinsulinaemic-euglycaemic clamp studies demonstrate that SIRT1 activators improve whole-body glucose homeostasis and insulin sensitivity in adipose tissue, skeletal muscle and liver. Thus, SIRT1 activation is a promising new therapeutic approach for treating diseases of ageing such as type 2 diabetes.     

A nuclear-receptor-dependent phosphatidylcholine pathway with antidiabetic effects

Nuclear hormone receptors regulate diverse metabolic pathways and the orphan nuclear receptor LRH-1 (also known as NR5A2) regulates bile acid biosynthesis. Structural studies have identified phospholipids as potential LRH-1 ligands, but their functional relevance is unclear. Here we show that an unusual phosphatidylcholine species with two saturated 12 carbon fatty acid acyl side chains (dilauroyl phosphatidylcholine (DLPC)) is an LRH-1 agonist ligand in vitro. DLPC treatment induces bile acid biosynthetic enzymes in mouse liver, increases bile acid levels, and lowers hepatic triglycerides and serum glucose. DLPC treatment also decreases hepatic steatosis and improves glucose homeostasis in two mouse models of insulin resistance. Both the antidiabetic and lipotropic effects are lost in liver-specific Lrh-1 knockouts. These findings identify an LRH-1 dependent phosphatidylcholine signalling pathway that regulates bile acid metabolism and glucose homeostasis.     

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.     

CREB regulates hepatic gluconeogenesis through the coactivator PGC-1

When mammals fast, glucose homeostasis is achieved by triggering expression of gluconeogenic genes in response to glucagon and glucocorticoids. The pathways act synergistically to induce gluconeogenesis (glucose synthesis), although the underlying mechanism has not been determined. Here we show that mice carrying a targeted disruption of the cyclic AMP (cAMP) response element binding (CREB) protein gene, or overexpressing a dominant-negative CREB inhibitor, exhibit fasting hypoglycaemia [corrected] and reduced expression of gluconeogenic enzymes. CREB was found to induce expression of the gluconeogenic programme through the nuclear receptor coactivator PGC-1, which is shown here to be a direct target for CREB regulation in vivo. Overexpression of PGC-1 in CREB-deficient mice restored glucose homeostasis and rescued expression of gluconeogenic genes. In transient assays, PGC-1 potentiated glucocorticoid induction of the gene for phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in gluconeogenesis. PGC-1 promotes cooperativity between cyclic AMP and glucocorticoid signalling pathways during hepatic gluconeogenesis. Fasting hyperglycaemia is strongly correlated with type II diabetes, so our results suggest that the activation of PGC-1 by CREB in liver contributes importantly to the pathogenesis of this disease.     

Ca2+-activated ATPase and ATP-dependent calmodulin-stimulated Ca2+ transport in islet cell plasma membrane

Calcium is known to play an essential part in the regulation of insulin secretion in the pancreatic beta cell. Calcium influx/efflux studies indicate that glucose promotes an accumulation of calcium by the beta cell. However, interpretation of such data is particularly difficult due to the complex compartmentalization of calcium within the cell. Although indirect evidence using chlorotetracycline suggests that control of calcium homeostasis at the plasma membrane may be central to insulin secretion, the mechanism by which secretagogues influence the handling of calcium remains unknown. Despite its continuous diffusive entry, intracellular calcium is maintained in the submicromolar range by energy-dependent mechanisms. One such process which has been well characterized in erythrocytes is a plasma membrane calcium extrusion pump whose enzymatic basis is a high affinity (Ca+2 + Mg+2)ATPase. A similar mechanism regulated by insulin has recently been identified in adipocyte plasma membranes. We report here the presence of a high affinity (Ca+2 + Mg+2)ATPase and ATP-dependent calmodulin-stimulated calcium transport system in rat pancreatic islet cell plasma membranes.     

Conserved mechanisms of glucose sensing and regulation by Drosophila corpora cardiaca cells

Antagonistic activities of glucagon and insulin control metabolism in mammals, and disruption of this balance underlies diabetes pathogenesis. Insulin-producing cells (IPCs) in the brain of insects such as Drosophila also regulate serum glucose, but it remains unclear whether insulin is the sole hormonal regulator of glucose homeostasis and whether mechanisms of glucose-sensing and response in IPCs resemble those in pancreatic islets. Here we show, by targeted cell ablation, that Drosophila corpora cardiaca (CC) cells of the ring gland are also essential for larval glucose homeostasis. Unlike IPCs, CC cells express Drosophila cognates of sulphonylurea receptor (Sur) and potassium channel (Ir), proteins that comprise ATP-sensitive potassium channels regulating hormone secretion by islets and other mammalian glucose-sensing cells. They also produce adipokinetic hormone, a polypeptide with glucagon-like functions. Glucose regulation by CC cells is impaired by exposure to sulphonylureas, drugs that target the Sur subunit. Furthermore, ubiquitous expression of an akh transgene reverses the effect of CC ablation on serum glucose. Thus, Drosophila CC cells are crucial regulators of glucose homeostasis and they use glucose-sensing and response mechanisms similar to islet cells.     

Regulation of human insulin gene expression in transgenic mice

Insulin is a polypeptide hormone of major physiological importance in the regulation of fuel homeostasis in animals (reviewed in refs 1,2). It is synthesized by the beta-cells of pancreatic islets, and circulating insulin levels are regulated by several small molecules, notably glucose, amino acids, fatty acids and certain pharmacological agents. Insulin consists of two polypeptide chains (A and B, linked by disulphide bonds) that are derived from the proteolytic cleavage of proinsulin, generating equimolar amounts of the mature insulin and a connecting peptide (C-peptide). Humans, like most vertebrates, contain one proinsulin gene, although several species, including mice and rats, have two highly homologous insulin genes. We have studied the regulation of serum insulin levels and of insulin gene expression by generating a series of transgenic mice containing the human insulin gene. We report here that the human insulin gene is expressed in a tissue-specific manner in the islets of these transgenic mice, and that serum human insulin levels are properly regulated by glucose, amino acids and tolbutamide, an oral hypoglycaemic agent.     

Remission in models of type 1 diabetes by gene therapy using a single-chain insulin analogue

A cure for diabetes has long been sought using several different approaches, including islet transplantation, regeneration of beta cells and insulin gene therapy. However, permanent remission of type 1 diabetes has not yet been satisfactorily achieved. The development of type 1 diabetes results from the almost total destruction of insulin-producing pancreatic beta cells by autoimmune responses specific to beta cells. Standard insulin therapy may not maintain blood glucose concentrations within the relatively narrow range that occurs in the presence of normal pancreatic beta cells. We used a recombinant adeno-associated virus (rAAV) that expresses a single-chain insulin analogue (SIA), which possesses biologically active insulin activity without enzymatic conversion, under the control of hepatocyte-specific L-type pyruvate kinase (LPK) promoter, which regulates SIA expression in response to blood glucose levels. Here we show that SIA produced from the gene construct rAAV-LPK-SIA caused remission of diabetes in streptozotocin-induced diabetic rats and autoimmune diabetic mice for a prolonged time without any apparent side effects. This new SIA gene therapy may have potential therapeutic value for the cure of autoimmune diabetes in humans.     

Insulin disrupts beta-adrenergic signalling to protein kinase A in adipocytes

Hormones mobilize intracellular second messengers and initiate signalling cascades involving protein kinases and phosphatases, which are often spatially compartmentalized by anchoring proteins to increase signalling specificity. These scaffold proteins may themselves be modulated by hormones. In adipocytes, stimulation of beta-adrenergic receptors increases cyclic AMP levels and activates protein kinase A (PKA), which stimulates lipolysis by phosphorylating hormone-sensitive lipase and perilipin. Acute insulin treatment activates phosphodiesterase 3B, reduces cAMP levels and quenches beta-adrenergic receptor signalling. In contrast, chronic hyperinsulinaemic conditions (typical of type 2 diabetes) enhance beta-adrenergic receptor-mediated cAMP production. This amplification of cAMP signalling is paradoxical because it should enhance lipolysis, the opposite of the known short-term effect of hyperinsulinaemia. Here we show that in adipocytes, chronically high insulin levels inhibit beta-adrenergic receptors (but not other cAMP-elevating stimuli) from activating PKA. We measured this using an improved fluorescent reporter and by phosphorylation of endogenous cAMP-response-element binding protein (CREB). Disruption of PKA scaffolding mimics the interference of insulin with beta-adrenergic receptor signalling. Chronically high insulin levels may disrupt the close apposition of beta-adrenergic receptors and PKA, identifying a new mechanism for crosstalk between heterologous signal transduction pathways.     

Hypothalamic K(ATP) channels control hepatic glucose production

Obesity is the driving force behind the worldwide increase in the prevalence of type 2 diabetes mellitus. Hyperglycaemia is a hallmark of diabetes and is largely due to increased hepatic gluconeogenesis. The medial hypothalamus is a major integrator of nutritional and hormonal signals, which play pivotal roles not only in the regulation of energy balance but also in the modulation of liver glucose output. Bidirectional changes in hypothalamic insulin signalling therefore result in parallel changes in both energy balance and glucose metabolism. Here we show that activation of ATP-sensitive potassium (K(ATP)) channels in the mediobasal hypothalamus is sufficient to lower blood glucose levels through inhibition of hepatic gluconeogenesis. Finally, the infusion of a K(ATP) blocker within the mediobasal hypothalamus, or the surgical resection of the hepatic branch of the vagus nerve, negates the effects of central insulin and halves the effects of systemic insulin on hepatic glucose production. Consistent with these results, mice lacking the SUR1 subunit of the K(ATP) channel are resistant to the inhibitory action of insulin on gluconeogenesis. These findings suggest that activation of hypothalamic K(ATP) channels normally restrains hepatic gluconeogenesis, and that any alteration within this central nervous system/liver circuit can contribute to diabetic hyperglycaemia.