Search

Cellular senescence is thought to contribute to age-associated deterioration of tissue physiology. The senescence Effector [p16.sup.Ink4a] is expressed in pancreatic beta cells during aging and limits their proliferative potential; however, its effects on beta cell function are poorly characterized. We found that beta cell-specific activation of [p16.sup.Ink4a] in transgenic mice enhances glucose-stimulated insulin secretion (GSIS). In mice with diabetes, this leads to improved glucose homeostasis, providing an unexpected functional benefit. Expression of [p16.sup.Ink4a] in beta cells induces hallmarks of senescence--including cell enlargement, and greater glucose uptake and mitochondrial activity--which promote increased insulin secretion. GSIS increases during the normal aging of mice and is driven by elevated [p16.sup.Ink4a] activity. We found that islets from human adults contain [p16.sup.Ink4a]-expressing senescent beta cells and that senescence induced by [p16.sup.Ink4a] in a human beta cell line increases insulin secretion in a manner dependent, in part, on the activity of the mechanistic target of rapamycin (mTOR) and the peroxisome proliferator-activated receptor (PPAR)-γ proteins. Our findings reveal a novel role for [p16.sup.Ink4a] and cellular senescence in promoting insulin secretion by beta cells and in regulating normal functional tissue maturation with age., Aged tissues typically show decreased regenerative capacity and deterioration in overall function. Cellular senescence is thought to contribute to tissue aging and associated pathologies through various means, including the limitation [...]

The ability of glucose to stimulate insulin secretion from the pancreatic islets of Langerhans is enhanced by the intestinal hormone glucagon-like peptide 1 (GLP-1), which is secreted from the gut in response to nutrient ingestion. This action, called the incretin effect, accounts for as much as half of the postprandial insulin response and is exploited therapeutically for diabetes treatment through the use of incretin mimetic drugs and inhibitors of dipeptidyl peptidase 4, which degrades GLP-1. Despite a prominent role for incretin mimetics in diabetes treatment, several key questions remain about GLP-1-induced insulin secretion. Most studies have examined the effects of GLP-1 at concentrations several orders of magnitude higher than those found in vivo; therefore, one might question the physiological (and perhaps even pharmacological) relevance of pathways identified in these studies and whether other important mechanisms might have been obscured. In this issue of theJCI, Shigeto and colleagues demonstrate that physiological GLP-1 does indeed amplify the insulin secretory response. Intriguingly, while much of this response is PKA dependent, as might be expected, the use of picomolar GLP-1 reveals a new and important mechanism that contributes to GLP-1-induced insulin secretion., GLP-1 amplification of glucose-stimulated insulin secretion It is well established that the incretin hormone glucagon-like peptide 1 (GLP-1) amplifies the insulin secretory response to glucose (1, 2). Specifically, nutrient sensing [...]

Strategies aimed at mimicking or enhancing the action of the incretin hormone glucagon-like peptide 1 (GLP-1) therapeutically improve glucose-stimulated insulin secretion (GSIS); however, it is not clear whether GLP-1 directly drives insulin secretion in pancreatic islets. Here, we examined the mechanisms by which GLP-1 stimulates insulin secretion in mouse and human islets. We found that GLP-1 enhances GSIS at a half-maximal effective concentration of 0.4 pM. Moreover, we determined that GLP-1 activates PLC, which increases submembrane diacylglycerol and thereby activates PKC, resulting in membrane depolarization and increased action potential firing and subsequent stimulation of insulin secretion. The depolarizing effect of GLP-1 on electrical activity was mimicked by the PKC activator PMA, occurred without activation of PKA, and persisted in the presence of PKA inhibitors, the [K.sub.ATP] channel blocker tolbutamide, and the L-type [Ca.sup.2+] channel blocker isradipine; however, depolarization was abolished by lowering extracellular [Na.sup.+]. The PKC-dependent effect of GLP-1 on membrane potential and electrical activity was mediated by activation of [Na.sup.+]-permeable TRPM4 and TRPM5 channels by mobilization of intracellular [Ca.sup.2+] from thapsigargin-sensitive [Ca.sup.2+] stores. Concordantly, GLP-1 effects were negligible in Trpm4 or TrpmS KO islets. These data provide important insight into the therapeutic action of GLP-1 and suggest that circulating levels of this hormone directly stimulate insulin secretion by β cells., Introduction Type 2 diabetes currently affects approximately 350 million individuals in the world (1). It is caused by insufficient insulin secretion, often in combination with impaired insulin action (2). The [...]

Alterations in insulin granule exocytosis and endocytosis are paramount to pancreatic β cell dysfunction in diabetes mellitus. Here, using temporally controlled gene ablation specifically in β cells in mice, we identified an essential role of dynamin 2 GTPase in preserving normal biphasic insulin secretion and blood glucose homeostasis. Dynamin 2 deletion in β cells caused glucose intolerance and substantial reduction of the second phase of glucose-stimulated insulin secretion (GSIS); however, mutant β cells still maintained abundant insulin granules, with no signs of cell surface expansion. Compared with control β cells, real-time capacitance measurements demonstrated that exocytosis- endocytosis coupling was less efficient but not abolished; clathrin-mediated endocytosis (CME) was severely impaired at the step of membrane fission, which resulted in accumulation of clathrin-coated endocytic intermediates on the plasma membrane. Moreover, dynamin 2 ablation in β cells led to striking reorganization and enhancement of actin filaments, and insulin granule recruitment and mobilization were impaired at the later stage of GSIS. Together, our results demonstrate that dynamin 2 regulates insulin secretory capacity and dynamics in vivo through a mechanism depending on CME and F-actin remodeling. Moreover, this study indicates a potential pathophysiological link between endocytosis and diabetes mellitus., Introduction Proper regulation of membrane trafficking balances material and signal exchange and is fundamental to cellular functions. Similar to nerve terminals, in which endocytosis supports high rates of synaptic vesicle [...]

Type 2 diabetes is a major health problem worldwide, and one of its key features is the inability of elevated glucose to stimulate the release of sufficient amounts of insulin from pancreatic beta cells to maintain normal blood glucose levels (1,2). New therapeutic strategies to improve beta cell function are therefore believed to be beneficial (3,4). Here we demonstrate that the short-chain fatty acid receptors FFA2 (encoded by FFAR2) and FFA3 (encoded by FFAR3) are expressed in mouse and human pancreatic beta cells and mediate an inhibition of insulin secretion by coupling to [G.sub.i]-type G proteins. We also provide evidence that mice with dietary-induced obesity and type 2 diabetes, as compared to non-obese control mice, have increased local formation by pancreatic islets of acetate, an endogenous agonist of FFA2 and FFA3, as well as increased systemic levels. This elevation may contribute to the insufficient capacity of beta cells to respond to hyperglycemia in obese states. Indeed, we found that genetic deletion of both receptors, either on the whole-body level or specifically in pancreatic beta cells, leads to greater insulin secretion and a profound improvement of glucose tolerance when mice are on a high-fat diet compared to controls. On the other hand, deletion of Ffar2 and Ffar3 in intestinal cells did not alter glucose tolerance in diabetic animals, suggesting these receptors act in a cell-autonomous manner in beta cells to regulate insulin secretion. In summary, under diabetic conditions elevated acetate acts on FFA2 and FFA3 to inhibit proper glucose-stimulated insulin secretion, and we expect antagonists of FFA2 and FFA3 to improve insulin secretion in type 2 diabetes., The most important stimulator of insulin release is glucose itself (5,6), and glucose-stimulated insulin secretion (GSIS) can be potentiated or inhibited by a variety of mediators, most of them acting [...]

Tissue-specific autoimmunity occurs when selected antigens presented by susceptible alleles of the major histocompatibility complex are recognized by T cells. However, the reason why certain specific self-antigens dominate the response and are indispensable for triggering autoreactivity is unclear. Spontaneous presentation of insulin is essential for initiating autoimmune type 1 diabetes in non-obese diabetic mice.sup.1,2. A major set of pathogenic CD4 T cells specifically recognizes the 12-20 segment of the insulin B-chain (B:12-20), an epitope that is generated from direct presentation of insulin peptides by antigen-presenting cells.sup.3,4. These T cells do not respond to antigen-presenting cells that have taken up insulin that, after processing, leads to presentation of a different segment representing a one-residue shift, B:13-21.sup.4. CD4 T cells that recognize B:12-20 escape negative selection in the thymus and cause diabetes, whereas those that recognize B:13-21 have only a minor role in autoimmunity.sup.3-5. Although presentation of B:12-20 is evident in the islets.sup.3,6, insulin-specific germinal centres can be formed in various lymphoid tissues, suggesting that insulin presentation is widespread.sup.7,8. Here we use live imaging to document the distribution of insulin recognition by CD4 T cells throughout various lymph nodes. Furthermore, we identify catabolized insulin peptide fragments containing defined pathogenic epitopes in [beta]-cell granules from mice and humans. Upon glucose challenge, these fragments are released into the circulation and are recognized by CD4 T cells, leading to an activation state that results in transcriptional reprogramming and enhanced diabetogenicity. Therefore, a tissue such as pancreatic islets, by releasing catabolized products, imposes a constant threat to self-tolerance. These findings reveal a self-recognition pathway underlying a primary autoantigen and provide a foundation for assessing antigenic targets that precipitate pathogenic outcomes by systemically sensitizing lymphoid tissues.A sensitive T cell tracking assay reveals immunogenic activity of specific catabolized peptide fragments of insulin and their effects on T cell activity in lymph nodes, highlighting communication between pancreatic islets and lymphoid tissue., Author(s): Xiaoxiao Wan [sup.1] , Bernd H. Zinselmeyer [sup.1] , Pavel N. Zakharov [sup.1] , Anthony N. Vomund [sup.1] , Ruth Taniguchi [sup.2] , Laura Santambrogio [sup.3] , Mark S. [...]

Insulin and [Zn.sup.2+] enjoy a multivalent relationship. [Zn.sup.2+] binds insulin in pancreatic β cells to form crystalline aggregates in dense core vesicles (DCVs), which are released in response to physiological [...]

In rodents, acute brain insulin action reduces blood glucose levels by suppressing the expression of enzymes in the hepatic gluconeogenic pathway, thereby reducing gluconeogenesis and endogenous glucose production (EGP). Whether a similar mechanism is functional in large animals, including humans, is unknown. Here, we demonstrated that in canines, physiologic brain hyperinsulinemia brought about by infusion of insulin into the head arteries (during a pancreatic clamp to maintain basal hepatic insulin and glucagon levels) activated hypothalamic Akt, altered STAT3 signaling in the liver, and suppressed hepatic gluconeogenic gene expression without altering EGP or gluconeogenesis. Rather, brain hyperinsulinemia slowly caused a modest reduction in net hepatic glucose output (NHGO) that was attributable to increased net hepatic glucose uptake and glycogen synthesis. This was associated with decreased levels of glycogen synthase kinase 3β (GSK3β) protein and mRNA and with decreased glycogen synthase phosphorylation, changes that were blocked by hypothalamic PI3K inhibition. Therefore, we conclude that the canine brain senses physiologic elevations in plasma insulin, and that this in turn regulates genetic events in the liver. In the context of basal insulin and glucagon levels at the liver, this input augments hepatic glucose uptake and glycogen synthesis, reducing NHGO without altering EGP., Introduction The ability of insulin to suppress hepatic glucose production (HGP) in vivo has been well defined (1), but the role of the CNS in this suppression remains controversial. The [...]

ARTICLE INFO Keywords: Cinnamon Cinnamomum cassia Cinnamomum japonica Diabetes mellitus Procyanidin oligomers ABSTRACT Procyanidin oligomers in Cinnamon are thought to be responsible for the biological activity in the treatment of [...]

Estrogen receptors (ERs) protect pancreatic islet survival in mice through rapid extranuclear actions. ER[alpha] also enhances insulin synthesis in cultured islets. Whether ERa stimulates insulin synthesis in vivo and, if so, through which mechanism(s) remain largely unknown. To address these issues, we generated a pancreas-specific ERa knockout mouse (PER[alpha][KO.sup.-/-]) using the Cre-loxP strategy and used a combination of genetic and pharmacologic tools in cultured islets and [beta] cells. Whereas 17[beta]-estradiol (E2) treatment up-regulates pancreatic insulin gene and protein content in control ERcdox/lox mice, these E2 effects are abolished in PER[alpha][KO.sup.-/-] mice. We find that E2-activated ER[alpha] increases insulin synthesis by enhancing glucose stimulation of the insulin promoter activity. Using a knock-in mouse with a mutated ER[alpha] eliminating binding to the estrogen response elements (EREs), we show that E2 stimulation of insulin synthesis is independent of the ERE. We find that the extranuclear ER[alpha] interacts with the tyrosine kinase Src, which activates extracellular signal-regulated [kinases.sub.1/2], to increase nuclear localization and binding to the insulin promoter of the transcription factor NeuroD1. This study supports the importance of ERa in [beta] cells as a regulator of insulin synthesis in vivo. diabetes | islet doi/ 10.1073/pnas.0914501107

Primary cultures of rainbow trout (Oncorhynchus mykiss) adipocytes were used to examine the main signaling pathways of insulin and insulin-like growth factor I (IGF-I) during adipogenesis. We first determined the presence of IGF-I receptors (IGF-IR) and insulin receptors (IR) in trout preadipocytes (day 5) and adipocytes (day 14). IGF-IRs were more abundant and appeared to be in higher levels in differentiated cells than in preadipocytes, whereas IRs were detected in lower but constant levels throughout the culture. The cells were immunoreactive against ERK 1/2 MAPK, and AKT/PI3K, components of the two main signal transduction pathways for insulin and IGF-I receptors. Stimulation of MAPK phosphorylation by IGF-I was higher in preadipocytes than in adipocytes, while no effects were observed in MAPK phosphorylation after incubation of cells with insulin. AKT phosphorylation increased in the presence of both insulin and IGF-I, with higher levels of stimulation in adipocytes than in preadipocytes. Activation of both pathways was blocked by the use of specific inhibitors of MAPK (PD98059) and AKT (wortmannin). We describe here, for the first time, the effects of IGF-I and insulin on 2-deoxyglucose uptake in primary culture of trout adipocytes. IGF-I was more potent in stimulating glucose uptake than insulin, and PD98059 and wortmannin inhibited the stimulation of glucose uptake by this growth factor, suggesting that IGF-I plays an important metabolic role in trout adipocytes. Our results suggest that differential activation of the MAPK and AKT pathways are involved in the IGF-I- and insulin-induced effects of trout adipocytes during the various stages of adipogenesis. fish adipocytes; insulin and IGF-I receptors; MAPK and AKT; glucose uptake; GLUT4 doi: 10.1152/ajpregu.00457.2009.

Although the role of the islets in the regulation of acinar cell function seemed a mystery to investigators who observed their dispersion among pancreatic acini, over time an appreciation for this intricate and unique structural arrangement has developed. The last three decades have witnessed a steadily growing understanding of the interrelationship of the endocrine and the exocrine pancreas. The islet innervation and vascular anatomy have been more fully characterized and provide an appropriate background for our current understanding. The interrelationship between the endocrine and exocrine pancreas is mediated by islet-derived hormones such as insulin and somatostatin, other humoral factors including pancreastatin and ghrelin, and also neurotransmitters (nitric oxide, peptide YY, substance P, and galanin) released by the nerves innervating the pancreas. Although considerable progress has been achieved, further work is required to fully delineate the complex interplay of the numerous mechanisms involved. This review aims to provide a comprehensive update of the current literature available, bringing together data gleaned from studies addressing the actions of individual hormones, humoral factors, and neurotransmitters on the regulation of amylase secretion from the acinar cell. This comprehensive view of the islet-acinar axis of the pancreas while acknowledging the dominant role played by insulin and somatostatin on exocrine secretion sheds light on the influence of the various neuropeptides on amylase secretion. neuropeptides; amylase; galanin; somatostatin doi: 10.1152/ajpgi.00077.2010.

Physical training affects insulin secretion and action, but there is a paucity of data on the direct effects in skeletal muscle and adipose tissue and on the effect of training in first-degree relatives (FDR) of patients with type 2 diabetes. We studied insulin action at the whole body level and peripherally in skeletal muscle and adipose tissue as well as insulin-secretory capacity in seven FDR and eight control (CON) subjects before and after 12 wk of endurance training. Training improved physical fitness. Insulin- mediated glucose uptake (GU) increased (whole body and leg; P < 0.05) after training in CON but not in FDR, whereas glucose-mediated GU increased (P < 0.05) in both groups. Adipose tissue GU was not affected by training, but it was higher (abdominal, P < 0.05; femoral, P = 0.09) in FDR compared with CON. Training increased skeletal muscle lipolysis (P < 0.05), and it was markedly higher (P < 0.05) in subcutaneous abdominal than in femoral adipose tissue and quadriceps muscle with no difference between FDR and CON. Glucosestimulated insulin secretion was lower in FDR compared with CON, but no effect of training was seen. Glucagon-like peptide-1 stimulated insulin secretion five- to sevenfold. We conclude that insulin-secretory capacity is lower in FDR than in CON and that there is dissociation between training-induced changes in insulin secretion and insulin-mediated GU. Maximal GU rates are similar between groups and increases with physical training. glucagon-like peptide-1; hyperglycemic clamp; blood flow; intravenous glucose tolerance test; hyperinsulinemic euglycemic clamp; microdialysis; arteriovenous balance doi: 10.1152/ajpendo.00765.2009.

Recombinant adenovirus (Ad) vectors can initiate an inflammatory response, limiting its use in gene therapy and basic research. Despite increased efforts to better understand Ad infection, little is known about how it affects cellular metabolic responses. In the current studies, we explored the effects of Ad vectors on insulin signaling molecules and glucose homeostasis. Nonreplicative Ad vectors were injected into rats through the tail vein, and at 4-13 days postinjection insulin signaling and glucose tolerance were examined. Ad vector infection significantly reduced total levels of the insulin receptor (IR), and insulin receptor substrates 1 and 2 (IRS-1, IRS-2) in the liver of rats, resulting in decreased insulin-induced tyrosine phosphorylation of IR, IRS-1, and IRS-2, and decreased interaction of IRS-1 and IRS-2 with phosphoinositide 3-kinase (PI3K). In addition, Ad infection resulted in impaired systemic glucose homeostasis, which recovered by 13 days, after the protein levels of IR, IRS-1, and IRS-2 had started to normalize. Expression of a TNF inhibitor or Kupffer cell depletion attenuated the Ad vector-induced decreases of insulin signaling molecules, indicating a potential role of Kupffer cell activation in this process. These studies provide evidence that systemic administration of Ad vectors can impair insulin signaling in liver, resulting in altered systemic glucose metabolism. Thus, effects of Ad vector infection on insulin action and glucose metabolism need to be considered when Ad vectors are used in research or gene therapy and may be more broadly applicable to other viral agents. liver; insulin receptor; insulin receptor substrates; tumor necrosis factor-[alpha]; Kupffer cells doi: 10.1152/ajpendo.00723.2009.

The C-terminal segment of the human insulin receptor a-chain (designated [alpha]CT) is critical to insulin binding as has been previously demonstrated by alanine scanning mutagenesis and photo-cross-linking. To date no information regarding the structure of this segment within the receptor has been available. We employ here the technique of thermal-factor sharpening to enhance the interpretability of the electron-density maps associated with the earlier crystal structure of the human insulin receptor ectodomain. The [alpha]CT segment is now resolved as being engaged with the central [beta]-sheet of the first leucine-rich repeat (L1) domain of the receptor. The segment is a-helical in conformation and extends 11 residues N-terminal of the classical [alpha]CT segment boundary originally defined by peptide mapping. This tandem structural element ([alpha]CT-L1) thus defines the intact primary insulin-binding surface of the apo-receptor. The structure, together with isothermal titration calorimetry data of mutant [alpha]CT peptides binding to an insulin minireceptor, leads to the conclusion that putative 'insulin-mimetic' peptides in the literature act at least in part as mimics of the [alpha]CT segment as well as of insulin. Photo-cross-linking by novel bifunctional insulin derivatives demonstrates that the interaction of insulin with the [alpha]CT segment and the L1 domain occurs in trans, i.e., these components of the primary binding site are contributed by alternate [alpha]-chains within the insulin receptor homodimer. The tandem structural element defines a new target for the design of insulin agonists for the treatment of diabetes mellitus. diabetes | IGF-1 receptor | insulin binding | photo-cross-linking | protein crystallography doi/ 10.1073/pnas.1001813107

In this study, we examined chronic norepinephrine suppression of insulin secretion in sheep fetuses with placental insufficiency-induced intrauterine growth restriction (IUGR). Glucose-stimulated insulin secretion (GSIS) was measured with a square-wave hyperglycemic clamp in the presence or absence of adrenergic receptor antagonists phentolamine ([alpha]) and propranolol ([beta]). IUGR fetuses were hypoglycemic and hypoxemic and had lower GSIS responsiveness (P [less than or equal to] 0.05) than control fetuses. IUGR fetuses also had elevated plasma norepinephrine (3,264 [+ or -] 614 vs. 570 [+ or -] 86 pg/ml; P [less than or equal to] 0.05) and epinephrine (164 [+ or -] 32 vs. 60 [+ or -] 12 pg/ml; P [less than or equal to] 0.05) concentrations. In control fetuses, adrenergic inhibition increased baseline plasma insulin concentrations (1.7-fold, P [less than or equal to] 0.05), whereas during hyperglycemia insulin was not different. A greater (P [less than or equal to] 0.05) response to adrenergic inhibition was found in IUGR fetuses, and the average plasma insulin concentrations increased 4.9-fold at baseline and 7.1-fold with hyperglycemia. Unlike controls, basal plasma glucose concentrations fell (P [less than or equal to] 0.05) with adrenergic antagonists. GSIS responsiveness, measured by the change in insulin, was higher (8.9-fold, P [less than or equal to] 0.05) in IUGR fetuses with adrenergic inhibition than controls (1.8-fold, not significant), showing that norepinephrine suppresses insulin secretion in IUGR fetuses. Strikingly, in IUGR fetuses, adrenergic inhibition resulted in a greater GSIS responsiveness, because [beta]-cell mass was 56% lower and the maximal stimulatory insulin response tended (P < 0.1) to be higher than controls. This persistent norepinephrine suppression appears to be partially explained by higher mRNA concentrations of adrenergic receptors [[alpha].sub.1D], [[alpha].sub.2A], and [[alpha].sub.2B] in a cohort of fetuses that were naive to the antagonists. Therefore, norepinephrine suppression of insulin secretion was maintained, in part, by upregulating adrenergic receptor expression, but the [beta]-cells also appeared to compensate with enhanced GSIS. These findings may begin to explain why IUGR infants have a propensity for increased glucose requirements if norepinephrine is suddenly decreased after birth. pancreas; islets of Langerhans; hypoxia; glucose-stimulated insulin secretion; fetal growth restriction doi: 10.1152/ajpendo.00494.2009.

Both insulin and leptin signaling converge on phosphatidylinositol 3-OH kinase [PI(3)K]/3-phosphoinositide-dependent protein kinase-1 (PDK-1)/ protein kinase B (PKB, also known as Akt) in proopiomelanocortin (POMC) neurons. Forkhead box-containing protein-O1 (FoxO1) is inactivated in a PI(3)K-dependent manner. However, the interrelationship between PI(3)K/PDK-1/Akt and FoxO1, and the chronic effects of the overexpression of FoxO1 in POMC neurons on energy homeostasis has not been elucidated. To determine the extent to which PDK-1 and FoxO1 signaling in POMC neurons was responsible for energy homeostasis, we generated POMC neuron-specific Pdk1 knockout mice ([POMCPdkl.sup.-/-]) and mice selectively expressing a constitutively nuclear (CN)FoxO 1 or transactivation-defective (A256)FoxO 1 in POMC neurons ([CNFoxO1.sup.POMC] or [[DELTA]256FoxO1.sup.POMC]). [POMCPdkl.sup. -/-] mice showed increased food intake and body weight accompanied by decreased expression of Pomc gene. The [CNFoxO1.sup.POMC] mice exhibited mild obesity and hyperphagia compared with [POMCPdkl.sup.-/-] mice. Although expression of the CNFoxO1 made [POMCPdkl.sup.-/-] mice more obese due to excessive suppression of Pomc gene, overexpression of A256FoxO1 in POMC neurons had no effects on metabolic phenotypes and Pomc expression levels of [POMCPdkl.sup.-/-] mice. These data suggest a requirement for PDK-1 and FoxO1 in transcriptional regulation of Pomc and food intake. phosphoinositide-dependent protein kinase-1; forkhead box-containing protein O1; proopiomelanocortin neurons; Pomc; food intake doi: 10.1152/ajpendo.00512.2009.

Background. Reduced insulin and insulin-like growth factor-1 (IGF-1) signaling extends the life span of invertebrate and mammals. Recently, reduced insulin receptor substrate-2 (IRS2) signaling was found associated with increased longevity in mice. The aim of our study was to evaluate whether a common polymorphism (Gly1057Asp) in human IRS2 gene is associated with human longevity. Methods. Six hundred seventy-seven participants (289 males and 388 females) between 16 and 104 years of age, categorized as long lived (LL; >85 years old) or controls (C; Results. In the whole population, homozygous [IRS2.sup.Asp/Asp] participants were more represented among LL versus C participants (16.7% vs 12.0%; p = .04). The association between IRS2 gene polymorphism with longevity (being LL) was independent of anthropometric and metabolic covariates (odds ratio: 2.07, 95% confidence interval [CI] = 1.38-3.12; p = .001). Categorizing participants into percentiles by age, [IRS2.sp.Asp/Asp] participants were more likely to reach extreme old age ([greater than or equal to] 90 percentile, 96-104 years; odds ratio: 2.03, 95% CI = 1.39-2.99; p = .0003). Conclusions. These results support the hypothesis that the IRS2 branch of the insulin and IGF signaling is associated with human longevity. Further studies will be necessary for replicating our finding in an independent larger population group with sufficient power before the association between IRS2 gene polymorphism and longevity can be regarded as proven. Furthermore, studies of genetic and/or environmental background interactions may be useful after basic replication is complete. Key Words: IRS2 gene polymorphism--Longevity--Insulin signaling. doi: 10.1093/gerona/glp154

Heterozygous coding mutations in the INS gene that encodes preproinsulin were recently shown to be an important cause of permanent neonatal diabetes. These dominantly acting mutations prevent normal folding of proinsulin, which leads to beta-cell death through endoplasmic reticulum stress and apoptosis. We now report 10 different recessive INS mutations in 15 probands with neonatal diabetes. Functional studies showed that recessive mutations resulted in diabetes because of decreased insulin biosynthesis through distinct mechanisms, including gene deletion, lack of the translation initiation signal, and altered mRNA stability because of the disruption of a polyadenylation signal. A subset of recessive mutations caused abnormal INS transcription, including the deletion of the C1 and E1 cis regulatory elements, or three different single base-pair substitutions in a CC dinucleotide sequence located between E1 and A1 elements. In keeping with an earlier and more severe beta-cell defect, patients with recessive INS mutations had a lower birth weight (-3.2 SD score vs. -2.0 SD score) and were diagnosed earlier (median 1 week vs. 10 weeks) compared to those with dominant INS mutations. Mutations in the insulin gene can therefore result in neonatal diabetes as a result of two contrasting pathogenic mechanisms. Moreover, the recessively inherited mutations provide a genetic demonstration of the essential role of multiple sequence elements that regulate the biosynthesis of insulin in man. gene regulation | genetic testing | gene expression regulation | RNA instability | promoter regions www.pnas.org/cgi/doi/10.1073/pnas.0910533107

In the rare medical condition termed injection amyloidosis, extracellular fibrils of insulin are observed. We found that the segment of the insulin B-chain with sequence LVEALYL is the smallest segment that both nucleates and inhibits the fibrillation of fulllength insulin in a molar ratio-dependent manner, suggesting that this segment is central to the cross-/3 spine of the insulin fibril. In isolation from the rest of the protein, LVEALYL forms microcrystalline aggregates with fibrillar morphology, the structure of which we determined to 1 A resolution. The LVEALYL segments are stacked into pairs of tightly interdigitated /3-sheets, each pair displaying the dry steric zipper interface typical of amyloid-like fibrils. This structure leads to a model for fibrils of human insulin consistent with electron microscopic, x-ray fiber diffraction, and biochemical studies. amyloid | fibril structure www.pnas.org/cgi/doi/10.1073/pnas.0910080106

Intracerebroventricular insulin decreases food intake (FI). The central bed nucleus of the amygdala (CeA), as other regions of the brain regulating feeding behavior, expresses insulin receptors. Our objectives were to show an insulin anorectic response in the amygdala, study the effect of high-fat diets on this response, and map the neural network activated by CeA insulin using c-Fos immunohistochemistry. Sprague-Dawley (SD) rats fitted with unilateral CeA cannulas were adapted to a low-fat (LFD) diet before they were fed a high-fat diet (HFD). Their feeding response to CeA saline or insulin (8 mU) was tested after 24 h, 72 h, or 7 days of being on a HFD. In a second experiment, SD rats were fed the HFD for 3, 7, or 49 days and were then refed with the LFD. They were tested for their insulin response before and after an HFD and every 3 days for the following weeks. Insulin tolerance tests were performed in a parallel group of rats. The CeA insulin stimulation c-Fos expression was studied to identify the distribution of activated neuronal populations. Feeding an HFD for 72 h or more induced a CeA, but not peripheral, insulin resistance, which was slowly reversed by LFD refeeding. The duration of HFD feeding determined the time frame for reversal of the insulin resistance. CeA insulin increased c-Fos in multiple brain regions, including the arcuate nucleus/paraventricular nucleus region of the hypothalamus. We conclude that the amygdala may be an important site for insulin regulation of food intake and may have a significant role in determining susceptibility to HFD-induced obesity. rat; food intake; central insulin sensitivity; limbic system doi: 10.1152/ajpregu.00252.2009.

Knowledge of how insulin (INS) gene expression is regulated will lead to better understanding of normal and abnormal pancreatic [beta] cell function. We have mapped histone modifications over the INS region, coupled with an expression profile, in freshly isolated islets from multiple human donors. Unlike many other human genes, in which active modifications tend to be concentrated within 1 kb around the transcription start site, these marks are distributed over the entire coding region of INS as well. Moreover, a region of [approximately equal to] 80 kb around the INS gene, which contains the {tyrosine hydroxylase (TH)--(INS)--insulin-like growth factor 2 antisense (IGF2AS)--insulin-like growth factor 2 (IGF2)} gene cluster, unusually is marked by almost uniformly elevated levels of histone acetylation and H3K4 dimethylation, extending both downstream into IGF2 and upstream beyond the TH gene. This is accompanied by islet specific coordinate expression with INS of the neighboring TH and IGF2 genes. The presence of islet specific intergenic transcripts suggests their possible function in the maintenance of this unusual large open chromatin domain. histone modifications | intergenic transcription | pancreatic islets doi/ 10.1073/pnas.0909288106

Background: The contemporary American diet figures centrally in the pathogenesis of numerous chronic diseases--&apos;diseases of civilization&apos;. We investigated in humans whether a diet similar to that consumed by our preagricultural hunter-gatherer ancestors (that is, a paleolithic type diet) confers health benefits. Methods: We performed an outpatient, metabolically controlled study, in nine nonobese sedentary healthy volunteers, ensuring no weight loss by daily weight. We compared the findings when the participants consumed their usual diet with those when they consumed a paleolithic type diet. The participants consumed their usual diet for 3 days, three ramp-up diets of increasing potassium and fiber for 7 days, then a paleolithic type diet comprising lean meat, fruits, vegetables and nuts, and excluding nonpaleolithic type foods, such as cereal grains, dairy or legumes, for 10 days. Outcomes included arterial blood pressure (BP); 24-h urine sodium and potassium excretion; plasma glucose and insulin areas under the curve (AUC) during a 2 h oral glucose tolerance test (OGTT); insulin sensitivity; plasma lipid concentrations; and brachial artery reactivity in response to ischemia. Results: Compared with the baseline (usual) diet, we observed (a) significant reductions in BP associated with improved arterial distensibility (-3.1 [+ or -] 2.9, P = 0.01 and + 0.19 [+ or -] 0.23, P = 0.05);(b) significant reduction in plasma insulin vs time AUC, during the OGTT (P=0.006); and (c) large significant reductions in total cholesterol, low-density lipoproteins (LDL) and triglycerides (-0.8 [+ or -] 0.6 (P = 0.007), -0.7 [+ or -] 0.5 (P = 0.003) and -0.3 [+ or -] 0.3 (P = 0.01) mmol/l respectively). In all these measured variables, either eight or all nine participants had identical directional responses when switched to paleolithic type diet, that is, near consistently improved status of circulatory, carbohydrate and lipid metabolism/physiology. Conclusions: Even short-term consumption of a paleolithic type diet improves BP and glucose tolerance, decreases insulin secretion, increases insulin sensitivity and improves lipid profiles without weight loss in healthy sedentary humans. doi:10.1038/ejcn.2009.4; published online 11 February 2009 Keywords: paleolithic diet; blood pressure; glucose tolerance; insulin sensitivity; lipids, Introduction In 1985, anthropologists Eaton and Konner (1985) introduced the general medical community in 'paleolithic nutrition. A consideration of its nature and current implications'. 'Paleolithic' refers to the period of [...]

Migration of airway epithelial cells (AEC) is an integral component of airway mucosal repair after injury. The inflammatory cytokine IL-4, abundant in chronic inflammatory airways diseases such as asthma, stimulates overproduction of mucins and secretion of chemokines from AEC; these actions enhance persistent airway inflammation. The effect of IL-4 on AEC migration and repair after injury, however, is not known. We examined migration in primary human AEC differentiated in air-liquid interface culture for 3 wk. Wounds were created by mechanical abrasion and followed to closure using digital microscopy. Concurrent treatment with IL-4 up to 10 ng/ml accelerated migration significantly in fully differentiated AEC. As expected, IL-4 treatment induced phosphorylation of the IL-4 receptor-associated protein STAT (signal transducer and activator of transcription)6, a transcription factor known to mediate several IL-4-induced AEC responses. Expressing a dominant negative STAT6 cDNA delivered by lentivirus infection, however, failed to block IL-4-stimulated migration. In contrast, decreasing expression of either insulin receptor substrate (IRS)-I or IRS-2 using a silencing hairpin RNA blocked IL-4-stimulated AEC migration completely. These data demonstrate that IL-4 can accelerate migration of differentiated AEC after injury. This reparative response does not require STAT6 activation, but rather requires IRS-1 and/or IRS-2. epithelium; interleukin-4; STAT6; IRS-1; IRS-2

The relative contributions of net hepatic glycogenolysis (NHG) and gluconeo-genesis to rates of glucose production during a physiological increment in plasma epinephrine concentrations, independent of changes in plasma insulin concentrations, were determined in seven fasting, healthy young subjects. Plasma insulin concentrations were kept constant by infusing somatostatin (0.1 [micro]g x [kg.sup.-1] x [min.sup.-1]) and replacing basal insulin (24 pmol x [m.sup.-2] x [min.sup.-1]). Epinephrine (1.2 [micro]g x [m.sup.-2] x [min.sup.-1]) was infused for 90 min while NHG was assessed directly by [sup.13]C magnetic resonance spectroscopy. The rate of glucose production was assessed using [6,6-[sup.2][H.sub.2]]glucose, and gluconeogenesis was calculated as the difference between the rate of glucose production and NHG. Plasma epinephrine concentrations increased rapidly from ~100 to ~2,000 pmol/l (P < 0.00001) accompanied by an increase in plasma glucose concentrations from 4.3 [+ or -] 0.2 to 13.3 [+ or -] 0.3 mmol/l at 90 min (P = 0.00001). This increase in plasma epinephrine concentration resulted in a 2.5-fold increase in glucose production (from 14.4 [+ or -] 1.0 [micro]mol x kg-1 x min-1 to 35.7 [+ or -] 2.0 [micro]mol x kg-1 x min-1, P < 0.0001), which lasted for ~60 min (phase 1), after which glucose production decreased to 31.2 [+ or -] 1.9 [micro]mol x kg-1 x min-1 (P < 0.0001 vs. basal) during the last 30 min of the epinephrine infusion (phase 2). Hepatic glycogen concentrations decreased almost linearly during phase 1, and rates of NHG were 19.9 [+ or -] 3.0 [micro]mol x kg-1 x min-1 (P = 0.005 vs. basal), which could account for ~60% of glucose production. During phase 2, NHG decreased to 7.3 [+ or -] 2.8 [micro]mol x kg-1 x min-1 (P = 0.02 vs. peak), accounting for only ~20% of glucose production. In conclusion, in the presence of basal plasma insulin and glucagon concentrations, a physiological increase in plasma epinephrine concentrations stimulates glucose production with an initial, 60-min transient phase caused by stimulation of NHG and a second phase that can mostly be attributed to a twofold increase in rates of gluconeogenesis. net hepatic glycogenolysis; gluconeogenesis; epinephrine; humans

Recent evidence suggests that leptin reduces food intake via actions in the brain circuitry of food reward, such as the ventral tegmental area (VTA), as leptin receptors are present in the VTA, and leptin injection in the VTA reduces food intake. In the hypothalamus, leptin-induced anorexia requires signaling via Janus kinase-signal transducer and activator of transcription (Jak-STAT), insulin receptor substrate (IRS)-phosphatidylinositol 3-kinase (PI 3-kinase), and mammalian target of rapamycin (mTOR). In this study, we determined whether leptin activates each of these signal transduction pathways in the VTA and whether these signaling pathways are required for VTA-leptin induced anorexia. Here, we show that pSTAT3-[Tyr.sup.705], a marker of leptin activation, was induced in a midbrain region containing the VTA and substantia nigra following either intracerebroventricular leptin or direct administration of leptin to the VTA, but these interventions failed to increase levels of either pAKT-[Ser.sup.473] or phospho-p70S6K-[Thr.sup.389], markers of IRS-PI 3-kinase and mTOR signaling, respectively. Moreover, the effect of intra-VTA leptin administration to reduce 4- and 20-h food intake and 20-h body weight was blocked by an inhibitor of Jak-2, at a dose that had no effect on food intake or body weight by itself, but not by local inhibition of either PI 3-kinase (LY-294002) or mTOR (rapamycin) in this timeframe. Taken together, these data support the hypothesis that leptin signaling in the VTA is involved in the regulation of energy balance, but, in contrast to the leptin signaling in the hypothalamus, these effects are mediated predominantly via Jak-2 signaling rather than via the IRS-PI 3-kinase or mTOR signaling pathway. leptin; food intake; ventral tegmental area; signal transducer and activator of transcription signaling

Ku HC, Chang HH, Liu HC, Hsiao CH, Lee M J, Hu Y J, Hung PF, Liu CW, Kao YH. Green tea (-)-epigallocatechin gallate inhibits insulin stimulation of 3T3-LI preadipocyte mitogenesis via the 67-kDa laminin receptor pathway. Am J Physiol Cell Physiol 297: C121-C132, 2009. First published January 28, 2009; doi:10.1152/ajpcell.00272.2008.--Insulin and (-)-epigallocatechin gallate (EGCG) have been reported to regulate fat cell mitogenesis and adipogenesis, respectively. This study investigated the pathways involved in EGCG modulation of insulin-stimulated mitogenesis in 3T3-LI preadipocytes. EGCG inhibited insulin stimulation of preadipocyte proliferation in a dose- and time-dependent manner. EGCG also suppressed insulin-stimulated phosphorylation of the insulin receptor-J3, insulin receptor (IR) substrates 1 and 2 (IRS1 and IRS2), and mitogen-activated protein kinase pathway proteins, RAFI, MEKI/2, and ERKI/2, but not JNK. Furthermore, EGCG inhibited the association of IR with the IRS1 and IRS2 proteins, but not with the IRS4 protein. These data suggest that EGCG selectively affects particular types of IRS and MAPK family members. Generally, EGCG was more effective than epicatechin, epicatechin gallate, and epigallocatechin in modulating insulin-stimulated mitogenic signaling. We identified the EGCG receptor [also known as the 67-kDa laminin receptor (67LR)] in fat cells and found that its expression was sensitive to growth phase, tissue type, and differentiation state. Pretreatment of preadipocytes with 67LR antiserum prevented the effects of EGCG on insulin-stimulated phosphorylation of IRS2, RAFI, and ERK1/2 and insulin-stimulated preadipocyte proliferation (cell number and bromodeoxyuridine incorporation). Moreover, EGCG tended to increase insulin-stimulated associations between the 67LR and IR, IRS1, IRS2, and IRS4 proteins. These data suggest that EGCG mediates anti-insulin signaling in preadipocyte mitogenesis via the 67LR pathway. insulin receptor: insulin receptor substrate; mitogen-activated protein kinase

Intracellular signaling by which pancreatic [beta]-cells synthesize and secrete insulin in control of glucose homeostasis is not fully understood. Here we show that Shp2, a cytoplasmic tyrosine phosphatase possessing 2 SH2 domains, coordinates signaling events required for insulin biosynthesis in [beta]-cells. Mice with conditional ablation of the Shp2/Ptpn11 gene in the pancreas exhibited defective glucose-stimulated insulin secretion and impaired glucose tolerance. Consistently, siRNA-mediated Shp2-knockdown in rat insulinoma INS-1 832/13 cells resulted in decreased insulin production and secretion despite ah increase in cellular ATP. Shp2 modulates the strength of signals flowing through Akt/FoxO1 and Erk pathways, culminating in control of Pdx1 expression and activity on Ins1 and Ins2 promoters, and forced Pdx1 expression rescued insulin production in Shp2-knockdown [beta]-cells. Therefore, Shp2 acts asa signal coordinator in [beta]-cells, orchestrating multiple pathways controlling insulin biosynthesis to maintain glucose homeostasis. Pdx1 | signal transduction | diabetes | insulin secretion | gene expression

The interaction of the antitumor drug cisplatin with insulin was studied using a top-down mass spectrometric approach. In vitro incubations were prepared under acidic and physiological conditions at different insulin/cisplatin molar ratios for different incubation times. Size exclusion chromatography--inductively coupled plasma mass spectrometry (SEC--ICPMS) analysis enabled the specific detection of platinum containing species attributed to the binding of the drug to the protein. Further analysis through matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and nanoelectrospray ionization mass spectrometry using a linear ion trap (nESI-LIT-MS) allowed the identification of platinated mono-, di-, and even triadducts in the incubations. Platinum binding sites were identified by [CID-MS.sup.n] as B chain N-terminus, His5, and probably His10 residues, which turned out to be the same, regardless of the incubation conditions. Evidence on the binding of Pt to B chain Cys7 was also observed. Working with the LIT zoom scan mode provides enough resolution to discern the isotopic pattern for both precursor and fragment ions, allowing the differentiation of platinum-containing ions. The elucidation of platinum binding sites in a native protein through a top-down approach has been performed for the first time with this type of instrument.

The 3 Akt protein kinase isoforms have critical and distinct functions in the regulation of metabolism, cell growth, and apoptosis, yet the mechanisms by which their signaling specificity is achieved remain largely unclear. Here, we investigated potential mechanisms underlying Akt isoform functional specificity by using Akt2-specific regulation of glucose transport in insulin-stimulated adipocytes asa model system. We found that insulin activates both Akt1 and Akt2 in adipocytes, but differentially regulates the subcellular distribution of these Akt isoforms. The greater accumulation of Akt2 at the plasma membrane (PM) of insulin-stimulated adipocytes correlates with Akt2-specific regulation of the trafficking of the GLUT4 glucose transporter. Consistent with this pattern, Akt constructs that do not accumulate at the PM to the same degree as Akt2 fail to regulate GLUT4 translocation to the PM, whereas enhancement of Akt1 PM association through mutation in Akt1 PH domain is sufficient to overcome Akt-isoform specificity in GLUT4 regulation. Indeed, we found that this distinct insulin-induced PM accumulation of Akt kinases is translated into a differential regulation by the Akt isoforms of AS160, a RabGAP that regulates GLUT4 trafficking. Our data show that Akt2 specifically regulates AS160 phosphorylation and membrane association providing molecular basis for Akt2 specificity in the modulation of GLUT4 trafficking. Together, our findings reveal the stimulus-induced subcellular compartmentalization of Akt kinases as a mechanism contributing to specify Akt isoform functions. GLUT4 | AS160 | Akt2 | TBClD4 | signaling compartmentalization

Quantification of insulin release from pancreatic islets of Langerhans is of interest for diabetes research. Typical insulin secretion experiments are performed using offline techniques that are expensive, slow, have low-throughput, and require multiple islets. We have developed a microfluidic device for high-throughput, automated, and online monitoring of insulin secretion from individual islets in parallel. This chip consists of 15 channel networks each capable of superfusing a single islet and mixing superfusate from each islet online with fluorescein isothiocyanate-labeled insulin and anti-insulin antibody for a competitive immunoassay. The resulting continuous reaction streams are periodically injected onto parallel electrophoresis channels where the mixtures are separated. The resulting traces are used to quantify relative insulin released from islets. Serial immunoassays were performed at 10 s intervals on all 15 channels, corresponding to 5400 immunoassays per hour, to create temporally resolved insulin release profiles that captured single islet secretion dynamics. The chip was used to demonstrate that free fatty acid induced lipotoxicity in islets eliminates pulsatile insulin secretion.

Chronically high insulin levels and increased circulating free fatty acids released from adipose tissue through lipolysis are two features associated with insulin resistance. The relationship between chronic insulin exposure and adipocyte lipolysis has been unclear. In the present study we found that chronic insulin exposure in 3T3-L1 adipocytes, as well as in mouse primary adipocytes, increased basal lipolysis rates. This effect of insulin on lipolysis was only observed when the mammalian target of rapamycin (mTOR) pathway was inhibited by rapamycin in the adipocytes. In addition, amino acid deprivation in adipocytes phenocopied the effect of rapamycin in permitting the stimulation of lipolysis by chronic insulin exposure. The phosphatidylinositol 3-kinase-Akt pathway does not appear to be involved in this insulin effect. Furthermore, we found that triacylglycerol hydrolase (TGH) activity was required for the stimulation of lipolysis by combined exposure to insulin and rapamycin. Therefore, we propose that nutrient sufficiency, mediated by an mTOR pathway, suppresses TGH-dependent lipolysis stimulated by chronic insulin exposure in adipocytes. mammalian target of rapamycin; insulin resistance; 3T3-L1 cells; nutrient sufficiency; triacylglycerol hydrolase

This study evaluated whether improved insulin-stimulated glucose uptake in recovery from acute exercise coincides with reduced malonyl-CoA (MCoA) content in human muscle. Furthermore, we investigated whether a high-fat diet [65 energy-% (Fat)] would alter the content of MCoA and insulin action compared with a high-carbohydrate diet [65 energy-% (CHO)]. After 4 days of isocaloric diet on two occasions (Fat/CHO), 12 male subjects performed 1 h of one-legged knee extensor exercise (~80% peak workload). Four hours after exercise, insulin-stimulated glucose uptake was determined in both legs during a euglycemic-hyperinsulinemic clamp. Muscle biopsies were obtained in both legs before and after the clamp. Four hours after exercise, insulin-stimulated glucose uptake was improved (~70%, P < 0.001) independent of diet composition and despite normal insulin-stimulated regulation of insulin receptor substrate-l-associated phosphatidylinositol 3-kinase, Akt, GSK-3, and glycogen synthase. Interestingly, exercise resulted in a sustained reduction (~20%, P < 0.05) in MCoA content 4 h after exercise that correlated (r = 0.65, P < 0.001) with improved insulin-stimulated glucose uptake. Four days of Fat diet resulted in an increased content of intramyocellular triacylglycerol (P < 0.01) but did not influence muscle MCoA content or whole body insulin-stimulated glucose uptake. However, at the muscular level proximal insulin signaling and insulin-stimulated glucose uptake appeared to be compromised, although to a minor extent, by the Fat diet. Collectively, this study indicates that reduced muscle MCoA content in recovery from exercise may be part of the adaptive response leading to improved insulin action on glucose uptake after exercise in human muscle. AMP-activated protein kinase; acetyl coenzyme A carboxylase; intramuscular triacylglycerol; Akt; diet

Insulin signaling at target tissues is essential for growth and development and for normal homeostasis of glucose, fat, and protein metabolism. Control over this process is therefore tightly regulated. It can be achieved by a negative feedback control mechanism whereby downstream components inhibit upstream elements along the insulin-signaling pathway (autoregulation) or by signals from apparently unrelated pathways that inhibit insulin signaling thus leading to insulin resistance. Phosphorylation of insulin receptor substrate (IRS) proteins on serine residues has emerged as a key step in these control processes under both physiological and pathological conditions. The list of IRS kinases implicated in the development of insulin resistance is growing rapidly, concomitant with the list of potential Ser/Thr phosphorylation sites in IRS proteins. Here, we review a range of conditions that activate IRS kinases to phosphorylate IRS proteins on 'hot spot' domains. The flexibility vs. specificity features of this reaction is discussed and its characteristic as an 'array' phosphorylation is suggested. Finally, its implications on insulin signaling, insulin resistance and type 2 diabetes, an emerging epidemic of the 21st century are outlined. insulin receptor substrate

Here, we report that Cdk5 activation is stimulated by insulin and plays a key role in the regulation of GLUT4-mediated glucose uptake in 3T3-L1 adipocytes. Insulin activation of Cdk5 requires PI3K signaling. Insulin-activated Cdk5 phosphorylates E-Syt1, a 5 C2-domain protein-related to the synaptotagmins that is induced during adipocyte differentiation. Phosphorylated E-Syt1 associates with GLUT4, an event inhibited by the Cdks inhibitor roscovitine. Cdk5 silencing inhibits glucose uptake by 3T3-L1 adipocytes. These studies elucidate a previously unknown activity of Cdk5 and demonstrate the involvement of this kinase in the regulation of insulin-dependent glucose uptake in adipocytes.

Normal [beta]-cells adjust their function to compensate for any decrease in insulin sensitivity. Our aim was to explore whether a prolonged fast would allow a study of the effects of changes in circulating free fatty acid (FFA) levels on insulin secretion and insulin sensitivity and whether any potential effects could be reversed by the antilipolytic agent acipimox. Fourteen (8 female, 6 male) healthy young adults (aged 22.8-26.9 yr) without a family history of diabetes and a body mass index of 22.6 [+ or -] 3.2 kg/[m.sup.2] were studied on three occasions in random order. Growth hormone and FFA levels were regularly measured overnight (2200-0759), and subjects underwent an intravenous glucose tolerance test in the morning (0800-1100) on each visit. Treatment A was an overnight fast, treatment B was a 24-h fast with regular administrations of a placebo, and treatment C was a 24-h fast with regular ingestions of 250 mg of acipimox. The 24-h fast increased overnight FFA levels (as measured by the area under the curve) 2.8-fold [51.3 (45.6-56.9) vs. 18.4 (14.4-22.5) *[10.sup.4] [micro]mol/l*min, P < 0.0001], and it led to decreases in insulin sensitivity [5.7 (3.6-8.9) vs. 2.6 (1.3-4.7) *[10.sup.-4] [min.sup.-1] per mU/l, P < 0.0001] and the acute insulin response [16.3 (10.9-21.6) vs. 12.7 (8.7-16.6) *[10.sup.2] pmol/ l*min, P = 0.02], and therefore a reduction in the disposition index [93.1 (64.8-121.4) vs. 35.5 (21.6-49.4) *[10.sup.2] pmol/mU, P < 0.0001]. Administration of acipimox during the 24-h fast lowered FFA levels by an average of 20% (range: -62 to +49%; P = 0.03), resulting in a mean increase in the disposition index of 31% (P = 0.03). In conclusion, the 24-h fast was accompanied by substantial increases in fasting FFA levels and induced reductions in the acute glucose-simulated insulin response and insulin sensitivity. The use of acipimox during the prolonged fast increased the disposition index, suggesting a partial reversal of the effects of fasting on the acute insulin response and insulin sensitivity. free fatty acid; growth hormone; first phase insulin secretion; disposition index; lipotoxicity

Background/Objective: Early insulin secretion following a meal is representative for normal physiology and may depend on meal composition. To compare the effects of a fat-rich and a carbohydrate-rich mixed meal on insulinogenic index as a measure of early insulin secretion in normoglycemic women (NGM) and in women with type 2 diabetes mellitus (DM2), and to assess the relationship of anthropometric and metabolic factors with insulinogenic index. Subjects/Methods: Postmenopausal women, 76 with NGM and 64 with DM2, received a fat-rich meal and a carbohydrate-rich meal on separate occasions. Early insulin response was estimated as insulinogenic index ([DELTA][insulin.sub.0-30 min]/ [DELTA][glucose.sub.0-30 min]) for each meal. Associations of fasting and postprandial triglycerides, body mass index, waist and hip circumference and alanine aminotransferase with insulinogenic indices were determined. Results: Women with NGM present with higher insulinogenic index than women with DM2. The insulinogenic index following the fat-rich meal ([DELTA][l.sub.30]/[DELTA][G.sub.30] (fat)) was higher than the index following the carbohydrate-rich meal ([DELTA][l.sub.3]/[DELTA][G.sub.30] (CH)) (P < 0.05 in women with DM2, and not significant in women with NGM). In women with DM2, homeostasis model assessment for insulin resistance was positively associated with [DELTA][l.sub.30]/ [DELTA][G.sub.30] (CH). In women with NGM, waist circumference was independently and inversely associated with [DELTA][l.sub.30]/[DELTA][G.sub.30] (fat) and with [DELTA][l.sub.30]/[DELTA][G.sub.30] (CH); hip circumference was positively associated with [DELTA][l.sub.30]/[DELTA][G.sub.30] (fat). Conclusions: The insulinogenic index following the fat-rich meal was higher than following the isocaloric carbohydrate-rich meal, which might favorably affect postprandial glucose excursions, especially in women with DM2. The association between a larger waist circumference and a lower meal-induced insulinogenic index in women with NGM requires further mechanistic studies. doi:10.1038/sj.ejcn.1602953; published online 7 November 2007 Keywords: carbohydrates; fat; hyperglycemia; insulin secretion; postprandial glucose regulation; type 2 diabetes, Introduction Insulin resistance and a decline in [beta]-cell function contribute to the development of type 2 diabetes mellitus (DM2) (Weyer et al., 1999; Festa et al., 2006). Especially impairment of [...]

When previously sedentary men and women follow exercise training programs with ad libitum feeding, men lose body fat, but women do not. The purpose of this study was to evaluate whether this observation could be related to sex differences in the way energy-regulating hormones and appetite perception respond to exercise. Eighteen (9 men, 9 women) overweight/ obese individuals completed four bouts of exercise with energy added to the baseline diet to maintain energy balance (BAL), and four bouts without energy added to induce energy deficit (DEF). Concentrations of acylated ghrelin, insulin, and leptin, as well as appetite ratings were measured in response to a meal after a no-exercise baseline and both exercise conditions. In men, acylated ghrelin area under the curve (AUC) was not different between conditions. In women, acylated ghrelin AUC was higher after DEF (+32%) and BAL (+25%), and the change from baseline was higher than men (P < 0.05). In men, insulin AUC was reduced (-17%) after DEF (P < 0.05), but not BAL. In women, insulin AUC was lower (P < 0.05) after DEF (-28%) and BAL (-15%). Leptin concentrations were not different across conditions in either sex. In men, but not in women, appetite was inhibited after BAL relative to DEF. The results indicate that, in women, exercise altered energy-regulating hormones in a direction expected to stimulate energy intake, regardless of energy status. In men, the response to exercise was abolished when energy balance was maintained. The data are consistent with the paradigm that mechanisms to maintain body fat are more effective in women. acylated ghrelin; leptin; insulin; physical activity; body fat; food intake

Insulin signals through several intracellular pathways. Here, we tested the hypothesis that insulin modulates [Na.sup.+]/[H.sup.+] exchange (NHE) activity in vascular cells through [H.sub.2][O.sub.2]-mediated inhibition of tyrosine phosphatase Src homology 2 domain containing tyrosine phosphatase 2 (SHP-2). We measured intracellular pH ([pH.sub.i]) in isolated mouse mesenteric arteries using fluorescence confocal and wide-field microscopy. In the absence of C[O.sub.2]/HC[O.sub.3], removal of bath [Na.sup.+] produced endothelial acidification ([DELTA][pH.sub.i] = -0.71 [+ or -] 0.12) inhibited by cariporide. Cariporide reduced endothelial steady-state [pH.sub.i] ([DELTA][pH.sub.i] = -0.28 [+ or -] 0.08). Insulin and H202 acidified endothelial cells 0.2-0.3 pH units and reduced the acidification upon [Na.sup.+] removal by ~65%. Cariporide abolished the effect of insulin and [H.sub.2][O.sub.2]. In vascular smooth muscle cells, [H.sub.2][O.sub.2] produced intracellular acidification ([DELTA][pH.sub.i] = -0.48 [+ or -] 0.06) as did high concentrations of insulin ([DELTA][pH.sub.i] = -0.03 [+ or -] 0.01). NHE activity after an N[H.sup.+.sub.4] prepulse was ~80% attenuated by [H.sub.2][O.sub.2] and ~40% by high insulin concentrations. [H.sub.2][O.sub.2] had no effect on [Na.sup.+]-HC[O.sup.-.sub.3] cotransport activity. NHE1 (slc9a1) was the only plasma membrane NHE isoform detected in mouse mesenteric arteries by RT-PCR analyses. In both cell types, polyethylene glycol catalase abolished the effect of insulin on [pH.sub.i]. Exposure to insulin increased the intracellular concentration of reactive oxygen species estimated with the fluorophore 5-(6)chloromethyl-2',7'-dichlorodihydrofluorescein. The SHP-2 selective inhibitor NSC-87877 and protein tyrosine phosphatase (PTP) inhibitor IV reduced steady-state [pH.sub.i] up to 0.3 pH units and inhibited NHE activity 60-80%; when applied in combination with insulin or [H.sub.2][O.sub.2], no further effect was obtained. We conclude that NHE contributes to phi regulation in arterial endothelial and smooth muscle cells in situ and is inhibited by insulin and [H.sub.2][O.sub.2]. We propose that insulin signaling involves [H.sub.2][O.sub.2] and inhibition of PTP SHP-2. sodium/hydrogen exchanger 1; protein tyrosine phosphatase; hydrogen peroxide; intracellular pH; mesenteric artery

Previous studies have demonstrated that some endocrine consequences of long-term caloric restriction persist after weight restoration in human subjects. Here we evaluate effects of chronic food restriction in rats that were restricted to 70% of control kcal for 4 wk and subsequently weight restored. Measures were taken from rats at 80% (chronically restricted; CR), 90% (partially weight restored; PR), 100% (fully weight restored; FR), and after 4 wk at 100% body weight of controls (extended weight restored; ER). Plasma insulin and leptin were decreased, and ghrelin was increased in CR compared with controls. Leptin and ghrelin normalized with weight restoration at PR, FR, and ER; however, baseline insulin was not normalized until the ER state. Hypothalamic mRNA expression levels for proopiomelanocortin (POMC), agouti-related protein (AgRP), and neuropeptide Y (NPY) revealed significantly less POMC mRNA expression in CR and PR rats, and significantly less arcuate NPY mRNA in PR and FR. In the dorsomedial hypothalamus, CR, PR, and FR rats had significantly increased NPY expression that was not normalized until the ER state. In response to a test meal, insulin and ghrelin release patterns were altered through the FR stage, and ghrelin remained affected at ER. Collectively, these data demonstrate that mere weight restoration is not sufficient to normalize hypothalamic gene expression levels and q endocrine responses to a meal, and that meal-related ghrelin responses persist despite weight restoration for up to 4 wk. ghrelin; insulin; leptin; hypothalamus

Insulin resistance associated with Type 2 diabetes contributes to impaired vasorelaxation. Previously, we showed the phosphorylation of myosin-bound phosphatase substrate MYPT1, a marker of the vascular smooth muscle cell (VSMC) contraction, was negatively regulated by Akt (protein kinase B) phosphorylation in response to insulin stimulation. In this study we examined the role of Akt phosphorylation on impaired insulin-induced vasodilation in the Goto-Kakizaki (GK) rat model of Type 2 diabetes. GK VSMCs had impaired basal and insulin-induced Akt phosphorylation as well as increases in basal MYPT1 phosphorylation, inducible nitric oxide synthase (iNOS) expression, and nitrite/nitrate production compared with Wistar-Kyoto controls. Both iNOS expression and the inhibition of angiotensin (ANG) II-induced MYPT1 phosphorylation were resistant to the effects of insulin in diabetic GK VSMC. We also measured the isometric tension of intact and denuded GK aorta using a myograph and observed significantly impaired insulin-induced vasodilation. Adenovirus-mediated overexpression of constitutively active Akt in GK VSMC led to significantly improved insulin sensitivity in terms of counteracting ANG II-induced contractile signaling via MYPT1, myosin light chain dephosphorylation, and reduced iNOS expression, S-nitrosylation and survivin expression. We demonstrated for the first time the presence of Akt-independent iNOS expression in the GK diabetic model and that the defective insulin-induced vasodilation observed in the diabetic vasculature can be restored by the overexpression of active Akt, which advocates a novel therapeutic strategy for treating diabetes. diabetes; insulin; inducible nitric oxide synthase; Akt; vasodilation

Stem cell maintenance depends on local signals provided by specialized microenvironments, or niches, in which they reside. The potential role of systemic factors in stem cell maintenance, however, has remained largely unexplored. Here, we show that insulin signaling integrates the effects of diet and age on germline stem cell (GSC) maintenance through the dual regulation of cap cell number (via Notch signaling) and cap cell-GSC interaction (via E-cadherin) and that the normal process of GSC and niche cell loss that occurs with age can be suppressed by increased levels of insulin-like peptides. These results underscore the importance of systemic factors for the regulation of stem cell niches and, thereby, of stem cell numbers. diet | oogenesis | Notch | E-cadherin | aging

The osteoblast-secreted molecule osteocalcin favors insulin secretion, but how this function is regulated in vivo by extracellular signals is for now unknown. In this study, we show that leptin, which instead inhibits insulin secretion, partly uses the sympathetic nervous system to fulfill this function. Remarkably, for our purpose, an osteoblast-specific ablation of sympathetic signaling results in a leptin-dependent hyperinsulinemia. In osteoblasts, sympathetic tone stimulates expression of Esp, a gene inhibiting the activity of osteocalcin, which is an insulin secretagogue. Accordingly, Esp inactivation doubles hyperinsulinemia and delays glucose intolerance in ob/ob mice, whereas Osteocalcin inactivation halves their hyperinsulinemia. By showing that leptin inhibits insulin secretion by decreasing osteocalcin bioactivity, this study illustrates the importance of the relationship existing between fat and skeleton for the regulation of glucose homeostasis.

Peroxisome proliferator-activated receptor-[gamma] coactivator (PGC)-1[alpha] has been shown to play critical roles in regulating mitochondria biogenesis, respiration, and muscle oxidative phenotype. Furthermore, reductions in the expression of PGC-1[alpha] in muscle have been implicated in the pathogenesis of type 2 diabetes. To determine the effect of increased muscle-specific PGC-1[alpha] expression on muscle mitochondrial function and glucose and lipid metabolism in vivo, we examined body composition, energy balance, and liver and muscle insulin sensitivity by hyperinsulinemic-euglycemic clamp studies and muscle energetics by using [sup.31]P magnetic resonance spectroscopy in transgenic mice. Increased expression of PGC-1[alpha] in muscle resulted in a 2.4-fold increase in mitochondrial density, which was associated with an [approximately or equal to] 60% increase in the unidirectional rate of ATP synthesis. Surprisingly, there was no effect of increased muscle PGC-1[alpha] expression on whole-body energy expenditure, and PGC-1[alpha] transgenic mice were more prone to fat-induced insulin resistance because of decreased insulin-stimulated muscle glucose uptake. The reduced insulin-stimulated muscle glucose uptake could most likely be attributed to a relative increase in fatty acid delivery/triglyceride reesterfication, as reflected by increased expression of CD36, acyl-CoA:diacylglycerol acyltransferase1, and mitochondrial acyl-CoA:glycerol-sn-3-phosphate acyltransferase, that may have exceeded mitochondrial fatty acid oxidation, resulting in increased intracellular lipid accumulation and an increase in the membrane to cytosol diacylglycerol content. This, in turn, caused activation of PKC[theta], decreased insulin signaling at the level of insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, and skeletal muscle insulin resistance. diacylglycerol | fatty acid oxidation | insulin resistance | magnetic resonance spectroscopy | mitochondria

Zinc sulfide (ZnS) semiconductor nanoparticles (NPs) capped with a variety of functional groups including bare ZnS NPs, 3-mercaptopropanoic acid (ZnS-3-MPA), sodium citrate (ZnS-citrate), cysteamine (ZnS-Cys), and 2-mercaptoethane sulfonate (ZnS-2-MES) have been investigated as the matrix and affinity probes for analysis of [alpha]-, [beta]-, and [gamma]-cyclodextrins (CDs), ubiquitin, and insulin in biological samples by using surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF-MS). Various parameters that would influence the ionization efficiency and sensitivity of these ZnS NPs in SALDI-TOF-MS were examined including the effect of capping agents, sample pH, ion abundance, and concentration of ZnS NPs. Among these ZnS NPs, our results have demonstrated that ZnS-3-MPA exhibited the highest efficiency toward CDs, ubiquitin, and insulin for high-sensitivity detection in SALDI-TOF-MS. The detection limits were 20-55 nM for CDs, 91 nM for ubiquitin, and 85 nM for insulin. The applicability of the present method is demonstrated by detection of ubiquitin-like proteins in oyster mushroom and also in the analysis of analytes in biological samples such as human urine and plasma. To our best knowledge, this is the first time semiconductor NPs were used as the matrix and affinity probes for high-sensitivity detection of organic and biomolecules in SALDI-TOF-MS. This approach exhibits the advantages of being simple, rapid, efficient, and straightforward for direct analysis of organic and biological samples in SALDI-TOF-MS without the need for time-consuming separation processes, tedious washing steps, or further laborious purification. In addition, it also can provide a sensitive and reliable quantitative assay for small- and large-molecule analysis with the detectable mass up to 8500 Da. We believe that this novel ZnS nanoprobe is simple, efficient, lower cost (compared with Au, Ag, and Pt NPs), fast, and with the potential for high-throughput analysis in SALDI-TOF-MS.

Insulin/insulin-like growth factor (IGF) signaling constitutes an evolutionarily conserved pathway that controls growth, energy homeostasis, and longevity. In Drosophila melanogaster, key components of this pathway are the insulin-like peptides (Dilps). The major source of Dilps is a cluster of large neurons in the brain, the insulin-producing cells (IPCs). The genetic control of IPC development and function is poorly understood. Here, we demonstrate that the Pax6 homolog Eyeless is required in the IPCs to control their differentiation and function. Loss of eyeless results in phenotypes associated with loss of insulin signaling, including decreased animal size and increased carbohydrate levels in larval hemolymph. We show that mutations in eyeless lead to defective differentiation and morphologically abnormal IPCs. We also demonstrate that Eyeless controls IPC function by the direct transcriptional control of one of the major Dilps, dilp5. We propose that Eyeless has an evolutionarily conserved role in IPCs with remarkable similarities to the role of vertebrate Pax6 in [beta] cells of the pancreas. energy homeostasis | evolutionarily conserved | growth control | target gene | transcription factor

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) regulate islet function after carbohydrate ingestion. Whether incretin hormones are of importance for islet function after ingestion of noncarbohydrate macronutrients is not known. This study therefore examined integrated incretin and islet hormone responses to ingestion of pure fat (oleic acid; 0.88 g/kg) or protein (milk and egg protein; 2 g/kg) over 5 h in healthy men, aged 20-25 yr (n = 12); plain water ingestion served as control. Both intact (active) and total GLP-1 and GIP levels were determined as was plasma activity of dipeptidyl peptidase-4 (DPP-4). Following water ingestion, glucose, insulin, glucagon, GLP-1, and GIP levels and DPP-4 activity were stable during the 5-h study period. Both fat and protein ingestion increased insulin, glucagon, GIP, and GLP-1 levels without affecting glucose levels or DPP-4 activity. The GLP-1 responses were similar after protein and fat, whereas the early (30 min) GIP response was higher after protein than after fat ingestion (P < 0.001). This was associated with sevenfold higher insulin and glucagon responses compared with fat ingestion (both P < 0.001). After protein, the early GIP, but not GLP-1, responses correlated to insulin ([r.sup.2] = 0.86; P = 0.0001) but not glucagon responses. In contrast, after fat ingestion, GLP-I and GIP did not correlate to islet hormones. We conclude that, whereas protein and fat release both incretin and islet hormones, the early GIP secretion after protein ingestion may be of primary importance to islet hormone secretion. insulin; glucagon; glucagon-like peptide-1; glucose-dependent insulinotropic polypeptide; incretins; man

Epidemiological and experimental studies have demonstrated that early postnatal nutrition has been associated with long-term effects on glucose homeostasis in adulthood. Recently, our group demonstrated that undernutrition during early lactation affects the expression and activation of key proteins of the insulin signaling cascade in rat skeletal muscle during postnatal development. To elucidate the molecular mechanisms by which undernutrition during early life leads to changes in insulin sensitivity in peripheral tissues, we investigated the insulin signaling in adipose tissue. Adipocytes were isolated from epididymal fat pads of adult male rats that were the offspring of dams fed either a normal or a protein-free diet during the first 10 days of lactation. The cells were incubated with 100 nM insulin before the assays for immunoblotting analysis, 2-deoxyglucose uptake, immunocytochemistry for GLUT4, and/or actin filaments. Following insulin stimulation, adipocytes isolated from undernourished rats presented reduced tyrosine phosphorylation of IR and IRS-1 and increased basal phosphorylation of IRS-2, Akt, and mTOR compared with controls. Basal glucose uptake was increased in adipocytes from the undernourished group, and the treatment with LY294002 induced only a partial inhibition both in basal and in insulin-stimulated glucose uptake, suggesting an involvement of phosphoinositide 3-kinase activity. These alterations were accompanied by higher GLUT4 content in the plasma membrane and alterations in the actin cytoskeleton dynamics. These data suggest that early postnatal undernutrition impairs insulin sensitivity in adulthood by promoting changes in critical steps of insulin signaling in adipose tissue, which may contribute to permanent changes in glucose homeostasis. undernutrition; glucose transporter 4; insulin signaling

Accumulated evidence over the last several years indicates that insulin regulates multiple steps in the overall translocation of GLUT4 vesicles to the fat/muscle cell surface, including formation of an intracellular storage pool of GLUT4 vesicles, its movement to the proximity of the cell surface, and the subsequent docking/fusion with the plasma membrane. Insulin-stimulated formation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)[P.sub.3]; and in some cases, of its catabolite PtdIns(3,4)[P.sub.2]] plays a pivotal role in this process. PtdIns(3,4,5)[P.sub.3] is synthesized by the activated wort-mannin-sensitive class IA phosphoinositide (PI) 3-kinase and controls the rate-limiting cell surface terminal stages of the GLUT4 journey. However, recent research is consistent with the conclusion that signals by each of the remaining five PIs, i.e., PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,5)[P.sub.2], and PtdIns(4,5)[P.sub.2], may act in concert with that of PtdIns(3,4,5)[P.sub.3] in integrating the insulin receptor-issued signals with GLUT4 surface translocation and glucose transport activation. This review summarizes the experimental evidence supporting the complementary function of these PIs in insulin responsiveness of fat and muscle cells, with particular reference to mechanistic insights and functional significance in the regulation of overall GLUT4 vesicle dynamics. glucose transporter 4; glucose transport; fat and muscle cells; phosphatidylinositol monophosphates; phosphatidylinositol bisphosphates

Objective: Moderate alcohol consumption is associated with a decreased risk of type II diabetes. This study investigates the effect of moderate alcohol consumption on adipokines and insulin sensitivity. Subjects: Twenty healthy, lean (body mass index (BMI) 18.5-25 kg/ [m.sup.2] ; n=11) or overweight (BMI>27kg/[m.sup.2]; n=9) men (18-25 years). Methods: Three cans of beer (40g alcohol) or alcohol-free beer daily during 3 weeks. Results: Adiponectin and ghrelin concentrations increased (P Conclusions: Moderate alcohol consumption increased adiponectin and ghrelin, while it decreased ASP concentrations both in lean and overweight men. These changes are in line with the hypothesized improvement of insulin sensitivity, but did not affect insulin sensitivity within 3 weeks of moderate alcohol consumption. doi:10.1038/sj.ejcn.1602821; published online 6 June 2007 Keywords: moderate alcohol consumption; adiponectin; leptin; acylation-stimulating protein; insulin sensitivity, Introduction Moderate alcohol consumption is associated with a decreased risk of type 11 diabetes (Koppes et al., 2005), which could be explained by improved insulin sensitivity after moderate alcohol consumption [...]