Macrophage-derived insulin antagonist ImpL2 induces lipoprotein mobilization upon bacterial infection.

The immune response is an energy-demanding process that must be coordinated with systemic metabolic changes redirecting nutrients from stores to the immune system. Although this interplay is fundamental for the function of the immune system, the underlying mechanisms remain elusive. Our data show that the pro-inflammatory polarization of Drosophila macrophages is coupled to the production of the insulin antagonist ImpL2 through the activity of the transcription factor HIF1α. ImpL2 production, reflecting nutritional demands of activated macrophages, subsequently impairs insulin signaling in the fat body, thereby triggering FOXO-driven mobilization of lipoproteins. This metabolic adaptation is fundamental for the function of the immune system and an individual's resistance to infection. We demonstrated that analogically to Drosophila, mammalian immune-activated macrophages produce ImpL2 homolog IGFBP7 in a HIF1α-dependent manner and that enhanced IGFBP7 production by these cells induces mobilization of lipoproteins from hepatocytes. Hence, the production of ImpL2/IGFBP7 by macrophages represents an evolutionarily conserved mechanism by which macrophages alleviate insulin signaling in the central metabolic organ to secure nutrients necessary for their function upon bacterial infection.

The mammalian insulin antagonist S961 does not exhibit insulin receptor antagonism in rainbow trout in vivo.

Due to their reported 'glucose-intolerant' phenotype, rainbow trout have been the focus of comparative studies probing underlying endocrine mechanisms at the organismal, tissue and molecular level. A particular focus has been placed on the investigation of the comparative role of insulin, an important glucoregulatory hormone, and its interaction with macronutrients. A limiting factor in the comparative investigation of insulin is the current lack of reliable assays to quantify circulating mature and thus bioactive insulin. To circumvent this limitation, tissue-specific responsiveness to postprandial or exogenous insulin has been quantified at the level of post-translational modifications of cell signalling proteins. These studies revealed that the insulin responsiveness of these proteins and their post-translational modifications are evolutionarily highly conserved and thus provide useful and quantifiable proxy indices to investigate insulin function in rainbow trout. While the involvement of specific branches of the intracellular insulin signalling pathway (e.g., mTor) in rainbow trout glucoregulation have been successfully probed through pharmacological approaches, it would be useful to have a functionally validated insulin receptor antagonist to characterize the glucoregulatory role of the insulin receptor pathway in its entirety for this species. Here, we report two separate in vivo experiments to test the ability of the mammalian insulin receptor antagonist, S961, to efficiently block insulin signalling in liver and muscle in response to endogenously released insulin and to exogenously infused bovine insulin. We found that, irrespective of the experimental treatment or dose, activation of the insulin pathway in liver and muscle was not inhibited by S961, showing that its antagonistic effect does not extend to rainbow trout.

Inceptor counteracts insulin signalling in ß-cells to control glycaemia.

Resistance to insulin and insulin-like growth factor 1 (IGF1) in pancreatic ß-cells causes overt diabetes in mice; thus, therapies that sensitize ß-cells to insulin may protect patients with diabetes against ß-cell failure1-3. Here we identify an inhibitor of insulin receptor (INSR) and IGF1 receptor (IGF1R) signalling in mouse ß-cells, which we name the insulin inhibitory receptor (inceptor; encoded by the gene Iir). Inceptor contains an extracellular cysteine-rich domain with similarities to INSR and IGF1R4, and a mannose 6-phosphate receptor domain that is also found in the IGF2 receptor (IGF2R)5. Knockout mice that lack inceptor (Iir-/-) exhibit signs of hyperinsulinaemia and hypoglycaemia, and die within a few hours of birth. Molecular and cellular analyses of embryonic and postnatal pancreases from Iir-/- mice showed an increase in the activation of INSR-IGF1R in Iir-/- pancreatic tissue, resulting in an increase in the proliferation and mass of ß-cells. Similarly, inducible ß-cell-specific Iir-/- knockout in adult mice and in ex vivo islets led to an increase in the activation of INSR-IGF1R and increased proliferation of ß-cells, resulting in improved glucose tolerance in vivo. Mechanistically, inceptor interacts with INSR-IGF1R to facilitate clathrin-mediated endocytosis for receptor desensitization. Blocking this physical interaction using monoclonal antibodies against the extracellular domain of inceptor resulted in the retention of inceptor and INSR at the plasma membrane to sustain the activation of INSR-IGF1R in ß-cells. Together, our findings show that inceptor shields insulin-producing ß-cells from constitutive pathway activation, and identify inceptor as a potential molecular target for INSR-IGF1R sensitization and diabetes therapy.

Phosphorylation status of fetuin-A is critical for inhibition of insulin action and is correlated with obesity and insulin resistance.

Fetuin-A (Fet-A), a hepatokine associated with insulin resistance, obesity, and incident type 2 diabetes, is shown to exist in both phosphorylated and dephosphorylated forms in circulation. However, studies on fetuin-A phosphorylation status in insulin-resistant conditions and its functional significance are limited. We demonstrate that serum phosphofetuin-A (Ser312) levels were significantly elevated in high-fat diet-induced obese mice, insulin-resistant Zucker diabetic fatty rats, and in individuals with obesity who are insulin resistant. Unlike serum total fetuin-A, serum phosphofetuin-A was associated with body weight, insulin, and markers of insulin resistance. To characterize potential mechanisms, fetuin-A was purified from Hep3B human hepatoma cells. Hep3B Fet-A was phosphorylated (Ser312) and inhibited insulin-stimulated glucose uptake and glycogen synthesis in L6GLUT4 myoblasts. Furthermore, single (Ser312Ala) and double (Ser312Ala + Ser120Ala) phosphorylation-defective Fet-A mutants were without effect on glucose uptake and glycogen synthesis in L6GLUT4 myoblasts. Together, our studies demonstrate that phosphorylation status of Fet-A (Ser312) is associated with obesity and insulin resistance and raise the possibility that Fet-A phosphorylation may play a role in regulation of insulin action.

Interference of selenium and selenoproteins with the insulin-regulated carbohydrate and lipid metabolism.

An assumed link between supranutritional intake of the micronutrient selenium (Se) and type 2 diabetes mellitus is discussed controversially. Se concentrations in the habitual diet and in dietary supplements are probably not sufficient to induce overt diabetes in healthy individuals. On the other hand, high plasma Se and selenoprotein P (Sepp1) levels have been found to be associated with biomarkers of an impaired carbohydrate and lipid homeostasis in humans. Moreover, abundant expression of antioxidant selenoproteins due to dietary Se oversupply resulted in hyperinsulinemia and decreased insulin sensitivity in animal models. This review discusses findings from animal and cell culture studies in search of molecular mechanisms underlying an interference of Se and selenproteins such as the Se transport and supply protein Sepp1 and the hydrogen peroxide-reducing selenoenzyme glutathione peroxidase 1 (GPx1) with insulin-controlled metabolic pathways. A probable rationale derives from the positive and negative regulation of both glucose-induced insulin secretion and insulin-induced signaling by hydrogen peroxide. Se status and GPx1 expression have been reported to affect the activity of insulin-antagonistic phosphatases that are regulated by hydrogen peroxide-mediated reversible oxidation of catalytic cysteine residues. GPx1 and/or Sepp1 inhibited phosphorylation (activation) of key mediators in energy metabolism such as protein kinase B (Akt) and AMP-activated protein kinase (AMPK) in liver and/or skeletal muscle. Conversely, a dys-regulated carbohydrate metabolism in diabetes might affect plasma Se and Sepp1 levels, as the hepatic biosynthesis of Sepp1 is suppressed by insulin and stimulated under hyperglycemic conditions.

Islet ß-cell ghrelin signaling for inhibition of insulin secretion.

Ghrelin, an acylated 28-amino acid peptide, was isolated from the stomach, where circulating ghrelin is produced predominantly. In addition to its unique role in regulating growth-hormone release, mealtime hunger, lipid metabolism, and the cardiovascular system, ghrelin is involved in the regulation of glucose metabolism. Ghrelin is expressed in pancreatic islets and released into pancreatic microcirculations. Ghrelin inhibits insulin release in mice, rats, and humans. Pharmacological and genetic blockades of islet-derived ghrelin markedly augment glucose-induced insulin release. The signal transduction mechanisms of ghrelin in islet ß-cells are very unique, being distinct from those utilized for growth-hormone release. Ghrelin attenuates the glucose-induced cAMP production and PKA activation, which drives activation of Kv channels and suppression of the glucose-induced [Ca(2+)](i) increase and insulin release in ß-cells. Insulinostatic function of the ghrelin-GHS-R system in islets is a potential therapeutic target for type 2 diabetes.

Differential insulin response to myo-inositol administration in obese polycystic ovary syndrome patients.

Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism, chronic anovulation, polycystic ovaries at ultrasound evaluation, and quite frequently by insulin resistance or compensatory hyperinsulinemia. Attention has been given to the role of inositol-phosphoglycan (IPG) mediators of insulin action and growing evidences suggest that a deficiency of D-chiro-inositol (DCI) containing IPG might be at the basis of insulin resistance, frequent in PCOS patients. On such basis, we investigated the efficacy on insulin sensitivity and hormonal parameters of 8 weeks treatment with myo-inositol (MYO) (Inofert, ItalPharmaco, Milano, Italy) at the dosage of 2 g day in a group (n = 42) of obese PCOS patients,. After the treatment interval body mass index (BMI) and insulin resistance decreased together with luteinizing hormone (LH), LH/FSH and insulin. When subdividing the patients according to their fasting insulin levels, Group A (n = 15) insulin below 12 µU/ml and Group B (n = 27) insulin above 12 µU/ml, MYO treatment induced similar changes in both groups but only patients of Group B showed the significant decrease of both fasting insulin plasma levels (from 20.3 ± 1.8 to 12.9 ± 1.8 µU/ml, p < 0.00001) and of area under the curve (AUC) of insulin under oral glucose tolerance test (OGTT). In conclusion, our study supports the hypothesis that MYO administration is more effective in obese patients with high fasting insulin plasma levels.

Catecholamines are the key for explaining the biological relevance of insulin-melatonin antagonisms in type 1 and type 2 diabetes.

In this paper, we analyze the biological relevance of melatonin in diabetogenesis. As has recently been demonstrated, melatonin decreases insulin secretion via specific melatonin receptor isoforms (MT1 and MT2) in the pancreatic ß-cells. In addition, type 2 diabetic rats, as well as patients, exhibit decreased melatonin levels, whereas the levels in type 1 diabetic rats are increased. The latter effects were normalized by insulin substitution, which signifies that a specific receptor-mediated insulin-melatonin antagonism exists. These results are in agreement with several recent genome-wide association studies, which have identified a number of single nucleotide polymorphisms in the MTNR1B gene, encoding the MT2 receptor, that were closely associated with a higher prognostic risk of developing type 2 diabetes. We hypothesize that catecholamines, which decrease insulin levels and stimulate melatonin synthesis, control insulin-melatonin interactions. The present results support this assertion as we show that catecholamines are increased in type 1 but are diminished in type 2 diabetes. Another important line of inquiry involves the fact that melatonin protects the ß-cells against functional overcharge and, consequently, hinders the development of type 2 diabetes. In this context, it is striking that at advanced ages, melatonin levels are reduced and the incidence of type 2 diabetes is increased. Thus, melatonin appears to have a protective biological role. Here, we strongly repudiate misconceptions, resulting from observations that melatonin reduces the plasma insulin level, that the blockage of melatonin receptors would be of benefit in the treatment of type 2 diabetes.

Molecular determinants of Grb14-mediated inhibition of insulin signaling.

Grb14 belongs to the Grb7 family of molecular adapters and was identified as an inhibitor of insulin signaling. Grb14 binds to activated insulin receptors (IR) and inhibits their catalytic activity. To gain more insight into the Grb14 molecular mechanism of action, we generated various mutants and studied the Grb14-IR interaction using coimmunoprecipitation and bioluminescence resonance energy transfer (BRET) experiments. Biological activity was further analyzed using the Xenopus oocyte model and a functional complementation assay measuring cellular proliferation rate in Grb14 knockout mouse embryonic fibroblasts. These studies identified two important interaction sites, Grb14 L404-IR L1038 and Grb14 R385-IR K1168, involving the IR alphaC-helix and activation loop, respectively. Interestingly, the former involves residues that are likely to be crucial for the specificity of IR binding with regard to other members of the Grb7 family. In addition, mutation of the Grb14-S370 residue suggested that its phosphorylation status controlled the biological activity of the protein. We further demonstrated that insulin-induced Grb14-PDK1 interaction is required in addition to Grb14-IR binding to mediate maximal inhibition of insulin signaling. This study provides important insights into the molecular determinants of Grb14 action by demonstrating that Grb14 regulates insulin action at two levels, through IR binding and by interfering with downstream pathways. Indeed, a precise knowledge of the molecular mechanism of insulin signaling inhibition by Grb14 is a prerequisite for the development of insulin-sensitizing molecules to treat pathophysiological states such as obesity or type 2 diabetes.

Activation of muscarinic M-3 receptor may decrease glucose uptake and lipolysis in adipose tissue of rats.

Role of muscarinic receptor in the regulation of glucose uptake or lipolysis in adipose tissue remained unclear. In epididymal white adipose tissue (WAT) isolated from Wistar rats, we observed that acetylcholine (ACh) attenuated the insulin-stimulated glucose uptake and the release of glycerol from WAT in a concentration-dependent manner. Using the blockade of specific antagonists, both actions of ACh were characterized mainly due to an activation of M3 receptors. In the presence of various inhibitors for PLC-PKC pathway, ACh-decreased glucose uptake was also reversed. Taken together, these results suggest that muscarinic M3 receptor is involved in the regulation of glucose uptake and/or lipolysis in adipose tissue.

A novel high-affinity peptide antagonist to the insulin receptor.

In this publication we describe a peptide insulin receptor antagonist, S661, which is a single chain peptide of 43 amino acids. The affinity of S661 for the insulin receptor is comparable to that of insulin and the selectivity for the insulin receptor versus the IGF-1 receptor is higher than that of insulin itself. S661 is also an antagonist of the insulin receptor of other species such as pig and rat, and it also has considerable affinity for hybrid insulin/IGF-1 receptors. S661 completely inhibits insulin action, both in cellular assays and in vivo in rats. A biosynthetic version called S961 which is identical to S661 except for being a C-terminal acid seems to have properties indistinguishable from those of S661. These antagonists provide a useful research tool for unraveling biochemical mechanisms involving the insulin receptor and could form the basis for treatment of hypoglycemic conditions.

Protocols to differentiate embryonic stem cells into insulin-producing cells

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Embryonic stem cells represent an interesting alternative to treat diabetesdue to their two main properties: self-renewal by symmetricdivisions, and the potential for cell differentiation into insulin-expressingcells under certain culture conditions. Achieving the latter impliesthe modification of the culture medium adding specific growth factorsand/or reprogramming the cells by transfecting specific DNAconstructs. Several laboratories have used one or a combination ofsuch strategies in order to obtain insulin-positive cells with differentdegrees of success. The main obstacles posed in the different protocolsconcern the amounts of intracellular insulin stored and secretedin response to different stimuli, the correct processing of thehormone, the risk of forming teratomas and the immune rejectiononce cells are transplanted. Promising results have been obtainedfrom protocols which try to recapitulate the pancreas ontogeny in theculture dish. To this end, it is instrumental to understand the embryonicdevelopment process of the human pancreas from definitiveendoderm to the adult, as well as its molecular markers and thefunctional traits of the obtained cells in order to achieve a transplantableproduct to cure diabetes. This manuscript explores publishedprotocols following this objective, analyzing the main features thatcould contribute to elaborate a more definitive strategy. The finalprotocol must be universal, easily transferable to the clinical practiceand, most importantly, liberate patients from the insulin injectionswithout the development of secondary complications

L-diabetes--causes, pathogenesis and therapy.

L-diabetes represents a subtype of non-autoimmunopathic and non-adipose diabetes mellitus. It is hypothesized that ATP-sensory brain centres measure the cerebral ATP concentration and announce a hypoglycaemia if the setpoint is undercut. The disease involves a decreased ATP formation in the CNS that is independent of blood glucose levels, and that leads to a "hypoglycaemia" false alarm. UGT1-polymorphisms, a sensitive sympathetic system, an IgM deficit and an increased porousity of the mucous membrane of the small intestine have been postulated in its etiology. These causative factors bring about increasing amounts of toxins and radicals which impair the ATP generation in the CNS so that through the announcement of a non-existing hypoglycaemia the release of the insulin antagonists hGH, cortisol and adrenaline is induced.

Toward a better understanding of Klotho.

klotho mutant mice were originally described as a short-lived mouse model with premature aging-like disorders. The klotho gene responsible for these phenotypes encodes a type I membrane protein with a considerable similarity to beta-glycosidase. klotho is predominantly expressed in tissues functioning in the regulation of calcium homeostasis. Suggested functions of Klotho are (i) a fundamental regulator of calcium homeostasis, namely, a cofactor for the fibroblast growth factor (FGF) receptor 1c in FGF23 signaling and a regulator of parathyroid hormone secretion; (ii) a hormone that interferes with the intracellular signaling of insulin and insulin-like growth factor-1; and (iii) a beta-glucuronidase that activates the transient receptor potential ion channel TRPV5 by trimming its sugar moiety. How can we reconcile these pleiotropic functions of Klotho? Is there any common mechanism? Further in vivo studies, and biochemical as well as physiological analyses, are required for a better understanding of the molecular aspects of Klotho.

Flies on steroids: the interplay between ecdysone and insulin signaling.

In the fruit fly Drosophila melanogaster, the insulin and ecdysone signaling pathways have long been known to regulate growth and developmental timing, respectively. Recent findings reveal that crosstalk between these pathways allows coordination of growth and developmental timing and thus determines final body size.

Is insulin resistance caused by defects in insulin's target cells or by a stressed mind?

The importance of understanding insulin action is emphasized by the increasing prevalence of insulin resistance in various populations and by the fact that it plays an important pathophysiological role in many common disorders, for example, diabetes, obesity, hypertension and dyslipidemia. The primary factors responsible for the development of insulin resistance are so far unknown, although both genetic and environmental factors are involved. The genetic defects responsible for the common forms of insulin resistance, for example, in type 2 diabetes, are largely unidentified. Some studies from our group as well as by other investigators suggest that cellular insulin resistance is reversible and that it may be secondary to factors in the in vivo environment. These may include insulin-antagonistic action of hormones like catecholamines, glucocorticoids, sex steroids and adipokines as well as dysregulation of autonomic nervous activity and they could contribute to the early development of insulin resistance. Some of these factors can directly impair glucose uptake capacity and this might be due to alterations in key proteins involved in insulin's intracellular signaling pathways. This article briefly summarizes proposed mechanisms behind cellular and whole-body insulin resistance. In particular, we question the role of intrinsic defects in insulin's target cells as primary mechanisms in the development of insulin resistance in type 2 diabetes and we suggest that metabolic and neurohormonal factors instead are the main culprits.

Requirements for pYXXM motifs in Cbl for binding to the p85 subunit of phosphatidylinositol 3-kinase and Crk, and activation of atypical protein kinase C and glucose transport during insulin action in 3T3/L1 adipocytes.

Cbl is phosphorylated by the insulin receptor and reportedly functions within the flotillin/CAP/Cbl/Crk/C3G/TC10 complex during insulin-stimulated glucose transport in 3T3/L1 adipocytes. Cbl, via pYXXM motifs at tyrosine-371 and tyrosine-731, also activates phosphatidylinositol (PI) 3-kinase, which is required to activate atypical protein kinase C (aPKC) and glucose transport during thiazolidinedione action in 3T3/L1 and human adipocytes [Miura et al. (2003) Biochemistry 42, 14335-14341]. Presently, we have examined the importance of Cbl in activating PI 3-kinase and aPKC during insulin action in 3T3/L1 adipocytes by expressing Y371F and Y731F Cbl mutants, which nullify pYXXM binding of Cbl to SH2 domains of downstream effectors. Interestingly, these mutants inhibited insulin-induced increases in (a) binding of Cbl to both Crk and the p85 subunit of PI 3-kinase, (b) activation of Cbl-dependent PI 3-kinase, (c) activation and translocation of aPKC to the plasma membrane, (d) translocation of Glut4 to the plasma membrane, (e) and glucose transport. Importantly, coexpression of wild-type Cbl reversed the inhibitory effects of Cbl mutants. In contrast to Cbl-dependent PI 3-kinase, Cbl mutants did not significantly inhibit the activation of PI 3-kinase by IRS-1, which is also required during insulin action. Our findings suggest that (a) Cbl uses pYXXM motifs to simultaneously activate PI 3-kinase and Crk/C3G/TC10 pathways and (b) Cbl, along with IRS-1, functions upstream of PI 3-kinase and aPKCs during insulin-stimulated glucose transport in 3T3/L1 adipocytes.

In vitro phosphorylation of insulin receptor substrate 1 by protein kinase C-zeta: functional analysis and identification of novel phosphorylation sites.

Protein kinase C-zeta (PKC-zeta) participates both in downstream insulin signaling and in the negative feedback control of insulin action. Here we used an in vitro approach to identify PKC-zeta phosphorylation sites within insulin receptor substrate 1 (IRS-1) and to characterize the functional implications. A recombinant IRS-1 fragment (rIRS-1(449)(-)(664)) containing major tyrosine motifs for interaction with phosphatidylinositol (PI) 3-kinase strongly associated to the p85alpha subunit of PI 3-kinase after Tyr phosphorylation by the insulin receptor. Phosphorylation of rIRS-1(449)(-)(664) by PKC-zeta induced a prominent inhibition of this process with a mixture of classical PKC isoforms being less effective. Both PKC-zeta and the classical isoforms phosphorylated rIRS-1(449)(-)(664) on Ser(612). However, modification of this residue did not reduce the affinity of p85alpha binding to pTyr-containing peptides (amino acids 605-615 of rat IRS-1), as determined by surface plasmon resonance. rIRS-1(449)(-)(664) was then phosphorylated by PKC-zeta using [(32)P]ATP and subjected to tryptic phosphopeptide mapping based on two-dimensional HPLC coupled to mass spectrometry. Ser(498) and Ser(570) were identified as novel phosphoserine sites targeted by PKC-zeta. Both sites were additionally confirmed by phosphopeptide mapping of the corresponding Ser --> Ala mutants of rIRS-1(449)(-)(664). Ser(570) was specifically targeted by PKC-zeta, as shown by immunoblotting with a phosphospecific antiserum against Ser(570) of IRS-1. Binding of p85alpha to the S570A mutant was less susceptible to inhibition by PKC-zeta, when compared to the S612A mutant. In conclusion, our in vitro data demonstrate a strong inhibitory action of PKC-zeta at the level of IRS-1/PI 3-kinase interaction involving multiple serine phosphorylation sites. Whereas Ser(612) appears not to participate in the negative control of insulin signaling, Ser(570) may at least partly contribute to this process.

Protein kinase C inhibits insulin-induced Akt activation in vascular smooth muscle cells.

Protein kinase C (PKC) activation, enhanced by hyperglycemia, is associated with many tissue abnormalities observed in diabetes. Akt is a serine/threonine kinase that mediates various biological responses induced by insulin. We hypothesized that the negative regulation of Akt in the vasculature by PKC could contribute to insulin resistant states and, may therefore play a role in the pathogenesis of cardiovascular disease. In this study, we specifically looked at the ability of PKC to inhibit Akt activation induced by insulin in cultured rat aortic vascular smooth muscle cells (VSMCs). Activation of Akt was determined by immunoblotting with a phospho-Akt antibody that selectively recognizes Ser473 phosphorylated Akt. A PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited insulin-dependent Akt phosphorylation. However, PMA did not inhibit platelet-derived growth factor (PDGF)-induced activation of Akt. We further showed that the PKC inhibitor, G06983, blocked the PMA-induced inhibition of Akt phosphorylation by insulin. In addition, we demonstrated that PMA inhibited the insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). From these data, we conclude that PKC is a potent negative regulator of the insulin signal in the vasculature, which indicate an important role of PKC in the development of insulin resistance in cardiovascular disease.