Cercosporamide inhibits bone morphogenetic protein receptor type I kinase activity in zebrafish.

Zebrafish models are well-established tools for investigating the underlying mechanisms of diseases. Here, we identified cercosporamide, a metabolite from the fungus Ascochyta aquiliqiae, as a potent bone morphogenetic protein receptor (BMPR) type I kinase inhibitor through a zebrafish embryo phenotypic screen. The developmental defects in zebrafish, including lack of the ventral fin, induced by cercosporamide were strikingly similar to the phenotypes caused by renowned small-molecule BMPR type I kinase inhibitors and inactivating mutations in zebrafish BMPRs. In mammalian cell-based assays, cercosporamide blocked BMP/SMAD-dependent transcriptional reporter activity and BMP-induced SMAD1/5-phosphorylation. Biochemical assays with a panel of purified recombinant kinases demonstrated that cercosporamide directly inhibited kinase activity of type I BMPRs [also called activin receptor-like kinases (ALKs)]. In mammalian cells, cercosporamide selectively inhibited constitutively active BMPR type I-induced SMAD1/5 phosphorylation. Importantly, cercosporamide rescued the developmental defects caused by constitutively active Alk2 in zebrafish embryos. We believe that cercosporamide could be the first of a new class of molecules with potential to be developed further for clinical use against diseases that are causally linked to overactivation of BMPR signaling, including fibrodysplasia ossificans progressiva and diffuse intrinsic pontine glioma.This article has an associated First Person interview with the first author of the paper.

Chronic Intermittent Hypoxia Impairs Insulin Sensitivity but Improves Whole-Body Glucose Tolerance by Activating Skeletal Muscle AMPK.

Obstructive sleep apnea syndrome is a highly prevalent disease resulting in transient respiratory arrest and chronic intermittent hypoxia (cIH). cIH is associated with insulin resistance and impaired metabolic homeostasis in rodents and humans, but the exact underlying mechanisms remain unclear. In the current study, we investigated the effects of 2 weeks of cIH (1-min cycle, fraction of inspired oxygen 21-5%, 8 h/day) on whole-body insulin sensitivity and glucose tolerance in lean mice. Although food intake and body weight were reduced compared with normoxia, cIH induced systemic insulin resistance in a hypoxia-inducible factor 1-independent manner and impaired insulin signaling in liver, white adipose tissue, and skeletal muscle. Unexpectedly, cIH improved whole-body glucose tolerance independently of changes in body weight and glucose-induced insulin response. This effect was associated with elevated phosphorylation of Thr172-AMPK and Ser237-TBC1 domain family member 1 (TBC1D1) in skeletal muscle, suggesting a tissue-specific AMPK-dependent increase in TBC1D1-driven glucose uptake. Remarkably, although food intake, body weight, and systemic insulin sensitivity were still affected, the improvement in glucose tolerance by cIH was abolished in muscle-specific AMPKα1α2-deficient mice. We conclude that cIH impairs insulin sensitivity while improving whole-body glucose tolerance by promoting specific activation of the skeletal muscle AMPK pathway.

Hypoxia-inducible factor prolyl hydroxylase 1 (PHD1) deficiency promotes hepatic steatosis and liver-specific insulin resistance in mice.

Obesity is associated with local tissue hypoxia and elevated hypoxia-inducible factor 1 alpha (HIF-1α) in metabolic tissues. Prolyl hydroxylases (PHDs) play an important role in regulating HIF-α isoform stability. In the present study, we investigated the consequence of whole-body PHD1 gene (Egln2) inactivation on metabolic homeostasis in mice. At baseline, PHD1-/- mice exhibited higher white adipose tissue (WAT) mass, despite lower body weight, and impaired insulin sensitivity and glucose tolerance when compared to age-matched wild-type (WT) mice. When fed a synthetic low-fat diet, PHD1-/- mice also exhibit a higher body weight gain and WAT mass along with glucose intolerance and systemic insulin resistance compared to WT mice. PHD1 deficiency led to increase in glycolytic gene expression, lipogenic proteins ACC and FAS, hepatic steatosis and liver-specific insulin resistance. Furthermore, gene markers of inflammation were also increased in the liver, but not in WAT or skeletal muscle, of PHD1-/- mice. As expected, high-fat diet (HFD) promoted obesity, hepatic steatosis, tissue-specific inflammation and systemic insulin resistance in WT mice but these diet-induced metabolic alterations were not exacerbated in PHD1-/- mice. In conclusion, PHD1 deficiency promotes hepatic steatosis and liver-specific insulin resistance but does not worsen the deleterious effects of HFD on metabolic homeostasis.

Chronic helminth infection and helminth-derived egg antigens promote adipose tissue M2 macrophages and improve insulin sensitivity in obese mice.

Chronic low-grade inflammation associated with obesity contributes to insulin resistance and type 2 diabetes. Helminth parasites are the strongest natural inducers of type 2 immune responses, and short-lived infection with rodent nematodes was reported to improve glucose tolerance in obese mice. Here, we investigated the effects of chronic infection (12 weeks) with Schistosoma mansoni, a helminth that infects millions of humans worldwide, on whole-body metabolic homeostasis and white adipose tissue (WAT) immune cell composition in high-fat diet-induced obese C57BL/6 male mice. Our data indicate that chronic helminth infection reduced body weight gain (-62%), fat mass gain (-89%), and adipocyte size; lowered whole-body insulin resistance (-23%) and glucose intolerance (-16%); and improved peripheral glucose uptake (+25%) and WAT insulin sensitivity. Analysis of immune cell composition by flow cytometry and quantitative PCR (qPCR) revealed that S. mansoni promoted strong increases in WAT eosinophils and alternatively activated (M2) macrophages. Importantly, injections with S. mansoni-soluble egg antigens (SEA) recapitulated the beneficial effect of parasite infection on whole-body metabolic homeostasis and induced type 2 immune responses in WAT and liver. Taken together, we provide novel data suggesting that chronic helminth infection and helminth-derived molecules protect against metabolic disorders by promoting a T helper 2 (Th2) response, eosinophilia, and WAT M2 polarization.

Middle-aged overweight South Asian men exhibit a different metabolic adaptation to short-term energy restriction compared with Europeans.

AIMS/HYPOTHESIS: South Asians have a higher risk of developing type 2 diabetes than Europeans. The underlying cause of this excess risk is still poorly understood but might be related to differences in the regulation of energy/nutrient-sensing pathways in metabolic tissues and subsequent changes in whole-body substrate metabolism. In this study, we investigated the whole-body and skeletal muscle metabolic adaptations to short-term energy restriction in South Asian and European volunteers. METHODS: Twenty-four middle-aged overweight South Asian and European men underwent a two-step hyperinsulinaemic-euglycaemic clamp, with skeletal muscle biopsies and indirect calorimetry before and after an 8 day diet very low in energy (very low calorie diet [VLCD]). Abdominal fat distribution and hepatic triacylglycerol content were assessed using MRI and MR spectroscopy. RESULTS: South Asian men had higher hepatic triacylglycerol content than European men, and exhibited elevated clamp insulin levels that probably reflect a lower insulin clearance rate. Despite higher insulin levels, endogenous glucose production rate was similar and glucose disposal rate (Rd) and nonoxidative glucose disposal rate (NOGD) were significantly lower in South Asian than European men, indicating impaired whole-body insulin sensitivity. Energy restriction decreased abdominal fat mass and hepatic triacylglycerol content in both groups. However, the shift induced by energy restriction from glucose towards lipid oxidation observed in European men was impaired in South Asian men, indicating whole-body metabolic inflexibility. Remarkably, although energy restriction improved hepatic insulin sensitivity in both groups, Rd improved only in South Asian men owing to higher NOGD. At the molecular level, an increase in insulin-induced activation of the skeletal muscle mTOR pathway was found in South Asian men, showing that skeletal muscle energy/nutrient-sensing pathways were differentially affected by energy restriction. CONCLUSIONS/INTERPRETATION: We conclude that South Asian men exhibit a different metabolic adaptation to short-term energy restriction than European men. TRIAL REGISTRATION: Dutch trial registry ( www.trialregister.nl ), trial number NTR 2473.

Regulation of skeletal muscle energy/nutrient-sensing pathways during metabolic adaptation to fasting in healthy humans.

During fasting, rapid metabolic adaptations are required to maintain energy homeostasis. This occurs by a coordinated regulation of energy/nutrient-sensing pathways leading to transcriptional activation and repression of specific sets of genes. The aim of the study was to investigate how short-term fasting affects whole body energy homeostasis and skeletal muscle energy/nutrient-sensing pathways and transcriptome in humans. For this purpose, 12 young healthy men were studied during a 24-h fast. Whole body glucose/lipid oxidation rates were determined by indirect calorimetry, and blood and skeletal muscle biopsies were collected and analyzed at baseline and after 10 and 24 h of fasting. As expected, fasting induced a time-dependent decrease in plasma insulin and leptin levels, whereas levels of ketone bodies and free fatty acids increased. This was associated with a metabolic shift from glucose toward lipid oxidation. At the molecular level, activation of the protein kinase B (PKB/Akt) and mammalian target of rapamycin pathways was time-dependently reduced in skeletal muscle during fasting, whereas the AMP-activated protein kinase activity remained unaffected. Furthermore, we report some changes in the phosphorylation and/or content of forkhead protein 1, sirtuin 1, and class IIa histone deacetylase 4, suggesting that these pathways might be involved in the transcriptional adaptation to fasting. Finally, transcriptome profiling identified genes that were significantly regulated by fasting in skeletal muscle at both early and late time points. Collectively, our study provides a comprehensive map of the main energy/nutrient-sensing pathways and transcriptomic changes during short-term adaptation to fasting in human skeletal muscle.

Metformin lowers plasma triglycerides by promoting VLDL-triglyceride clearance by brown adipose tissue in mice.

Metformin is the first-line drug for the treatment of type 2 diabetes. Besides its well-characterized antihyperglycemic properties, metformin also lowers plasma VLDL triglyceride (TG). In this study, we investigated the underlying mechanisms in APOE*3-Leiden.CETP mice, a well-established model for human-like lipoprotein metabolism. We found that metformin markedly lowered plasma total cholesterol and TG levels, an effect mostly due to a decrease in VLDL-TG, whereas HDL was slightly increased. Strikingly, metformin did not affect hepatic VLDL-TG production, VLDL particle composition, and hepatic lipid composition but selectively enhanced clearance of glycerol tri[(3)H]oleate-labeled VLDL-like emulsion particles into brown adipose tissue (BAT). BAT mass and lipid droplet content were reduced in metformin-treated mice, pointing to increased BAT activation. In addition, both AMP-activated protein kinase α1 (AMPKα1) expression and activity and HSL and mitochondrial content were increased in BAT. Furthermore, therapeutic concentrations of metformin increased AMPK and HSL activities and promoted lipolysis in T37i differentiated brown adipocytes. Collectively, our results identify BAT as an important player in the TG-lowering effect of metformin by enhancing VLDL-TG uptake, intracellular TG lipolysis, and subsequent mitochondrial fatty acid oxidation. Targeting BAT might therefore be considered as a future therapeutic strategy for the treatment of dyslipidemia.

A 5-day high-fat, high-calorie diet impairs insulin sensitivity in healthy, young South Asian men but not in Caucasian men.

South Asians (SAs) develop type 2 diabetes at a younger age and lower BMI compared with Caucasians (Cs). The underlying cause is still poorly understood but might result from an innate inability to adapt to the Westernized diet. This study aimed to compare the metabolic adaptation to a high-fat, high-calorie (HFHC) diet between both ethnicities. Twelve healthy, young lean male SAs and 12 matched Cs underwent a two-step hyperinsulinemic-euglycemic clamp with skeletal muscle biopsies and indirect calorimetry before and after a 5-day HFHC diet. Hepatic triglyceride content (HTG) and abdominal fat distribution were assessed using magnetic resonance imaging and spectroscopy. At baseline, SAs had higher insulin clamp levels than Cs, indicating reduced insulin clearance rate. Despite the higher insulin levels, endogenous glucose production was comparable between groups, suggesting lower hepatic insulin sensitivity in SAs. Furthermore, a 5-day HFHC diet decreased the insulin-stimulated (nonoxidative) glucose disposal rate only in SA. In skeletal muscle, no significant differences were found between groups in insulin/mammalian target of rapamycin signaling, metabolic gene expression, and mitochondrial respiratory chain content. Furthermore, no differences in (mobilization of) HTG and abdominal fat were detected. We conclude that HFHC feeding rapidly induces insulin resistance only in SAs. Thus, distinct adaptation to Western food may partly explain their propensity to develop type 2 diabetes.

Rapamycin and omega-1: mTOR-dependent and -independent Th2 skewing by human dendritic cells.

Recent reports have attributed an immunoregulatory role to the mammalian target of rapamycin (mTOR), a key serine/threonine protein kinase integrating input from growth factors and nutrients to promote cell growth and differentiation. In the present study, we investigated the role of the mTOR pathway in Th2 induction by human monocyte-derived dendritic cells (moDCs). Using a co-culture system of human lipopolysaccharide (LPS)-matured moDCs and allogeneic naive CD4(+) T cells, we show that inhibition of mTOR by the immunosuppressive drug rapamycin reduced moDC maturation and promoted Th2 skewing. Next, we investigated whether antigens from helminth parasites, the strongest natural inducers of Th2 responses, modulate moDCs via the mTOR pathway. In contrast to rapamycin, neither Schistosoma mansoni-soluble egg antigens (SEA) nor its major immunomodulatory component omega-1 affected the phosphorylation of S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1), downstream targets of mTORC1. Finally, we found that the effects of rapamycin and SEA/omega-1 on Th2 skewing were additive, suggesting two distinct underlying molecular mechanisms. We conclude that conditioning human moDCs to skew immune responses towards Th2 can be achieved via an mTOR-dependent and -independent pathway triggered by rapamycin and helminth antigens, respectively.

Proline-rich Akt substrate of 40-kDa contains a nuclear export signal.

The proline-rich Akt substrate of 40-kDa (PRAS40) has been linked to the regulation of the activity of the mammalian target of rapamycin complex 1 as well as insulin action. Despite these cytosolic functions, PRAS40 was originally identified as nuclear phosphoprotein in Hela cells. This study aimed to detail mechanisms and consequences of the nucleocytosolic trafficking of PRAS40. Sequence analysis identified a potential leucine-rich nuclear export signal (NES) within PRAS40. Incubation of A14 fibroblasts overexpressing human PRAS40 (hPRAS40) resulted in nuclear accumulation of the protein. Furthermore, mutation of the NES mimicked the effects of leptomycin B, a specific inhibitor of nuclear export, on the subcellular localization of hPRAS40. Finally, A14 cells expressing the NES-mutant showed impaired activation of components of the Akt-pathway as well as of the mTORC1 substrate p70 S6 kinase after insulin stimulation. This impaired insulin signaling could be ascribed to reduced protein levels of insulin receptor substrate 1 in cells expressing mutant NES. In conclusion, PRAS40 contains a functional nuclear export signal. Furthermore, enforced nuclear accumulation of PRAS40 impairs insulin action, thereby substantiating the function of this protein in the regulation of insulin sensitivity.

Effects of prolonged fasting on AMPK signaling, gene expression, and mitochondrial respiratory chain content in skeletal muscle from lean and obese individuals.

Obesity in humans is often associated with metabolic inflexibility, but the underlying molecular mechanisms remain incompletely understood. The aim of the present study was to investigate how adaptation to prolonged fasting affects energy/nutrient-sensing pathways and metabolic gene expression in skeletal muscle from lean and obese individuals. Twelve lean and 14 nondiabetic obese subjects were fasted for 48 h. Whole body glucose/lipid oxidation rates were determined by indirect calorimetry, and blood and skeletal muscle biopsies were collected and analyzed. In response to fasting, body weight loss was similar in both groups, but the decrease in plasma insulin and leptin and the concomitant increase in growth hormone were significantly attenuated in obese subjects. The fasting-induced shift from glucose toward lipid oxidation was also severely blunted. At the molecular level, the expression of insulin receptor-ß (IRß) was lower in skeletal muscle from obese subjects at baseline, whereas the fasting-induced reductions in insulin signaling were similar in both groups. The protein expression of mitochondrial respiratory chain components, although not modified by fasting, was significantly reduced in obese subjects. Some minor differences in metabolic gene expression were observed at baseline and in response to fasting. Surprisingly, fasting reduced AMPK activity in lean but not in obese subjects, whereas the expression of AMPK subunits was not affected. We conclude that whole body metabolic inflexibility in response to prolonged fasting in obese humans is associated with lower skeletal muscle IRß and mitochondrial respiratory chain content as well as a blunted decline of AMPK activity.

Prednisolone induces the Wnt signalling pathway in 3T3-L1 adipocytes.

Synthetic glucocorticoids are potent anti-inflammatory drugs but show dose-dependent metabolic side effects such as the development of insulin resistance and obesity. The precise mechanisms involved in these glucocorticoid-induced side effects, and especially the participation of adipose tissue in this are not completely understood. We used a combination of transcriptomics, antibody arrays and bioinformatics approaches to characterize prednisolone-induced alterations in gene expression and adipokine secretion, which could underlie metabolic dysfunction in 3T3-L1 adipocytes. Several pathways, including cytokine signalling, Akt signalling, and Wnt signalling were found to be regulated at multiple levels, showing that these processes are targeted by prednisolone. These results suggest that mechanisms by which prednisolone induce insulin resistance include dysregulation of wnt signalling and immune response processes. These pathways may provide interesting targets for the development of improved glucocorticoids.

Prednisolone-induced beta cell dysfunction is associated with impaired endoplasmic reticulum homeostasis in INS-1E cells.

Glucocorticoids (GCs), such as prednisolone (PRED), are widely prescribed anti-inflammatory drugs, but their use may induce glucose intolerance and diabetes. GC-induced beta cell dysfunction contributes to these diabetogenic effects through mechanisms that remain to be elucidated. In this study, we hypothesized that activation of the unfolded protein response (UPR) following endoplasmic reticulum (ER) stress could be one of the underlying mechanisms involved in GC-induced beta cell dysfunction. We report here that PRED did not affect basal insulin release but time-dependently inhibited glucose-stimulated insulin secretion in INS-1E cells. PRED treatment also decreased both PDX1 and insulin expression, leading to a marked reduction in cellular insulin content. These PRED-induced detrimental effects were found to be prevented by prior treatment with the glucocorticoid receptor (GR) antagonist RU486 and associated with activation of two of the three branches of the UPR. Indeed, PRED induced a GR-mediated activation of both ATF6 and IRE1/XBP1 pathways but was found to reduce the phosphorylation of PERK and its downstream substrate eIF2α. These modulations of ER stress pathways were accompanied by upregulation of calpain 10 and increased cleaved caspase 3, indicating that long term exposure to PRED ultimately promotes apoptosis. Taken together, our data suggest that the inhibition of insulin biosynthesis by PRED in the insulin-secreting INS-1E cells results, at least in part, from a GR-mediated impairment in ER homeostasis which may lead to apoptotic cell death.

Phosphorylation of PRAS40 on Thr246 by PKB/AKT facilitates efficient phosphorylation of Ser183 by mTORC1.

Type 2 diabetes is associated with alterations in protein kinase B (PKB/Akt) and mammalian target of rapamycin complex 1 (mTORC1) signalling. The proline-rich Akt substrate of 40-kDa (PRAS40) is a component of mTORC1, which has a regulatory function at the intersection of the PKB/Akt and mTORC1 signalling pathway. Phosphorylation of PRAS40-Thr246 by PKB/Akt, and PRAS40-Ser183 and PRAS40-Ser221 by mTORC1 results in dissociation from mTORC1, and its binding to 14-3-3 proteins. Although all phosphorylation sites within PRAS40 have been implicated in 14-3-3 binding, substitution of Thr246 by Ala alone is sufficient to abolish 14-3-3 binding under conditions of intact mTORC1 signalling. This suggests that phosphorylation of PRAS40-Thr246 may facilitate efficient phosphorylation of PRAS40 on its mTORC1-dependent sites. In the present study, we investigated the mechanism of PRAS40-Ser183 phosphorylation in response to insulin. Insulin promoted PRAS40-Ser183 phosphorylation after a euglycaemic-hyperinsulinaemic clamp in human skeletal muscle. The insulin-induced PRAS40-Ser183 phosphorylation was further evidenced in vivo in rat skeletal and cardiac muscle, and in vitro in A14 fibroblasts, 3T3L1 adipocytes and L6 myotubes. Inhibition of mTORC1 by rapamycin or amino acid deprivation partially abrogated insulin-mediated PRAS40-Ser183 phosphorylation in cultured cell lines. However, lowering insulin-induced PRAS40-Thr246 phosphorylation using wortmannin or palmitate in cell lines, or by feeding rats a high-fat diet, completely abolished insulin-mediated PRAS40-Ser183 phosphorylation. In addition, replacement of Thr246 by Ala reduced insulin-mediated PRAS40-Ser183 phosphorylation. We conclude that PRAS40-Ser183 is a component of insulin action, and that efficient phosphorylation of PRAS40-Ser183 by mTORC1 requires the phosphorylation of PRAS40-Thr246 by PKB/Akt.

The nuclear appearance of ERK1/2 and p38 determines the sequential induction of ATF2-Thr71 and ATF2-Thr69 phosphorylation by serum in JNK-deficient cells.

Growth factors activate ATF2 via sequential phosphorylation of Thr69 and Thr71, where the ATF2-Thr71-phosphorylation precedes the induction of ATF2-Thr69+71-phosphorylation. Here, we studied the mechanisms contributing to serum-induced two-step ATF2-phosphorylation in JNK1,2-deficient embryonic fibroblasts. Using anion exchange chromatography, ERK1/2 and p38 were identified as ATF2-kinases in vitro. Inhibitor studies as well as nuclear localization experiments show that the sequential nuclear appearance of ERK1/2 and p38 determines the induction of ATF2-Thr71 and ATF2-Thr69+71-phosphorylation in response to serum.

Permissive action of protein kinase C-zeta in insulin-induced CD36- and GLUT4 translocation in cardiac myocytes.

Insulin stimulates cardiac long-chain fatty acid (LCFA) and glucose uptake via translocation of human homolog of rat fatty acid translocase (CD36) and GLUT4 respectively, from intracellular membrane compartments to the sarcolemma, a process dependent on the activation of phosphatidylinositol-3 kinase. To identify downstream kinases of insulin signaling involved in translocation of CD36 and GLUT4 in the heart, we tested i) which cardiac protein kinase C (PKC) isoforms (alpha, delta, epsilon or zeta) are activated by insulin, and ii) whether PKC isoform-specific inhibition affects insulin-stimulated substrate uptake in the heart. Insulin-stimulated LCFA and glucose uptake were completely blunted by inhibition of PKC-zeta, but not by inhibition of conventional or novel PKCs. Concomitantly, translocation of CD36 and GLUT4 to the sarcolemma was completely blunted upon inhibition of PKC-zeta. However, insulin, in contrast to the diacylglycerol-analog phorbol-12-myristate-13-acetate (PMA), did not induce membrane-attachment of the conventional and novel PKCs-alpha, -delta, and -epsilon. PKC-zeta was already entirely membrane-bound in non-stimulated cells, and neither insulin nor PMA treatment had any effect on the subcellular localization of PKC-zeta. Furthermore, insulin treatment did not change phosphorylation of PKC-alpha, -delta, and -zeta or enzymatic activity of PKC-zeta towards a PKC-zeta substrate peptide. It is concluded that PKC-zeta, but not any other PKC isoform, is necessary for insulin-induced translocation of GLUT4 and CD36. However, PKC-zeta is already fully active under basal conditions and not further activated by insulin, indicating that its role in insulin-stimulated uptake of both glucose and LCFA is permissive rather than regulatory.

Insulin-mediated phosphorylation of the proline-rich Akt substrate PRAS40 is impaired in insulin target tissues of high-fat diet-fed rats.

Clinical insulin resistance is associated with decreased activation of phosphatidylinositol 3'-kinase (PI3K) and its downstream substrate protein kinase B (PKB)/Akt. However, its physiological protein substrates remain poorly characterized. In the present study, the effect of in vivo insulin action on phosphorylation of the PKB/Akt substrate 40 (PRAS40) was examined. In rat and mice, insulin stimulated PRAS40-Thr246 phosphorylation in skeletal and cardiac muscle, the liver, and adipose tissue in vivo. Physiological hyperinsulinemia increased PRAS40-Thr246 phosphorylation in human skeletal muscle biopsies. In cultured cell lines, insulin-mediated PRAS40 phosphorylation was prevented by the PI3K inhibitors wortmannin and LY294002. Immunohistochemical and immunofluorescence studies showed that phosphorylated PRAS40 is predominantly localized to the nucleus. Finally, in rats fed a high-fat diet (HFD), phosphorylation of PRAS40 was markedly reduced compared with low-fat diet-fed animals in all tissues examined. In conclusion, the current study identifies PRAS40 as a physiological target of in vivo insulin action. Phosphorylation of PRAS40 is increased by insulin in human, rat, and mouse insulin target tissues. In rats, this response is reduced under conditions of HFD-induced insulin resistance.

The role of c-Jun N-terminal kinase, p38, and extracellular signal-regulated kinase in insulin-induced Thr69 and Thr71 phosphorylation of activating transcription factor 2.

The stimulation of cells with physiological concentrations of insulin induces a variety of responses, e.g. an increase in glucose uptake, induction of glycogen and protein synthesis, and gene expression. One of the determinants regulating insulin-mediated gene expression may be activating transcription factor 2 (ATF2). Insulin activates ATF2 by phosphorylation of Thr69 and Thr71 via a two-step mechanism, in which ATF2-Thr71 phosphorylation precedes the induction of ATF2-Thr69+71 phosphorylation by several minutes. We previously found that in c-Jun N-terminal kinase (JNK)-/- fibroblasts, cooperation of the ERK1/2 and p38 pathways is required for two-step ATF2-Thr69+71 phosphorylation in response to growth factors. Because JNK is also capable of phosphorylating ATF2, we assessed the involvement of JNK, ERK1/2 and p38 in the insulin-induced two-step ATF2 phosphorylation in JNK-expressing A14 fibroblasts and 3T3L1-adipocytes. The induction of ATF2-Thr71 phosphorylation was sensitive to MAPK kinase (MEK) 1/2-inhibition with U0126, and this phosphorylation coincided with nuclear translocation of phosphorylated ERK1/2. Use of the JNK inhibitor SP600125 or expression of dominant-negative JNK-activator SAPK kinase (SEK1) prevented the induction of ATF2-Thr69+71, but not ATF2-Thr71 phosphorylation by insulin. ATF2-dependent transcription was also sensitive to SP-treatment. Abrogation of p38 activation with SB203580 or expression of dominant-negative MKK6 had no inhibitory effect on these events. In agreement with this, the onset of ATF2-Thr69+71 phosphorylation coincided with the nuclear translocation of phosphorylated JNK. Finally, in vitro kinase assays using nuclear extracts indicated that ERK1/2 preceded JNK translocation. We conclude that sequential activation and nuclear appearance of ERK1/2 and JNK, rather than p38, underlies the two-step insulin-induced ATF2 phosphorylation in JNK-expressing cells.

Rottlerin inhibits multiple steps involved in insulin-induced glucose uptake in 3T3-L1 adipocytes.

Recently, it was shown that rottlerin inhibits insulin-stimulated glucose uptake and reduces intracellular adenosine triphosphate (ATP) levels in 3T3-L1 adipocytes, suggesting that these two events are causally linked. However, several other reports show that ATP-depletion induces glucose uptake in both muscle cells and adipocytes. In the present study, the mechanism of inhibition by rottlerin was studied in detail, in order to resolve this apparent discrepancy. It was found that rottlerin strongly reduces insulin-stimulated 2-deoxyglucose (2-DOG) uptake in 3T3-L1 adipocytes by a partial inhibition of the translocation of the insulin-responsive GLUT4 glucose transporter towards the plasma membrane (PM). Whereas the insulin-induced phosphatidyl-inositol-3' (PI-3') kinase signaling pathway is unaffected by rottlerin, Cbl tyrosine phosphorylation, which provides an essential, PI-3' kinase-independent signal towards GLUT4 translocation, is markedly attenuated. Furthermore, we also observed a direct inhibitory effect of rottlerin on insulin-induced glucose uptake in 3T3-L1 adipocytes. The direct inhibition of insulin-stimulated 2-DOG uptake by rottlerin displayed characteristics of uncompetitive inhibition: with the K(m(app)) of glucose uptake reduced from 1.6 to 0.9 mM and the V(max(app)) reduced from 5.2 to 1.0 nmol/minmg in the presence of rottlerin. In conclusion, rottlerin inhibits multiple steps involved in insulin-stimulated 2-DOG uptake in 3T3-L1 adipocytes. The observed reduction in GLUT4 translocation towards the PM and the uncompetitive inhibition of the glucose transport process provide alternative explanations for the inhibitory effects of rottlerin aside from the effects of rottlerin on intracellular levels of ATP.

Identification and functional assessment of novel and known insulin receptor mutations in five patients with syndromes of severe insulin resistance.

We analyzed the insulin receptor gene in four patients with leprechaunism and one with type A insulin resistance. We detected novel and previously reported mutations. The novel mutants were expressed in Chinese hamster ovary cells to evaluate the consequences for insulin receptor function. A type A insulin resistance patient from Morocco was homozygous for Arg252His mutation, similar to a previously described type A patient from Japan. A patient with leprechaunism was homozygous for the Ser323Leu mutation, previously identified in homozygous form in two patients with Rabson-Mendenhall syndrome. Phenotypic expression of this mutation is variable. A patient with leprechaunism is compound heterozygous for the previously described Arg1092Trp mutation and a nonsense mutation in codon 897. Another patient with leprechaunism was homozygous for a novel Asn431Asp mutation, which only partially reduces insulin proreceptor processing and activation of signaling cascades. The novel Leu93Gln mutation that fully disrupts proreceptor processing was found in one allele in a patient with leprechaunism. A nonsense mutation at codon 1122 was in the other allele. These results expand the number of pathogenic insulin receptor mutations and demonstrate the variability in their phenotypic expression. The biochemical analysis of mutant insulin receptors does not reliably predict whether the phenotype will be leprechaunism, the Rabson-Mendenhall syndrome, or type A insulin resistance. The previously reported correlation between fibroblast insulin binding and duration of patient survival was not observed.