Insulin-stimulated Akt Phosphorylation Research Articles

Tryptophanylation of insulin receptor by WARS attenuates insulin signaling.

Increased circulating amino acid levels have been linked to insulin resistance and development of type 2 diabetes (T2D), but the underlying mechanism remains largely unknown. Herein, we show that tryptophan modifies insulin receptor (IR) to attenuate insulin signaling and impair glucose uptake. Mice fed with tryptophan-rich chow developed insulin resistance. Excessive tryptophan promoted tryptophanyl-tRNA synthetase (WARS) to tryptophanylate lysine 1209 of IR (W-K1209), which induced insulin resistance by inhibiting the insulin-stimulated phosphorylation of IR, AKT, and AS160. SIRT1, but not other sirtuins, detryptophanylated IRW-K1209 to increase the insulin sensitivity. Collectively, we unveiled the mechanisms of how tryptophan impaired insulin signaling, and our data suggested that WARS might be a target to attenuate insulin resistance in T2D patients.

LncRNA H19 inhibition impairs endoplasmic reticulum-mitochondria contact in hepatic cells and augments gluconeogenesis by increasing VDAC1 levels

Inspite of exerting independent cellular functions, the endoplasmic-reticulum (ER) and the mitochondria also physically connect at specific sites termed mitochondria-associated ER membranes (MAMs) and these sites consist of several tethering proteins that play varied roles in diverse cellular processes. However, the regulation of these tethering proteins within the cell is relatively less studied. Here, we show that several MAM proteins are significantly altered in the liver during diabetes and among these, the lncRNA, H19 regulates the levels of VDAC1. Inhibition of H19 expression using H19 specific siRNA altered VDAC1, mitochondrial Ca2+ and oxygen consumption rate, ATP and ROS levels and enhanced ER and mitochondria coupling in Hepa 1–6 cells. While H19 inhibition did not impact lipid accumulation, levels of gluconeogenic genes were significantly increased. JNK-phosphorylation and IRS1-Ser307-phosphorylation were increased by H19 inhibition and this was associated with abrogation of insulin-stimulated AKT (Ser-473) phosphorylation and glucose uptake in Hepa 1–6 cells. While inhibition of VDAC1 expression using siRNAs and with metformin significantly rescued the effects of H19 inhibition, VDAC1 overexpression alone exerted effects similar to H19 inhibition, suggesting that VDAC1 increase mediates the adverse effects of H19. In-vivo H19 inhibition using specific siRNAs increased hepatic VDAC1, pJNK and pIRS1 (Ser307) levels and decreased AKT (Ser-473) phosphorylation in mice. These suggest an important role of the H19-VDAC1 axis in ER-mitochondria coupling and regulation of gluconeogenesis in the liver during diabetes.

Simulated weightlessness induces hippocampal insulin resistance and cognitive impairment

Growing evidence highlights the potential consequences of long-term spaceflight, including gray matter volume reduction and cognitive dysfunction with subclinical manifestations of diabetes mellitus among astronauts, but the underlying mechanisms remain unknown. In this study, we found that long-term simulated weightlessness induced hippocampal insulin resistance and subsequent neuronal damage and cognitive impairment in rats. Rats subjected to 4-week tail suspension exhibited peripheral insulin resistance, evidenced by increased fasting blood glucose and abnormal glucose tolerance and insulin tolerance, alongside reduced spontaneous activity and impaired recognition memory. In addition, 4 weeks of simulated weightlessness induced neuronal apoptosis and degeneration in the hippocampus, as evidenced by increased TUNEL and Fluoro-Jade B staining-positive neurons. Mechanistically, insulin-stimulated hippocampal Akt phosphorylation was decreased, while PTEN, the negative regulator of insulin signaling, was increased in the hippocampus in tail-suspended rats. Interestingly, treatment with berberine, an insulin sensitizer, partly reversed the above-mentioned effects induced by simulated weightlessness. These data suggest that long-term simulated weightlessness induces cognitive impairment as well as neuronal apoptosis and neural degeneration, partially through hippocampal insulin resistance via PTEN up-regulation. Berberine treatment attenuates hippocampal insulin resistance and improves cognitive function.

236-LB: FHL2-/- Perivascular Adipose Tissue Improves Vessel Tone and Insulin-Stimulated Vasodilation by Inhibiting ERK1/2

Background: Type 2 diabetes, obesity and aging increase risk of developing cardiovascular diseases. Perivascular adipose tissue (PVAT) is a crucial regulator of vascular function. Four and Half Lim domains 2 (FHL2) is a protein upregulated in aging which targets various proteins with established links to adipose tissue growth, potentially regulating metabolic signaling. Objective: we hypothesized that FHL2 regulates adipose tissue regulation of vascular function. Methods: Femoral arteries were isolated from Wild type (WT) and FHL2-/- mice and vasoreactivity to insulin and acetylcholine was studied with and without perivascular adipose tissue. Results: FHL2-/- vessels with PVAT had increased tone compared to WT vessels with PVAT as well as improved insulin-stimulated vasodilation. Exchange experiments using vessels with PVAT from the opposite genotype showed that FHL2-PVAT enhanced tone and insulin-stimulated vasodilatation compared to WT-PVAT. Gene expression analysis showed downregulation of adiponectin in FHL2-PVAT. Western blot analysis of insulin-stimulated phosphorylation of ERK and Akt in human microvascular endothelial cells (HMECs) stimulated with PVAT-conditioned medium revealed that FHL2-PVAT but not WT-PVAT reduced insulin-stimulated phosphorylation of ERK but not activation of Akt. In line with the effect of FHL2 on insulin-induced vasodilatation and activation of ERK1/2, analysis of publicly available RNA sequencing data of peri-aortic PVAT showed that FHL2 expression in PVAT positively correlates with expression of the endothelial vasoconstrictor peptide endothelin-1 (edn1). Conclusion: FHL2 regulates vascular function through enhancement of smooth muscle tone, regulation of adipokine secretion affecting insulin-stimulated ERK1/2 activation and Edn1 expression in endothelial cells. These finding provide a new mechanism for vascular dysfunction in type 2 diabetes. Disclosure J. Habibe: None. D. van Raalte: Consultant; Boehringer Ingelheim and Eli Lilly Alliance, AstraZeneca, Merck & Co., Inc. Research Support; Boehringer Ingelheim and Eli Lilly Alliance, AstraZeneca, Merck & Co., Inc. M. Nieuwdorp: Advisory Panel; caelus health. C. de Vries: None. E. C. Eringa: None. Funding Amsterdam Cardiovascular Sciences (ACS201703)

Heptadecanoic Acid Is Not a Key Mediator in the Prevention of Diet-Induced Hepatic Steatosis and Insulin Resistance in Mice.

Epidemiological studies found that the intake of dairy products is associated with an increased amount of circulating odd-chain fatty acids (OCFA, C15:0 and C17:0) in humans and further indicate that especially C17:0 is associated with a lower incidence of type 2 diabetes. However, causal relationships are not elucidated. To provide a mechanistic link, mice were fed high-fat (HF) diets supplemented with either milk fat or C17:0 for 20 weeks. Cultured primary mouse hepatocytes were used to distinguish differential effects mediated by C15:0 or C17:0. Despite an induction of OCFA after both dietary interventions, neither long-term milk fat intake nor C17:0 supplementation improved diet-induced hepatic lipid accumulation and insulin resistance in mice. HF feeding with milk fat actually deteriorates liver inflammation. Treatment of primary hepatocytes with C15:0 and C17:0 suppressed JAK2/STAT3 signaling, but only C15:0 enhanced insulin-stimulated phosphorylation of AKT. Overall, the data indicate that the intake of milk fat and C17:0 do not mediate health benefits, whereas C15:0 might be promising in further studies.

Chronic cortisol elevation restricts glucose uptake but not insulin responsiveness in zebrafish skeletal muscle

Although teleosts show an elevated insulin response to hyperglycemia, the circulating glucose levels are not normalized as rapidly as in mammals. While this may suggest a lack of target tissue insulin responsiveness, the underlying mechanisms are unclear. We investigated whether changes in skeletal muscle insulin sensitivity and glucose uptake underlie the cortisol-mediated elevated blood glucose levels. Adult zebrafish (Danio rerio) were exposed to water-borne cortisol for 3 days followed by an intraperitoneal injection of glucose with or without insulin. Cortisol treatment resulted in a temporal delay in the reduction in blood glucose levels, and this corresponded with a reduced glucose uptake capacity and lower glycogen content in the skeletal muscle. The transcript abundance of slc2a1b (which encodes for GLUT1b) and a suite of genes encoding enzymes involved in muscle glycogenesis and glycolysis were upregulated in the cortisol group. Both the control and cortisol groups showed higher whole body insulin expression in response to blood glucose elevation, which also resulted in enhanced insulin-stimulated phosphorylation of AKT in the skeletal muscle. The insulin-mediated phosphorylation of S6 kinase was lower in the cortisol group. Altogether, chronic cortisol stimulation restricts glucose uptake and enhances the glycolytic capacity without affecting insulin responsiveness in zebrafish skeletal muscle.

A role for β-catenin in diet-induced skeletal muscle insulin resistance.

A central characteristic of insulin resistance is the impaired ability for insulin to stimulate glucose uptake into skeletal muscle. While insulin resistance can occur distal to the canonical insulin receptor-PI3k-Akt signaling pathway, the signaling intermediates involved in the dysfunction are yet to be fully elucidated. β-catenin is an emerging distal regulator of skeletal muscle and adipocyte insulin-stimulated GLUT4 trafficking. Here, we investigate its role in skeletal muscle insulin resistance. Short-term (5-week) high-fat diet (HFD) decreased skeletal muscle β-catenin protein expression 27% (p= 0.03), and perturbed insulin-stimulated β-cateninS552 phosphorylation 21% (p= 0.009) without affecting insulin-stimulated Akt phosphorylation relative to chow-fed controls. Under chow conditions, mice with muscle-specific β-catenin deletion had impaired insulin responsiveness, whereas under HFD, both mice exhibited similar levels of insulin resistance (interaction effect of genotype × diet p< 0.05). Treatment of L6-GLUT4-myc myocytes with palmitate lower β-catenin protein expression by 75% (p= 0.02), and attenuated insulin-stimulated β-catenin phosphorylationS552 and actin remodeling (interaction effect of insulin × palmitate p< 0.05). Finally, β-cateninS552 phosphorylation was 45% lower in muscle biopsies from men with type 2 diabetes while total β-catenin expression was unchanged. These findings suggest that β-catenin dysfunction is associated with the development of insulin resistance.

BPA exposure in L6 myotubes increased basal glucose metabolism in an estrogen receptor-dependent manner but induced insulin resistance

Exposure to bisphenol A (BPA) is associated with insulin resistance and type 2 diabetes (T2D). Since muscle insulin resistance is the primary defect in T2D, we aimed to determine whether BPA alters glucose metabolism in L6 muscle cells. L6 or L6-GLUT4-myc cells were exposed to 1-104 nM BPA or the vehicle (0.1% DMSO) for 7 days. BPA at 103-104 nM significantly decreased the levels of the muscle differentiation markers troponin- T and myosin heavy chain 3. Insulin-stimulated phosphorylation of Akt and GSK3, insulin-stimulated glucose uptake, and insulin-stimulated GLUT4 translocation were significantly decreased with 103-104 nM BPA. Basal glucose uptake and glycolysis (extracellular acidification rates measured by Seahorse XFe96) were increased with 103-104 nM BPA. Levels of ROS detoxifying enzymes were increased with BPA >10nM, while catalase activity was increased with 103-104 nM BPA. However, BPA did not induce oxidative stress (measured by protein carbonylation and lipid peroxidation) nor mitochondrial dysfunction. The effects of BPA on basal glucose uptake and catalase activity, but not on insulin sensitivity, were restored when estrogen receptors (ERs) were inhibited with ICI. These findings suggest that high concentrations of BPA increase muscle glucose uptake through the ERs but induce insulin resistance through another pathway.

Pancreatic Cancer Cell-Conditioned, Human-Derived Primary Myotubes Display Increased Leucine Turnover, Increased Lipid Accumulation, and Reduced Glucose Uptake.

Metabolic alterations occurring in cancer cells have been seen to also occur in other tissues than cancerous tissue. For instance, cachexia, peripheral insulin resistance, or both are commonly seen in patients with cancer. We explored differences in substrate use in myotubes conditioned with the medium from a pancreatic cancer cell line, PANC-1, or primary human pancreatic cells, hPECs. Protein turnover was assessed using scintillation proximity assay, glucose and oleic acid handling were analyzed by substrate oxidation assay. We performed qPCR to study gene expression and immunoblotting and proteomic analyses to study protein expression. PANC-1-conditioned myotubes had an imbalance in protein turnover with decreased accumulation, increased decay, and decreased MYH2 gene expression. Glucose uptake decreased despite increased insulin-stimulated Akt phosphorylation. Fatty acid uptake increased, whereas fatty acid oxidation was unchanged, leading to accumulation of intracellular lipids (TAG) in PANC-1-conditioned myotubes. Secretome analyses revealed increased release of growth factors and growth factor receptor from PANC-1 cells, potentially affecting muscle cell metabolism. Myotubes exposed to pancreatic cancer cell medium displayed altered energy metabolism with increased protein/leucine turnover and lipid accumulation, while glucose uptake and oxidation reduced. This indicates production and release of substances from pancreatic cancer cells affecting skeletal muscle.

Curcumin improves insulin sensitivity in high-fat diet-fed mice through gut microbiota

BackgroundInsulin resistance precedes metabolic syndrome which increases the risk of type 2 diabetes and cardiovascular disease. However, there is a lack of safe and long-lasting methods for the prevention and treatment of insulin resistance. Gut microbiota dysbiosis can lead to insulin resistance and associated glucose and lipid metabolic dysfunction. Thus, the role of gut microbiota in metabolic diseases has garnered growing interest. Curcumin, the active ingredient of tropical plant Curcuma longa, has excellent prospects for the prevention and treatment of metabolic diseases. However, due to the extremely low bioavailability of curcumin, the mechanisms by which curcumin increases insulin sensitivity remains to be elucidated. This study aimed to elucidate the role of gut microbiota in mediating the effects of curcumin on improving insulin sensitivity in high-fat diet (HFD)-fed mice.MethodsGlucose, insulin, and pyruvate tolerance were tested and hepatic triglycerides (TGs) content was measured in HFD-fed mice treated with curcumin (100 mg kg−1 d−1, p.o.) or vehicle for 4 weeks and aforementioned mice after gut microbiota depletion via antibiotic treatment for 4 weeks. Fecal microbiota transplantation (FMT) was conducted in endogenous gut microbiota-depleted HFD-fed mice. Glucose and lipid metabolic phenotypes were also measured in recipient mice colonized microbiota from vehicle- or curcumin-treated HFD-fed mice. The mechanisms underlying the effects of curcumin on increasing insulin sensitivity were testified by Western blotting, real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA).ResultsCurcumin ameliorated HFD-induced glucose intolerance, insulin resistance, pyruvate intolerance, and hepatic TGs accumulation, while these effects were mediated by gut microbiota. Curcumin induced insulin-stimulated Akt phosphorylation levels in insulin-regulated peripheral tissues. The inhibitory effects of curcumin on the expressions of genes involved in hepatic gluconeogenesis and de novo lipogenesis were dependent on gut microbiota. Meanwhile, curcumin upregulated the expression of fibroblast growth factor 15 (FGF15) through gut microbiota.ConclusionsThe effects of curcumin on promoting insulin sensitivity were dependent on gut microbiota in HFD-fed mice. Moreover, curcumin at least partly exerted its effects on increasing insulin sensitivity via FGF15 upregulation. This study provided new ideas on nutritional manipulations of gut microbiota for the treatment of metabolic diseases.

Anti-diabetic effect of hesperidin on palmitate (PA)-treated HepG2 cells and high fat diet-induced obese mice

The present study examined the relationship between the anti-diabetic effect of hesperidin (HES) and the differential gene expression in HES treated high fat diet (HFD)-induced obese mice. Based on the glucose uptake assay, the treatment of HES restored the glucose uptake to control level in an insulin-independent manner in PA-treated HepG2 cells. Western blot analysis confirmed that the treatment of HES increased the insulin-stimulated phosphorylation of Akt and GSK3β in insulin-resistant PA-treated HepG2 cells. HFD-induced obese mice treated with HES significantly reduced serum insulin, blood glucose, and homeostatic model assessment for insulin resistance (HOMA-IR) values. In addition, both glucose tolerance and insulin tolerance were significantly improved to normal level by HES in HFD-induced obese mice. RNA sequencing analysis disclosed that the expression levels of up-regulated 12 genes and down-regulated 6 genes related to insulin signaling and glucose metabolism were restored to normal level by HES in the liver of HFD-induced obese mice. A protein–protein interaction (PPI) network was constructed via search tool for the retrieval of interacting genes/proteins (STRING) analysis, and Eno1, Pik3cd, Hk2, Trib3, Myc, Nos3, Ppargc1a, and Igf2 were located in the functional hubs of the PPI network of glucose metabolism. Furthermore, Western blot analysis confirmed that HES improved insulin sensitivity and glucose homeostasis by normalizing the expression levels of hexokinase-II, enolase-1, and PI3 kinase p110δ to normal level. The overall results suggest that HES possess a potential anti-diabetic effect by normalizing the expression levels of the insulin signaling and glucose metabolism related genes which were perturbed in the liver of HFD-induced obese mice.

DCAF7 regulates cell proliferation through IRS1-FOXO1 signaling

SummaryCell proliferation is dependent on growth factors insulin and IGF1. We sought to identify interactors of IRS1, the most proximal mediator of insulin/IGF1 signaling, that regulate cell proliferation. Using proximity-dependent biotin identification (BioID), we detected 40 proteins displaying proximal interactions with IRS1, including DCAF7 and its interacting partners DYRK1A and DYRK1B. In HepG2 cells, DCAF7 knockdown attenuated cell proliferation by inducing cell cycle arrest at G2. DCAF7 expression was required for insulin-stimulated AKT phosphorylation, and its absence promoted nuclear localization of the transcription factor FOXO1. DCAF7 knockdown induced expression of FOXO1-target genes implicated in G2 cell cycle inhibition, correlating with G2 cell cycle arrest. In Drosophila melanogaster, wing-specific knockdown of DCAF7/wap caused smaller wing size and lower wing cell number; the latter recovered upon double knockdown of wap and dfoxo. We propose that DCAF7 regulates cell proliferation and cell cycle via IRS1-FOXO1 signaling, of relevance to whole organism growth.

Cellular senescence in hepatocytes contributes to metabolic disturbances in NASH.

Cellular senescence is a state of irreversible cell cycle arrest and has been shown to play a key role in many diseases, including metabolic diseases. To investigate the potential contribution of hepatocyte cellular senescence to the metabolic derangements associated with non-alcoholic steatohepatitis (NASH), we treated human hepatocyte cell lines HepG2 and IHH with the senescence-inducing drugs nutlin-3a, doxorubicin and etoposide. The senescence-associated markers p16, p21, p53 and beta galactosidase were induced upon drug treatment, and this was associated with increased lipid storage, increased expression of lipid transporters and the development of hepatic steatosis. Drug-induced senescence also led to increased glycogen content, and increased VLDL secretion from hepatocytes. Senescence was also associated with an increase in glucose and fatty acid oxidation capacity, while de novo lipogenesis was decreased. Surprisingly, cellular senescence caused an overall increase in insulin signaling in hepatocytes, with increased insulin-stimulated phosphorylation of IR, Akt, and MAPK. Together, these data indicate that hepatic senescence plays a causal role in the development of NASH pathogenesis, by modulating glucose and lipid metabolism, favoring steatosis. Our findings contribute to a better understanding of the mechanisms linking cellular senescence and fatty liver disease and support the development of new therapies targeting senescent cells for the treatment of NASH.

Derhamnosylmaysin Inhibits Adipogenesis via Inhibiting Expression of PPARγ and C/EBPα in 3T3-L1 Cells.

We investigated the effects of derhamnosylmaysin (DM) on adipogenesis and lipid accumulation in 3T3-L1 adipocytes. Our data showed that DM inhibited lipid accumulation and adipocyte differentiation in 3T3-L1 cells. Treatment of 3T3-L1 adipocytes with DM decreased the expression of major transcription factors, such as sterol regulatory element-binding protein-1c (SREBP-1c), the CCAAT-enhancer-binding protein (CEBP) family, and peroxisome proliferator-activated receptor gamma (PPARγ), in the regulation of adipocyte differentiation. Moreover, the expression of their downstream target genes related to adipogenesis and lipogenesis, including adipocyte fatty acid-binding protein (aP2), lipoprotein lipase (LPL), stearyl-CoA-desaturase-1 (SCD-1), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS), was also decreased by treatment with DM during adipogenesis. Additionally, DM attenuated insulin-stimulated phosphorylation of Akt. These results first demonstrated that DM inhibited adipogenesis and lipogenesis through downregulation of the key adipogenic transcription factors SREBP-1c, the CEBP family, and PPARγ and inactivation of the major adipogenesis signaling factor Akt, which is intermediated in insulin. These studies demonstrated that DM is a new bioactive compound for antiadipogenic reagents for controlling overweight and obesity.

Cixutumumab reveals a critical role for IGF-1 in adipose and hepatic tissue remodelling during the development of diet-induced obesity

ABSTRACT High fat diet (HFD)-induced obesity leads to perturbation in the storage function of white adipose tissue (WAT) resulting in deposition of lipids in tissues ill-equipped to deal with this challenge. The role of insulin like growth factor-1 (IGF-1) in the systemic and organ-specific responses to HFD is unclear. Using cixutumumab, a monoclonal antibody that internalizes and degrades cell surface IGF-1 receptors (IGF-1 R), leaving insulin receptor expression unchanged we aimed to establish the role of IGF-1 R in the response to a HFD. Mice treated with cixutumumab fed standard chow developed mild hyperinsulinemia with no change in WAT. When challenged by HFD mice treated with cixutumumab had reduced weight gain, reduced WAT expansion, and reduced hepatic lipid vacuole formation. In HFD-fed mice, cixutumumab led to reduced levels of genes encoding proteins important in fatty acid metabolism in WAT and liver. Cixutumumab protected against blunting of insulin-stimulated phosphorylation of Akt in liver of HFD fed mice. These data reveal an important role for IGF-1 R in the WAT and hepatic response to short-term nutrient excess. IGF-1 R inhibition during HFD leads to a lipodystrophic phenotype with a failure of WAT lipid storage and protection from HFD-induced hepatic insulin resistance.

Adipose tissue LECT2 expression is associated with obesity and insulin resistance in Korean women.

Leukocyte cell-derived chemotaxin 2 (LECT2) is an obesity-upregulated hepatokine inducing skeletal muscle insulin resistance. The study's aim was to explore whether LECT2 is expressed in human adipose tissue and whether the expression is dysregulated during obesity and associated with obesity-related metabolic disorders. This study measured metabolic parameters, serum LECT2, and expression of LECT2 and CD209, a gene encoding a putative receptor for LECT2, in abdominal subcutaneous and visceral adipose tissues in women with obesity (with or without type 2 diabetes) and women with normal weight. The expression/secretion of LECT2 and its putative effects were assessed in human adipocytes. Adipose tissue LECT2 mRNA and serum LECT2 were higher in women with obesity and were significantly correlated with parameters related to insulin resistance. LECT2 was mainly expressed by adipocytes. Both LECT2 and CD209 expression was higher in adipocytes from women with obesity. Incubating adipocytes with substances mimicking the microenvironment of obesity adipose tissue increased LECT2 expression/secretion. LECT2 treatment of adipocytes suppressed insulin-stimulated Akt phosphorylation; it reduced adiponectin (ADIPOQ) and increased leptin (LEP) expression in a CD209-dependent manner. This study demonstrates that LECT2 expression in adipose tissue is high in patients with obesity and associated with insulin resistance and suggests that adipocyte-derived LECT2 may contribute to adipose tissue dysfunction.

Skeletal muscle RUNX1 is related to insulin sensitivity through its effect on myogenic potential.

Skeletal muscle is the major site of insulin action. There are limited data on the relationship between insulin action and skeletal muscle myogenic/regenerative potential. RUNX1 is a transcription factor which plays a role in muscle development and regeneration. The aim of our study was to assess the role of skeletal muscle myogenic/regenerative potential in the development of insulin resistance through the studies on RUNX1 transcription factor. This study is a cross-sectional study. Experimental part with myoblast cell line culture. We examined 41 young healthy volunteers, 21 normal weight and 20 with overweight or obesity. Hyperinsulinemic-euglycemic clamp and vastus lateralis muscle biopsy were performed. In L6 myoblast and human skeletal muscle myoblasts (hSkMM) cell cultures, RUNX1 was silenced at two stages of development. Cell growth, the expression of markers of myogenesis, nuclei fusion index, Akt phosphorylation and glucose uptake were measured. Skeletal muscle RUNX1 expression was decreased in overweight/obese individuals in comparison with normal-weight individuals and was positively related to insulin sensitivity, independently of BMI. Runx1 loss-of-function at the stage of myoblast inhibited myoblast proliferation and differentiation and reduced insulin-stimulated Akt phosphorylation and insulin-stimulated glucose uptake. In contrast, Runx1 knockdown in myotubes did not affect Akt phosphorylation, glucose uptake and other parameters studied. Myogenic/regenerative potential of adult skeletal muscle may be an important determinant of insulin action. Our data suggest that muscle RUNX1 may play a role in the modulation of insulin action through its effect on myogenesis.

839-P: Pharmacological Inhibition of Mammalian INDY Ameliorates Western Diet–Induced NASH in Mice: Possible Implication of Fgf21-AMPK Signaling

Background: Recent data proposed a role of the citrate transporter INDY (I’m Not Dead Yet) in the development of obesity, insulin resistance and NAFLD suggesting its potential as a therapeutic target for metabolic-related disorders. The aim of our study is to determine if mammalian INDY (mINDY) inhibitors can be introduced as a new therapeutic option for NAFLD/NASH. Methods: Six-week-old C57Bl/6N mice were fed a western diet (WD) for 18 weeks to induce NASH. After 12 weeks of feeding, mice received mINDY inhibitor (PF-06649298, mINDYi; 100 mg/kg) or vehicle bid until end of week 18. During the treatment period, body weight and composition were monitored. ipGTT and FACS analysis of liver inflammatory cells were performed. Results: mINDYi-treated mice exhibited lower body weight, fat mass and higher lean mass compared to vehicle-treated mice. Results from ipGTT revealed improved glucose tolerance and insulin sensitivity. The latter was further confirmed through increased insulin-stimulated Akt phosphorylation in liver, gonadal WAT and BAT. Treatment with mINDYi attenuated WD-induced hepatic injury, steatosis and inflammation as shown histologically and biochemically by reduction in plasma ALT and AST, decrease in hepatic triglyceride accumulation and reduced hepatic lymphoid and myeloid immune cell populations. Interestingly, these effects were associated with a significant rise in plasma and hepatic Fgf21 together with increased hepatic phosphorylation of LKB1 and AMPK and decreased mTOR. Results from primary mouse hepatocytes revealed that mINDYi-induction of Fgf21 is partially dependent on AMPK activation. Conclusion: Our study shows for the first time that mINDYi attenuates diet-induced steatohepatitis; offering a promising novel treatment strategy for NAFLD. Further investigations are currently ongoing to elucidate the molecular mechanisms underlying mINDY inhibition and whether it is dependent on Fgf21-AMPK signaling. Disclosure N.El-agroudy: None. G.Zahn: Employee; Eternygen GmbH. C.Herrmann: None. G.Mingrone: Advisory Panel; Fractyl Health, Inc., Novo Nordisk, Consultant; ReCor Medical, Inc. T.C.Alves: None. A.L.Birkenfeld: None.

ALY688 elicits adiponectin-mimetic signaling and improves insulin action in skeletal muscle cells

Adiponectin is well established to mediate many beneficial metabolic effects, and this has stimulated great interest in development and validation of adiponectin receptor agonists as pharmaceutical tools. This study investigated the effects of ALY688, a peptide-based adiponectin receptor agonist, in rat L6 skeletal muscle cells. ALY688 significantly increased phosphorylation of several adiponectin downstream effectors, including AMPK, ACC, and p38MAPK, assessed by immunoblotting and immunofluorescence microscopy. Temporal analysis using cells expressing an Akt biosensor demonstrated that ALY688 enhanced insulin sensitivity. This effect was associated with increased insulin-stimulated Akt and IRS-1 phosphorylation. The functional metabolic significance of these signaling effects was examined by measuring glucose uptake in myoblasts stably overexpressing the glucose transporter GLUT4. ALY688 treatment increased basal glucose uptake and enhanced insulin-stimulated glucose uptake. In the model of high-glucose/high-insulin (HGHI)-induced insulin-resistant cells, both temporal studies using the Akt biosensor as well as immunoblotting to assess Akt and IRS-1 phosphorylation indicated that ALY688 significantly reduced insulin resistance. Importantly, we observed that ALY688 administration to high-fat high-sucrose-fed mice also improves glucose handling, validating its efficacy in vivo. In summary, these data indicate that ALY688 activates adiponectin signaling pathways in skeletal muscle, leading to improved insulin sensitivity and beneficial metabolic effects.

Coffee Bioactive N-Methylpyridinium Attenuates Tumor Necrosis Factor (TNF)-α-Mediated Insulin Resistance and Inflammation in Human Adipocytes.

Although coffee consumption has been historically associated with negative health outcomes, recent evidence suggests a lower risk of metabolic syndrome, obesity and diabetes among regular coffee drinkers. Among the plethora of minor organic compounds assessed as potential mediators of coffee health benefits, trigonelline and its pyrolysis product N-methylpyridinium (NMP) were preliminary shown to promote glucose uptake and exert anti-adipogenic properties. Against this background, we aimed at characterizing the effects of trigonelline and NMP in inflamed and dysfunctional human adipocytes. Human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes were treated with NMP or, for comparison, trigonelline, for 5 h before stimulation with tumor necrosis factor (TNF)-α. NMP at concentrations as low as 1 µmol/L reduced the stimulated expression of several pro-inflammatory mediators, including C-C Motif chemokine ligand (CCL)-2, C-X-C Motif chemokine ligand (CXCL)-10, and intercellular adhesion Molecule (ICAM)-1, but left the induction of prostaglandin G/H synthase (PTGS)2, interleukin (IL)-1β, and colony stimulating factor (CSF)1 unaffected. Furthermore, NMP restored the downregulated expression of adiponectin (ADIPOQ). These effects were functionally associated with downregulation of the adhesion of monocytes to inflamed adipocytes. Under the same conditions, NMP also reversed the TNF-α-mediated suppression of insulin-stimulated Ser473 Akt phosphorylation and attenuated the induction of TNF-α-stimulated lipolysis restoring cell fat content. In an attempt to preliminarily explore the underlying mechanisms of its action, we show that NMP restores the expression of the master regulator of adipocyte differentiation peroxisome proliferator-activated receptor (PPAR)γ and downregulates activation of the pro-inflammatory mitogen-activated protein jun N-terminal kinase (JNK). In conclusion, NMP reduces adipose dysfunction in pro-inflammatory activated adipocytes. These data suggest that bioactive NMP in coffee may improve the inflammatory and dysmetabolic milieu associated with obesity.