Insulin-stimulated 2-deoxyglucose Research Articles

Effect of Exogenous Hydrogen Sulfide and Polysulfide Donors on Insulin Sensitivity of the Adipose Tissue.

Hydrogen sulfide (H2S) and inorganic polysulfides are important signaling molecules; however, little is known about their role in adipose tissue. We examined the effect of H2S and polysulfides on insulin sensitivity of the adipose tissue in rats. Plasma glucose, insulin, non-esterified fatty acids, and glycerol were measured after administration of H2S and the polysulfide donors, Na2S and Na2S4, respectively. In addition, the effect of Na2S and Na2S4 on insulin-induced glucose uptake and inhibition of lipolysis was studied in adipose tissue explants ex vivo. Na2S and Na2S4 administered in vivo at a single dose of 100 μmol/kg had no effect on plasma glucose and insulin concentrations. In addition, Na2S and Na2S4 did not modify the effect of insulin on plasma glucose, fatty acids, and glycerol concentrations. Na2S and Na2S4had no effect on the antilipolytic effect of insulin in adipose tissue explants ex vivo. The effect of insulin on 2-deoxyglucose uptake by adipose tissue was impaired in obese rats which was accompanied by lower insulin-induced tyrosine phosphorylation of IRS-1 and Akt. Na2S4, but not Na2S, improved insulin signaling and increased insulin-stimulated 2-deoxyglucose uptake by adipose tissue of obese rats. The results suggest that polysulfides may normalize insulin sensitivity, at least in the adipose tissue, in obesity/metabolic syndrome.

Colchicine treatment impairs skeletal muscle mitochondrial function and insulin sensitivity in an age-specific manner.

The aim of the study was to investigate the impact of autophagy inhibition on skeletal muscle mitochondrial function and glucose homeostasis in young and aged mice. The transcriptional co-activator PGC-1α regulates muscle oxidative phenotype which has been shown to be linked with basal autophagic capacity. Therefore, young and aged inducible muscle-specific PGC-1α knockout (iMKO) mice and littermate lox/lox controls were used in three separate experiments performed after either saline or colchicine injections on two consecutive days: (1) Euthanization in the basal state obtaining skeletal muscle for mitochondrial respirometry, (2) whole body glucose tolerance test, and (3) in vivo insulin-stimulated 2-deoxyglucose (2-DG) uptake into skeletal muscle. Muscle PGC-1α was not required for maintaining basal autophagy flux, regardless of age. Colchicine-induced inhibition of autophagy was associated with impairments of skeletal muscle mitochondrial function, including reduced ADP sensitivity and altered mitochondrial redox balance in both young and aged mice. Colchicine treatment reduced the glucose tolerance in aged, but not young mice, and similarly in iMKO and lox/lox mice. Colchicine reduced insulin-stimulated 2-DG uptake in soleus muscle in aged mice, independently of PGC-1α, and without affecting insulin-regulated phosphorylation of proximal or distal mediators of insulin signaling. In conclusion, the results indicate that autophagy regulates the mitochondrial ADP sensitivity and redox balance as well as whole body glucose tolerance and skeletal muscle insulin sensitivity in aged mice, with no additional effects of inducible PGC-1α deletion.

DNA methylation status influences insulin-induced glucose transport in 3T3-L1 adipocytes by mediating p53 expression

Researchers frequently use 3T3-L1 adipocytes as a fat cell line, but the capacity of this line for insulin-mediated glucose transport is lower than that of primary isolated fat cells. In this study, we found that 5-azacytidine (5-aza-C), DNA methyltransferase 1 inhibitor, enhanced insulin-stimulated 2-deoxyglucose (2-DG) transport in 3T3-L1 cells after adipogenic differentiation. We next examined the expression of the genes related to glucose transport and insulin signal transduction. The insulin independent glucose transporter, glucose transporter 1 (GLUT1), showed lower expression in 5-aza-C pre-treated 3T3-L1 adipocytes, than in un-treated control adipocytes, while the expression of insulin dependent transporter GLUT4 remained unchanged. In addition, insulin receptor substrate-1 (IRS-1) was highly expressed in 5-aza-C pre-treated adipocytes. Based on DNA microarray and functional annotation analysis, we noticed that 5-aza-C pretreatment activated the tumor suppressor p53 pathway. We confirmed that in 5-aza-C adipocytes, p53 expression was markedly higher, and the methylation level of CpGs in its promoter region was lower than in un-treated control adipocytes. Moreover, pharmacological inhibition of p53 restored GLUT1 and IRS-1 expression to the same level as in un-treated 3T3-L1 adipocytes, and significantly decreased insulin-mediated 2-DG uptake. These results suggest that glucose transport capacity in adipocytes is influenced by DNA methylation status, and demethylation induced by 5-aza-C increased it possibly through the p53 signaling pathway.

Adipocytes initiate an adipose-cerebral-peripheral sympathetic reflex to induce insulin resistance during high-fat feeding.

The underlying mechanism by which amassing of white adipose tissue in obesity regulates sympathetic nerve system (SNS) drive to the tissues responsible for glucose disposal, and causes insulin resistance (IR), remains unknown. We tested the hypothesis that high-fat (HF) feeding increases afferent impulses from white adipose tissue that reflexively elevate efferent nerve activity to skeletal muscle (SM) and adipose tissue to impair their local glucose uptake. We also investigated how salt-intake can enhance IR. HF-fed rats received a normal salt (0.4%) or high salt (4%) diet for 3 weeks. High-salt intake in HF fed rats decreased insulin-stimulated 2-deoxyglucose uptake by over 30% in white adipose tissue and SM, exacerbated inflammation, and impaired their insulin signaling and glucose transporter 4 (Glut4) trafficking. Dietary salt in HF fed rats also increased the activity of the adipose-cerebral-muscle renin-angiotensin system (RAS) axes, SNS, and reactive oxygen species (ROS). Insulin sensitivity was reduced by 32% in HF rats during high-salt intake, but was improved by over 62% by interruption of central RAS and SNS drive, and by over 45% by denervation or deafferentation of epididymal fat (all P<0.05). Our study suggest that a HF diet engages a sympathetic reflex from the white adipose tissue that activates adipose-cerebral-muscle RAS/ROS axes and coordinates a reduction in peripheral glucose uptake. These are all enhanced by salt-loading. These findings provide new insight into the role of a reflex initiated in adipose tissue in the regulation of glucose homeostasis during HF feeding that could lead to new therapeutic approaches to IR.

OR31-4 Neonatal Vitamin D Deficiency and Insulin Resistance

Background: Vitamin D deficiency is prevalent in pregnant women and their offspring. Maternal vitamin D deficiency is associated with insulin resistance in children and can induce a proinflammatory macrophage phenotype [1, 2]. Purpose: We hypothesized that maternal vitamin D deficiency induces immune cell reprogramming that promotes adipose insulin resistance in the offspring and is detectable in the newborn period. Methods: We enrolled 32 normal pregnant women prior to delivery of AGA, term infants. Cord blood was obtained and used to determine vitamin D [25(OH)D] levels and qPCR of microRNA levels. Cord blood monocytes were co-cultured with 3T3L1 adipocytes for insulin-stimulated 2-deoxyglucose uptake studies, and the monocyte media and co-cultured adipocytes were analyzed for microRNA expression. 3T3L1 adipocytes were transfected with miR106b mimic and antagonist to confirm the effect of the 2-deoxyglucose results. Results: 83% of the newborn’s venous cord blood showed vitamin D deficiency with 25(OH)D <20 ng/mL (n=24, total n=29). Adipocytes co-cultured with cord blood monocytes showed an inverse correlation between insulin stimulated 2-deoxyglucose uptake and cord blood 25(OH)D levels (n=29, Spearman r=0.43, p=0.02). Adipocytes exposed to monocytes or to monocyte media elevated miR106b by 7 to 12-fold (n=8, p<0.01). Interestingly, cord blood plasma miR106b levels were found to inversely correlate with 25(OH)D levels (n=27, Spearman r=-0.41, p<0.04). Transfection of adipocytes with miR106b mimic reduced the 2-deoxyglucose uptake after insulin stimulation, whereas transfection with anti-miR-106b increased 2-deoxyglucose uptake after insulin stimulation, suggesting that monocyte secretion of miR106b induced adipose insulin resistance (n=8, p<0.05). Conclusions: This study provides evidence of a novel intercellular miRNA communication mechanism enabling monocytes programmed in utero by vitamin D deficiency to induce adipose insulin resistance. Identification of circulating miR106b as a mechanism of insulin resistance in offspring born with vitamin D deficiency is a potential advance toward precision medicine as it allows for optimal targeting of those at risk for diabetes later in life within a heterogeneous population. These findings serve as background for interventional studies evaluating the effect of maternal vitamin D supplementation on offspring insulin resistance later in life.

A renal-cerebral-peripheral sympathetic reflex mediates insulin resistance in chronic kidney disease.

BackgroundInsulin resistance (IR) complicates chronic kidney disease (CKD). We tested the hypothesis that CKD activates a broad reflex response from the kidneys and the white adipose tissue to impair peripheral glucose uptake and investigated the role of salt intake in this process.Methods5/6-nephrectomized rats were administered normal- or high-salt for 3 weeks. Conclusions were tested in 100 non-diabetic patients with stage 3–5 CKD.FindingsHigh-salt in 5/6-nephrectomized rats decreased insulin-stimulated 2-deoxyglucose uptake >25% via a sympathetic nervous system (SNS) reflex that linked the IR to reactive oxygen species (ROS) and the renin-angiotensin system (RAS) in brain and peripheral tissues. Salt-loading in CKD enhanced inflammation in adipose tissue and skeletal muscle, and enhanced the impairment of insulin signaling and Glut4 trafficking. Denervation of the kidneys or adipose tissue or deafferentation of adipose tissue improved IR >40%. In patients with non-diabetic CKD, IR was positively correlated with salt intake after controlling for cofounders (r = 0.334, P = 0.001) and was linked to activation of the RAS/SNS and to impaired glucose uptake in adipose tissue and skeletal muscle, all of which depended on salt intake.InterpretationCKD engages a renal/adipose-cerebral-peripheral sympathetic reflex that activates the RAS/ROS axes to promote IR via local inflammation and impaired Glut4 trafficking that are enhanced by high-salt intake. The findings point to a role for blockade of RAS or α-and-β-adrenergic receptors to reduce IR in patients with CKD.FundNational Natural Science Foundation of China.

Immobilization rapidly induces thioredoxin-interacting protein gene expression together with insulin resistance in rat skeletal muscle.

Acute short duration of disuse induces the development of insulin resistance for glucose uptake in rodent skeletal muscle. Because thioredoxin-interacting protein (TXNIP) has been implicated in the downregulation of insulin signaling and glucose uptake, we examined the possibility that muscle disuse rapidly induces insulin resistance via increased TXNIP mRNA and protein expression. Male Wistar rats were subjected to unilateral 6-h hindlimb immobilization by plaster cast. At the end of this period, the soleus muscles from both immobilized and contralateral nonimmobilized hindlimbs were excised and examined. The 6-h immobilization resulted in an increase in TXNIP mRNA and protein expressions together with a decrease in insulin-stimulated 2-deoxyglucose uptake in the rat soleus muscle. Additionally, in the rats euthanized 6 h after the plaster cast removal, TXNIP protein expression and insulin-stimulated glucose uptake in the immobilized muscle had both been restored to a normal level. Various interventions (pretreatment with transcription inhibitor actinomycin D or AMP-dependent protein kinase activator 5-aminoimidazole-4-carboxamide ribonucleotide) also suppressed the increase in TXNIP protein expression in 6-h-immobilized muscle together with partial prevention of insulin resistance for glucose uptake. These results suggested the possibility that increased TXNIP protein expression in immobilized rat soleus muscles was associated with the rapid induction of insulin resistance for glucose uptake in that tissue. NEW & NOTEWORTHY The cellular mechanism by which disuse rapidly induces muscle insulin resistance for glucose uptake remains to be identified. Using a rat hindlimb immobilization model, our findings suggest the possibility that transcriptional upregulation of thioredoxin-interacting protein is associated with the immobilization-induced rapid development of insulin resistance in skeletal muscle.

The transcriptional response to oxidative stress is part of, but not sufficient for, insulin resistance in adipocytes

Insulin resistance is a major risk factor for metabolic diseases such as Type 2 diabetes. Although the underlying mechanisms of insulin resistance remain elusive, oxidative stress is a unifying driver by which numerous extrinsic signals and cellular stresses trigger insulin resistance. Consequently, we sought to understand the cellular response to oxidative stress and its role in insulin resistance. Using cultured 3T3-L1 adipocytes, we established a model of physiologically-derived oxidative stress by inhibiting the cycling of glutathione and thioredoxin, which induced insulin resistance as measured by impaired insulin-stimulated 2-deoxyglucose uptake. Using time-resolved transcriptomics, we found > 2000 genes differentially-expressed over 24 hours, with specific metabolic and signalling pathways enriched at different times. We explored this coordination using a knowledge-based hierarchical-clustering approach to generate a temporal transcriptional cascade and identify key transcription factors responding to oxidative stress. This response shared many similarities with changes observed in distinct insulin resistance models. However, an anti-oxidant reversed insulin resistance phenotypically but not transcriptionally, implying that the transcriptional response to oxidative stress is insufficient for insulin resistance. This suggests that the primary site by which oxidative stress impairs insulin action occurs post-transcriptionally, warranting a multi-level ‘trans-omic’ approach when studying time-resolved responses to cellular perturbations.

Pharmacological Mechanisms of Boiogito and Bofutsushosan in Diabetes and Obesity Models

The antihyperglycemic activities of extracts of boiogito (BOT) and bofutsushosan (BTS) were investigated in streptozotocin-induced (STZ)-diabetic mice. BOT extract containing Stephania tetrandra S. MOORE root (stephania), has more potent antihyperglycemic activity than BOT extract containing sinomenium stem (sinomenium). Extracts of stephania and astragalus root (astragalus) exert combined effects in the antihyperglycemic and insulinotropic activities of BOT extract. Fangchinoline, but not tetrandrine, in stephania plays a role in its activity. Formononetin in astragalus potentiates the actions of fangchinoline. Tetrandrine has antiangiogenic effects on choroidal vessels in STZ-diabetic rats, which are associated with the inhibition of tumor necrosis factor (TNF)-α-induced nuclear factor (NF)-κB activation. BTS extract has shown antihyperglycemic and insulinotropic activities whereas gardenia fruit (gardenia) extract in BTS has antihyperglycemic, but not insulinotropic, activity in the diabetic mice. Gardenia extract decreased the HOMA-IR level and increased insulin-stimulated 2-deoxyglucose (2-DG) uptake to skeletal muscle. The effects of gardenia extract on 2-DG uptake were associated with the upregulation of glucose transporter type 4 and Akt phosphorylation. Gardenia extract was also shown to have antihyperglycemic and insulinotropic actions in high-fat diet (HFD)-fed and STZ-diabetic mice. In addition, gardenia extract decreased the production of TNF-α and leptin, and increased the production of adiponectin in the visceral adipose tissues. In the early administration period, BTS extract increased mRNA expression levels of leptin, adiponectin, and UCP1 in brown adipose tissues in HFD-fed obese mice. With a longer duration of administration, BTS extract improved insulin resistance and subsequently reduced serum leptin and triglyceride levels in parallel with visceral adipose tissue volume and size.

Rac1 in Muscle Is Dispensable for Improved Insulin Action After Exercise in Mice.

Exercise has a potent insulin-sensitivity enhancing effect on skeletal muscle, but the intracellular mechanisms that mediate this effect are not well understood. In muscle, Ras-related C3 botulinum toxin substrate 1 (Rac1) regulates both insulin- and contraction-stimulated glucose transport and is dysregulated in insulin resistant muscle. However, whether Rac1 is involved in mediating enhanced insulin sensitivity after an acute bout of exercise is unresolved. To address this question, we investigated after exercise whole-body (insulin tolerance test) as well as muscle (insulin-stimulated 2-deoxyglucose transport in isolated soleus muscle) insulin sensitivity in inducible muscle-specific Rac1 knockout (mKO) and wild-type (WT) littermate mice. Previous exercise enhanced whole-body insulin sensitivity by 40% in WT mice and rescued the insulin intolerance in Rac1 mKO mice by improving whole-body insulin sensitivity by 230%. In agreement, previous exercise significantly improved insulin sensitivity by 20% in WT and by 40% in Rac1 mKO soleus muscles. These findings suggest that muscle Rac1 is dispensable for the insulin sensitizing effect of exercise. Moreover, insulin resistance in Rac1 mKO mice can be completely normalized by previous exercise explaining why insulin resistant patients can increase insulin action with exercise despite dysfunctional Rac1 activity in muscle.

Exercise-induced increase in IL-6 level enhances GLUT4 expression and insulin sensitivity in mouse skeletal muscle

A single bout of exercise is known to increase the insulin sensitivity of skeletal muscle; however, the underlying mechanism of this phenomenon is not fully understood. Because a single bout of exercise induces a transient increase in blood interleukin-6 (IL-6) level, we hypothesized that the enhancement of insulin sensitivity after a single bout of exercise in skeletal muscle is mediated at least in part through IL-6-dependent mechanisms. To test this hypothesis, C57BL6J mice were intravenously injected with normal IgG or an IL-6 neutralizing antibody before exercise. Twenty-four hours after a single bout of exercise, the plantaris muscle was harvested to measure insulin sensitivity and glucose transporter (GLUT)-4 expression levels by ex-vivo insulin-stimulated 2-deoxyglucose (2-DG) uptake and Western blotting, respectively. Compared with sedentary mice, mice that performed exercise showed enhanced IL-6 concentration, insulin-stimulated 2-DG uptake, and GLUT-4 expression in the plantaris muscle. The enhanced insulin sensitivity and GLUT4 expression were canceled by injection of the IL-6 neutralizing antibody before exercise. In addition, IL-6 injection increased GLUT4 expression, both in the plantaris muscle and the soleus muscle in C57BL6J mice. Furthermore, a short period of incubation with IL-6 increased GLUT4 expression in differentiated C2C12 myotubes. In summary, these results suggested that IL-6 increased GLUT4 expression in muscle and that this phenomenon may play a role in the post-exercise enhancement of insulin sensitivity in skeletal muscle.

Exercise-Induced Transient Increase in IL-6 Stimulates GLUT4 Expression and Enhances Insulin Sensitivity in Mouse Skeletal Muscle

A single bout of exercise induces transient increase in blood interleukin-6 (IL-6) level in human and rodents, however, the role of exercise-induced IL-6 is poorly understand. Prolonged, chronic increase in IL-6 reflects low-grade inflammation, which decrease insulin sensitivity in adipose tissue, liver and skeletal muscle. On the other hand, acute, short-period of IL-6 enhances insulin sensitivity. Because, the increase in IL-6 after exercise is transient, we hypothesized that transient increase in IL-6 after exercise enhances insulin sensitivity in skeletal muscle. C57BL6J mouse were i.v. injected normal IgG or IL-6 antibody before exercise. Twenty-four hours after a single bout of exercise (treadmill running: 20 m/min, 90 min with 10 degree incline), plantaris muscle was harvested and incubated in oxygenized KRB buffer to measure insulin-stimulated 2-deoxyglucose (2-DG) uptake. Compared with sedentary mouse, insulin-stimulated 2-DG uptake in plantaris muscle was increased 24 h after exercise in IgG-injected mouse, however, the increase induced by exercise was not observed in IL-6 antibody-injected mouse. Concomitant with this results, GLUT4 expression was increased 24 h after exercise in IgG-injected mouse, the increase was canceled in IL-6 antibody-injected mouse. Recombinant mouse IL-6 injection increased GLUT4 expression both fast-twitch plantaris muscle and slow-twitch soleus muscle in C57BL6J mouse. Furthermore, short period incubation of IL-6 (3-12 hours) increased GLUT4 expression in differentiated C2C12 myotubes, however long period (24 h) did not. These results suggests that exercise-induced transient increase in IL-6 affects skeletal muscle in autocrine/paracrine manner, which enhances GLUT4 expression leading to increase insulin sensitivity in skeletal muscle.

Tctex1d2 Is a Negative Regulator of GLUT4 Translocation and Glucose Uptake.

Tctex1d2 (Tctex1 domain containing 2) is an open reading frame that encodes for a functionally unknown protein that contains a Tctex1 domain found in dynein light chain family members. Examination of gene expression during adipogenesis demonstrated a marked increase in Tctex1d2 protein expression that was essentially undetectable in preadipocytes and markedly induced during 3T3-L1 adipocyte differentiation. Tctex1d2 overexpression significantly inhibited insulin-stimulated glucose transporter 4 (GLUT4) translocation and 2-deoxyglucose uptake. In contrast, Tctex1d2 knockdown significantly increased insulin-stimulated GLUT4 translocation and 2-deoxyglucose uptake. However, acute insulin stimulation (up to 30 min) in 3T3-L1 adipocytes with overexpression or knockdown of Tctex1d2 had no effect on Akt phosphorylation, a critical signal transduction target required for GLUT4 translocation. Although overexpression of Tctex1d2 had no significant effect on GLUT4 internalization, Tctex1d2 was found to associate with syntaxin 4 in an insulin-dependent manner and inhibit Doc2b binding to syntaxin 4. In addition, glucose-dependent insulinotropic polypeptide rescued the Tctex1d2 inhibition of insulin-stimulated GLUT4 translocation by suppressing the Tctex1d2-syntaxin 4 interaction and increasing Doc2b-Synatxin4 interactions. Taking these results together, we hypothesized that Tctex1d2 is a novel syntaxin 4 binding protein that functions as a negative regulator of GLUT4 plasma membrane translocation through inhibition of the Doc2b-syntaxin 4 interaction.

Neonatal streptozotocin-induced diabetes in mothers promotes metabolic programming of adipose tissue in male rat offspring.

Maternal hyperglycemia during pregnancy can lead to fetal changes, like macrosomia or obesity in adultlife. Experimentalmodels of diabetes have been studied to evaluate the consequences of offspring lipidmetabolism. This study aimed to investigate the metabolic changes in adipose tissue of offspring of streptozotocininduced diabetic mothers during neonatal period. Diabetes was induced in female rats by streptozotocin administration on 5th day of life. In adulthood, female rats were bred with control male rats. Male puppies were sacrificed on 12th week of life and epididymal (EP) and subcutaneous (SC) adipose fat pads were excised and weighted. Adipocytes were isolated and evaluated for basal and insulin-stimulated 2-deoxyglucose uptake, oxidation of glucose into CO2, and incorporationof glucose into lipids and lipolytic capacity. Bodyweight, EP fat padweight and diameter of adipocytes fromoffspring of diabeticmothers were increased in comparison to offspring of control mothers. EP adipocytes from offspring of diabetic mothers presented increased basal and insulin stimulated glucose uptake in comparison to control ones. Similar pattern was observed for glucose oxidation into CO2 and incorporation into lipids. However, significant difference in lipolytic capacity in vitrowas not observed. Protein content of GLUT4, insulin receptor and acetyl-CoA carboxylase was significantly increased in EP fat pad of offspring of diabetic mothers in relation to control group. Metabolic programming occurred in the adipose tissue of offspring of diabetic mothers, increasing its capacity to store lipids with no changes in lipolytic capacity.

Mechanisms Of Insulin Resistance After 24 Hours Physical Inactivity In Murine Soleus Muscle.

BACKGROUND AND AIMS: Long term physical inactivity induces obesity and lead to insulin resistance. We have recently demonstrated that only 24 hours physical inactivity by leg immobilization induces insulin resistance in murine skeletal muscle. To elucidate its mechanism, we performed hind-limb cast immobilization (HCI) for 24 hours in C57BL6J mice and investigated ex-vivo insulin-stimulated 2-deoxy glucose uptake, insulin signal transduction and lipid composition in soleus muscle. RESULTS: Twenty-four hours HCI significantly decreased insulin-stimulated 2-deoxy glucose uptake by 41%. In parallel with this change, we observed decreased insulin stimulated serine phosphorylation of Akt and tyrosine phosphorylation of insulin receptor substrate (IRS)-1 after 24h HCI. It has been hypothesized that intramyocellular diacylglycerol (IMDG) accumulation and PKC activation impairs insulin signal transduction. Consistent with this hypothesis, we found that 24h HCI dramatically increased IMDG by 192%, while the amount of intramyocellular triacylglycerol (IMTG) was not changed. In addition, we also observed increase of PKCe translocation to the plasma membrane after 24 HCI. Associated with the IMDG accumulation, expression level of diacylglycerol acyltransferase (DGAT)-1, which converts diacylglycerol to triacylglycerol, was decreased by 53%, and activity of Lipin1, which converts phosphatidic acid to diacylglycerol, was increased by 141% after 24h HCI. Finally, overexpression of dominant negative Lipin1 in soleus muscle partially inhibited diacylglycerol accumulation after 24h HCI. CONCLUSION: These data suggested that 24h HCI induce insulin resistance through IMDG accumulation and trans-location of PKCe to the plasma membrane in skeletal muscle. Given that increased Lipin1 activity and decreased DGAT-1 expression might be involved in the mechanism of IMDG accumulation, suppression of lipin1 can be a drug target for insulin resistance induced by physical inactivity.

Adipose tissue p50α/p55α abundance may drive acute high fat diet induced insulin resistance

Insulin resistance (IR), a metabolic feature of obesity, drives a number of diseases. Recently, the focus of obesity research has been adipocyte‐immune cell crosstalk and its influence on systemic insulin sensitivity (IS). Currently, obesity induced IR is thought to arise secondary to inflammation due to infiltration of adipose tissue (AT) with pro‐inflammatory immune cells. Obesity is a progressive disease, and metabolic alterations in AT following acute nutrient excess, prior to development of obesity, are not well understood. Phosphotidylinositol 3‐kinase (PI3K) has been proposed as the integration point between immune cell infiltration and adipocyte insulin action. Global knockdown of pik3r1 (gene encoding regulatory subunits (p85α/p55α/p50α) of PI3K) resulted in enhanced adipocyte and systemic IS, with parallel decrease in AT macrophage infiltration despite marked obesity. Here we demonstrate that acute (3d) high fat diet (HFD) significantly increases bodyweight due to a significant increase in food intake in wild type mice. Body composition analysis by NMR indicates a significant increase in percent body fat with a concomitant decrease in lean mass. Energy expenditure measured by indirect calorimetry was also significantly decreased after 3d HFD. Importantly, both systemic IS measured by oral glucose challenge and insulin‐stimulated adipocyte 2‐deoxyglucose uptake was significantly reduced. No difference was found in adipocyte chemokine or cytokine secretion. We propose that reduction in adipocyte IS and subsequent systemic IS, is due to an increase in AT p50α/p55α abundance and occurs prior to initiation of a pro‐inflammatory response. Identifying primary steps in development of IR may guide treatments for obesity related metabolic diseases.NIH DK095926 (CEM)

Effects of Short Chain Fatty Acids on Glucose and Lipid metabolism in Adipocytes

Short chain fatty acids (SCFA) are major products of bacterial fermentation by gut microbiota. Recent literature suggests that microbiota contribute to regulate glucose homeostasis and insulin sensitivity in the host. The purpose of the study was to determine whether the SCFAs acetate, butyrate and propionate impact on glucose and lipid metabolism and insulin signalling in adipose cells in vitro. We cultured differentiated 3T3L1 adipocytes in the presence of acetate, butyrate or propionate for 48 hours. We determined glucose transporter expression and activation of insulin signalling cascades by western blotting. We measured insulin‐stimulated 2‐deoxyglucose uptake and determined total Triglyceride content and expression and activation of Hormone Sensitive lipase (HSL) and Adipose Triglyceride Lipase (ATGL) by western blotting. None of the SCFAs had an effect on Glut1 or Glut4 glucose transporter expression or elicited an effect on basal or insulin‐stimulated glucose uptake. Insulin‐mediated activation of AKT and ERK were similar to that obtained in control untreated cells. While acetate and propionate did not have any effect in total triglyceride accumulation compared to control cells, butyrate treated cells showed lower lipid content compared to untreated cells. Butyrate treated cells showed increased levels of phosphorylated HSL (Ser 660) in basal state (no insulin) whereas the response to insulin was similar to that of untreated cells. We did not detect any changes in the expression levels of HSL, but cells treated with butyrate had increased ATGL levels compared to untreated cells. These data suggests that SCFA do not directly modify glucose uptake, and that butyrate but not other SCFA reduces lipid content by increasing the basal activity of HSL and expression of ATGL in adipocytes.

Hypochlorous acid via peroxynitrite activates protein kinase Cθ and insulin resistance in adipocytes.

We recently reported that genetic deletion of myeloperoxidase (MPO) alleviates obesity-related insulin resistance in mice in vivo. How MPO impairs insulin sensitivity in adipocytes is poorly characterized. As hypochlorous acid (HOCl) is a principal oxidant product generated by MPO, we evaluated the effects of HOCl on insulin signaling in adipocytes differentiated from 3T3-L1 cells. Exposure of 3T3-L1 adipocytes to exogenous HOCl (200 μmol/l) attenuated insulin-stimulated 2-deoxyglucose uptake, GLUT4 translocation, and insulin signals, including tyrosine phosphorylation of insulin receptor substrate 1 (IRS1) and phosphorylation of Akt. Furthermore, treatment with HOCl induced phosphorylation of IRS1 at serine 307, inhibitor κB kinase (IKK), c-Jun NH2-terminal kinase (JNK), and phosphorylation of PKCθ (PKCθ). In addition, genetic and pharmacological inhibition of IKK and JNK abolished serine phosphorylation of IRS1 and impairment of insulin signaling by HOCl. Furthermore, knockdown of PKCθ using siRNA transfection suppressed phosphorylation of IKK and JNK and consequently attenuated the HOCl-impaired insulin signaling pathway. Moreover, activation of PKCθ by peroxynitrite was accompanied by increased phosphorylation of IKK, JNK, and IRS1-serine 307. In contrast, ONOO− inhibitors abolished HOCl-induced phosphorylation of PKCθ, IKK, JNK, and IRS1-serine 307, as well as insulin resistance. Finally, high-fat diet (HFD)-induced insulin resistance was associated with enhanced phosphorylation of PKCθ, IKK, JNK, and IRS1 at serine 307 in white adipose tissues from WT mice, all of which were not found in Mpo knockout mice fed HFDs. We conclude that HOCl impairs insulin signaling pathway by increasing ONOO− mediated phosphorylation of PKCθ, resulting in phosphorylation of IKK/JNK and consequent serine phosphorylation of IRS1 in adipocytes.

CYP2E1 Impairs GLUT4 Gene Expression and Function: NRF2 as a Possible Mediator

Impaired GLUT4 function/expression in insulin target tissues is well-documented in diabetes and obesity. Cytochrome P450 isoform 2E1 (CYP2E1) induces oxidative stress, leading to impaired insulin action. CYP2E1 knockout mice are protected against high fat diet-induced insulin resistance and obesity; however the molecular mechanisms are still unclear. We examined whether CYP2E1 impairs GLUT4 gene expression and function in adipose and muscle cells. CYP2E1 overexpression in skeletal muscle-derived L6 cells inhibited insulin-stimulated Glut4 translocation and 2-deoxyglucose uptake, with the latter inhibition being blocked by vitamin E. CYP2E1 overexpression in L6 and primary rat adipose (PRA) cells suppressed GLUT4 gene expression at promoter and mRNA levels, whereas CYP2E1 silencing had opposite effects. In PRA, CYP2E1-induced suppression of GLUT4 expression was blocked by chlormethiazole (CYP2E1-specific inhibitor) and the antioxidants vitamin E and N-acetyl-l-cysteine. CYP2E1 effect was mediated by the transcription factor NF-E2-related factor 2 (NRF2), as evident from its complete reversal by a coexpressed dominant-negative, but not wild-type NRF2. GLUT4 transcription was suppressed by NRF2 overexpression, and enhanced by NRF2 silencing. Promoter and ChIP analysis showed a direct and specific binding of NRF2 to a 58-326 GLUT4 promoter region that was required to maintain CYP2E1 suppression; this binding was enhanced by CYP2E1 overexpression. We suggest a mechanism for CYP2E1 action that involves: a) suppression of GLUT4 gene expression that is mediated by NRF2; b) impairment of insulin-stimulated Glut4 translocation and function. CYP2E1 and NRF2 are introduced as negative regulators of GLUT4 expression and function in insulin-sensitive cells.

Lowered extracellular pH is involved in the pathogenesis of skeletal muscle insulin resistance

Insulin resistance in the skeletal muscle is manifested by diminished insulin-stimulated glucose uptake and is a core factor in the pathogenesis of type 2 diabetes mellitus (DM), but the mechanism causing insulin resistance is still unknown. Our recent study has shown that pH of interstitial fluids was lowered in early developmental stage of insulin resistance in OLETF rats, a model of type 2 DM. Therefore, in the present study, we confirmed effects of the extracellular pH on the insulin signaling pathway in a rat skeletal muscle-derived cell line, L6 cell. The phosphorylation level (activation) of the insulin receptor was significantly diminished in low pH media. The phosphorylation level of Akt, which is a downstream target of the insulin signaling pathway, also decreased in low pH media. Moreover, the insulin binding to its receptor was reduced by lowering extracellular pH, while the expression of insulin receptors on the plasma membrane was not affected by the extracellular pH. Finally, insulin-stimulated 2-deoxyglucose uptake in L6 cells was diminished in low pH media. Our present study suggests that lowered extracellular pH conditions may produce the pathogenesis of insulin resistance in skeletal muscle cells.