Translocation Of Glucose Transporter Research Articles

Gastro-Resistant Insulin Receptor-Binding Peptide from Momordica charantia Improved the Glucose Tolerance in Streptozotocin-Induced Diabetic Mice via Insulin Receptor Signaling Pathway.

Momordica charantia is a commonly used food and has been used for the management of diabetes. Our previous study has identified an insulin receptor (IR)-binding protein (mcIRBP) from Momordica charantia. Here we identified the gastro-resistant hypoglycemic bioactive peptides from protease-digested mcIRBP. By in vitro digestion and IR kinase activity assay, we found that a 9-amino-acid-residue peptide, mcIRBP-9, was a gastro-resistant peptide that enhanced IR kinase activities. mcIRBP-9 activated IR signaling transduction pathway, which resulted in the phosphorylation of IR, the translocation of glucose transporter 4, and the uptake of glucose in cells. Intraperitoneal and oral administration of mcIRBP-9 stimulated the glucose clearance by 30.91 ± 0.39% and 32.09 ± 0.38%, respectively, in streptozotocin-induced diabetic mice. Moreover, a pilot study showed that daily ingestion of mcIRBP-9 for 30 days decreased the fasting blood glucose levels and glycated hemoglobin (HbA1c) levels by 23.62 ± 6.14% and 24.06 ± 1.53%, respectively. In conclusion, mcIRBP-9 is a unique gastro-resistant bioactive peptide generated after the digestion of mcIRBP. Furthermore, oral administration of mcIRBP-9 improves both the glucose tolerance and the HbA1c levels in diabetic mice via targeting IR signaling transduction pathway.

Mahanine enhances the glucose-lowering mechanisms in skeletal muscle and adipocyte cells

Insulin resistance is a major defect underlying type 2 diabetes development. Skeletal muscle tissue and adipocyte tissue are the major sites of postprandial glucose disposal, and enhancing glucose uptake into this tissue may decrease insulin resistance in type 2 diabetes patients. Mahanine (3,11-dihydro-3,5-dimethyl-3-(4-methyl-3-pentenyl)pyrano[3,2-a]carbazol-9-ol) has been reported to be a major bioactive carbazole alkaloid that has many biological activities including antitumor, anti-inflammatory, antioxidant and anti-diabetic activities. However, the molecular mechanism and signaling pathways mediating the anti-diabetic effects of mahanine require further investigation. Therefore, the aim of this study was to investigate the effects of mahanine, a carbazole alkaloid from Murraya koenigii, on glucose uptake and glucose transporter 4 (GLUT4) translocation in skeletal muscle and adipocyte cells. Mahanine treatment promoted a dose dependent increased in glucose uptake in L6 myotubes and adipocyte cells via activation of the Akt signaling pathway. Mahanine induced Akt-activation was reversed by co-treatment with wortmannin, an Akt inhibitor. Moreover, it was found that mahanine significantly enhanced GLUT4 translocation to the plasma membrane in L6 myotubes. These results suggest that increased activation of the Akt signaling pathway lead to increased plasma membrane GLUT4 content and increased glucose uptake. These data strongly suggest that mahanine has anti-diabetic potential for treating diabetes.

Antidiabetic activities of entagenic acid in type 2 diabetic db/db mice and L6 myotubes via AMPK/GLUT4 pathway

Ethnopharmacological relevanceEntada phaseoloides (L.) Merr., a traditional Chinese folk medicine, has been used in treating diabetes and other inflammatory disorders. Our previous study revealed that the triterpene saponins in E.Phaseoloides possessed an antidiabetic effect in type 2 diabetic rats by activating AMP-activated protein kinase (AMPK). Entagenic acid, the principal aglycon, isolated from the seed kernels of E. phaseoloides, has been proposed to possess a significant role in the antidiabetic effect, however, its actual effect and pertinent mechanisms are still unknown. Aim of the studyThe aim of the present study was to investigate the antidiabetic effect of entagenic acid in a type 2 diabetic animal model (C57BIKsj db/db mice) and its role in the regulation of glucose uptake in L6 myotubes, and to explore the possible molecular mechanisms. Materials and methodsIn vivo, average weekly body weight, daily water, food intake and postprandial blood glucose levels, the intraperitoneal insulin tolerance test, glucose tolerance test, serum lipid profiles and pancreatic histopathological changes in db/db mice treated with entagenic acid orally at different doses (5, 10 and 20mg/kg) were assessed and compared with wild-type littermates or vehicle- and metformin-treated db/db mice. In vitro, effects of entagenic acid on the glucose consumption and the phosphorylation of protein kinase B (AKT) and AMPK in L6 myotubes were evaluated. ResultsIn vivo, entagenic acid significantly lowered postprandial blood glucose levels but not the body weight, normalized the serum lipid imbalance, improved the impaired glucose tolerance, insulin resistance, as well as the pathological changes in pancreatic islets. In vitro, entagenic acid dose-dependently promoted glucose utilization and enhanced the translocation and expression of glucose transporter 4 (GLUT4), and phosphorylation of AMPK but not AKT. ConclusionsThe present study demonstrated that entagenic acid can markedly maintain the glucose homeostasis, improve insulin resistance and ameliorate dyslipidemia. Its antihyperglycemic effect could be caused by promoting AMPK mediated cellular signaling and GLUT4 translocation in muscles.

Cardiosphere-Derived Cells Demonstrate Metabolic Flexibility That Is Influenced by Adhesion Status

Adult stem cells demonstrate metabolic flexibility that is regulated by cell adhesion status. The authors demonstrate that adherent cells primarily utilize glycolysis, whereas suspended cells rely on oxidative phosphorylation for their ATP needs. Akt phosphorylation transduces adhesion-mediated regulation of energy metabolism, by regulating translocation of glucose transporters (GLUT1) to the cell membrane and thus, cellular glucose uptake and glycolysis. Cell dissociation, a pre-requisite for cell transplantation, leads to energetic stress, which is mediated by Akt dephosphorylation, downregulation of glucose uptake, and glycolysis. They designed hydrogels that promote rapid cell adhesion of encapsulated cells, Akt phosphorylation, restore glycolysis, and cellular ATP levels.

Cyclocarya paliurus extract activates insulin signaling via Sirtuin1 in C2C12 myotubes and decreases blood glucose level in mice with impaired insulin secretion.

Diabetes is caused by the lack of release or action of insulin. Some foods and supplements can compensate for this deficiency; thus, they can aid in the prevention or treatment of diabetes. The aim of this study was to investigate the effects of Cyclocarya paliurus extract (CPE) on insulin signaling and its capacity to correct hyperglycemia in the absence of insulin. To investigate the hypoglycemic effects of CPE, C2C12 cells were exposed to CPE (50 and 100 μg/mL). CPE promoted 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2NBDG) uptake into the cells via translocation of glucose transporter 4 (Glut4) to the plasma membrane. In addition, CPE enhanced tyrosine phosphorylation of insulin receptor substrate and activated phosphatidylinositol 3-kinase and protein kinase B (Akt) via sirtuin1 in C2C12 cells. Moreover, we found that oral administration of CPE (1 g/kg) to streptozotocin-induced hyperglycemic mice produced a progressive decrease in plasma glucose levels at 1 h after single dosing. At that point, CPE significantly increased the expression of skeletal muscle membrane Glut4 and enhanced the phosphorylation of Akt. These results suggest that CPE exerts antidiabetic effects similar to those of insulin, and may be an oral therapeutic alternative for the management of diabetes.

Enhancement of Glucose Uptake by Meso-Dihydroguaiaretic Acid through GLUT4 Up-Regulation in 3T3-L1 Adipocytes.

Type 2 diabetes is characterized by insulin resistance, which leads to increased blood glucose levels. Adipocytes are involved in the development of insulin resistance, resulting from the dysfunction of the insulin signaling pathway. In this study, we investigated whether meso-dihydroguaiaretic acid (MDGA) may modulate glucose uptake in adipocytes, and examined its mechanism of action. MDGA enhanced adipogenesis through up-regulation of peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α in 3T3-L1 adipocytes partially differentiated with sub-optimal concentrations of insulin. MDGA also increased glucose uptake by stimulating expression and translocation of glucose transporter 4 (GLUT4) in adipocytes. These results suggest that MDGA may increase GLUT4 expression and its translocation by promoting insulin sensitivity, leading to enhanced glucose uptake.

Polyphenols activate energy sensing network in insulin resistant models

Unhealthy diet deficient in fruits and vegetables but rich in calories is considered to be one factor responsible for the increased prevalence of insulin resistance and type 2 diabetes (T2D). The consumption of fast foods and soft drinks increases fructose consumption per se and this is of major concern since prolonged fructose intake induces insulin resistance and thereby T2D. The energy homeostasis is regulated by a network consisting of “fuel gauze” called AMP-activated protein kinase (AMPK), the NAD+ dependent type III deacetylase (SIRT1) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) which is disrupted in T2D. The present study was aimed to investigate the action of naringenin and quercetin on energy sensing molecules in insulin resistant models. L6 myotubes and albino Wistar rats were rendered insulin resistant with palmitate and fructose respectively. Naringenin, quercetin or metformin were used for treatment. Fructose and palmitate treatment resulted in insulin resistance as evidenced by decreased glucose transporter 4 (GLUT4) translocation. The translocation of GLUT4, phosphorylation of AMPK and the expression of SIRT1 and PGC-1α which were reduced in insulin resistant cells, were increased upon treatment with polyphenols. Further, naringenin and quercetin showed binding affinity with energy sensing molecules. We conclude that drugs from natural resources that target energy sensing molecules might be helpful to prevent insulin resistance.

Abstract 124: Chronic Beta-Adrenergic Stimulation Prevents Protective Insulin-Induced Increases in Cardiomyocyte Respiratory Capacity

Cardiac injury or stress increases circulating catecholamines (CA), stimulating cardiac β adrenergic receptors (βAR) and adaptive positive inotropy. Under such conditions of increased energy demand, cardiomyocyte metabolism and bioenergetics are altered to maintain cellular function and survival. During chronic CA stimulation, however, receptor desensitization occurs, contributing to loss of contractile reserve and heart failure (HF) development. This progression to HF is again marked by metabolic and bioenergetic changes that remain incompletely understood. Notably, chronic CA exposure induces insulin resistance (IR) in the heart, which decreases substrate uptake and prevents protective signaling. Our laboratory has shown that G protein-coupled receptor kinase 2 induces IR in the heart following myocardial infarction. However, neither the molecular mechanisms of CA-induced IR, nor its effects on cardiomyocyte bioenergetics are well characterized. We hypothesize that βAR stimulation induces IR by decreasing insulin-stimulated mitochondrial respiratory capacity, thus, compromising cardiomyocyte survival during stress. Bioenergetics were evaluated by measuring cellular respiration under coupled (basal) and uncoupled (maximal) conditions. Our results show that acute insulin treatment selectively increases (25%) maximal and reserve respiratory capacity in the presence of glucose, which positively correlates with cellular survival. This effect is completely inhibited with chronic βAR stimulation using isoproterenol or clenbuterol, as is glucose transporter (GLUT4) translocation to the membrane. A similar effect is reproduced by high glucose and/or palmitate-induced IR. Notably, chronic βAR stimulation does not affect basal or maximal respiration, while acute stimulation increases basal respiration (25%). In short, the data demonstrate a unique insulin-induced increase in mitochondrial respiratory capacity in cardiomyocytes that is antagonized by chronic CA stimulation. We propose that this effect contributes to the detrimental effects of βAR stimulation during HF. Thus, understanding this phenomenon and its mechanisms is critical for inhibiting IR and improving cardiac metabolism and function during HF.

Isoalantolactone derivative promotes glucose utilization in skeletal muscle cells and increases energy expenditure in db/db mice via activating AMPK-dependent signaling

Augmenting glucose utilization and energy expenditure in skeletal muscle via AMP-activated protein kinase (AMPK) is an imperative mechanism for the management of type 2 diabetes. Chemical derivatives (2a-2h, 3, 4a-4d, 5) of the isoalantolactone (K007), a bioactive molecule from roots of Inula racemosa were synthesized to optimize the bioactivity profile to stimulate glucose utilization in skeletal muscle cells. Interestingly, 4a augmented glucose uptake, driven by enhanced translocation of glucose transporter 4 (GLUT4) to cell periphery in L6 rat skeletal muscle cells. The effect of 4a was independent to phosphatidylinositide-3-kinase (PI-3-K)/Akt pathway, but mediated through Liver kinase B1 (LKB1)/AMPK-dependent signaling, leading to activation of downstream targets acetyl coenzyme A carboxylase (ACC) and sterol regulatory element binding protein 1c (SREBP-1c). In db/db mice, 4a administration decreased blood glucose level and improved body mass index, lipid parameters and glucose tolerance associated with elevation of GLUT4 expression in skeletal muscle. Moreover, 4a increased energy expenditure via activating substrate utilization and upregulated the expression of thermogenic transcription factors and mitochondrial proteins in skeletal muscle, suggesting the regulation of energy balance. These findings suggest the potential implication of isoalantolactone derivatives for the management of diabetes.

Heterotypic endosomal fusion as an initial trigger for insulin-induced glucose transporter 4 (GLUT4) translocation in skeletal muscle.

Comprehensive imaging analyses of glucose transporter 4 (GLUT4) behaviour in mouse skeletal muscle was conducted. Quantum dot-based single molecule nanometry revealed that GLUT4 molecules in skeletal myofibres are governed by regulatory systems involving 'static retention' and 'stimulus-dependent liberation'. Vital imaging analyses and super-resolution microscopy-based morphometry demonstrated that insulin liberates the GLUT4 molecule from its static state by triggering acute heterotypic endomembrane fusion arising from the very small GLUT4-containing vesicles in skeletal myofibres. Prior exposure to exercise-mimetic stimuli potentiated this insulin-responsive endomembrane fusion event involving GLUT4-containing vesicles, suggesting that this endomembranous regulation process is a potential site related to the effects of exercise. Skeletal muscle is the major systemic glucose disposal site. Both insulin and exercise facilitate translocation of the glucose transporter glucose transporter 4 (GLUT4) via distinct signalling pathways and exercise also enhances insulin sensitivity. However, the trafficking mechanisms controlling GLUT4 mobilization in skeletal muscle remain poorly understood as a resuly of technical limitations. In the present study, which employs various imaging techniques on isolated skeletal myofibres, we show that one of the initial triggers of insulin-induced GLUT4 translocation is heterotypic endomembrane fusion arising from very small static GLUT4-containing vesicles with a subset of transferrin receptor-containing endosomes. Importantly, pretreatment with exercise-mimetic stimuli potentiated the susceptibility to insulin responsiveness, as indicated by these acute endomembranous activities. We also found that AS160 exhibited stripe-like localization close to sarcomeric α-actinin and that insulin induced a reduction of the stripe-like localization accompanying changes in its detergent solubility. The results of the present study thus provide a conceptual framework indicating that GLUT4 protein trafficking via heterotypic fusion is a critical feature of GLUT4 translocation in skeletal muscles and also suggest that the efficacy of the endomembranous fusion process in response to insulin is involved in the benefits of exercise.

Pulmonary vascular effect of insulin in a rodent model of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is associated with metabolic derangements including insulin resistance, although their effects on the cardiopulmonary disease are unclear. We hypothesized that insulin resistance promotes pulmonary hypertension (PH) development and mutations in type 2 bone morphogenetic protein receptor (BMPR2) cause cellular insulin resistance. Using a BMPR2 transgenic murine model of PAH and two models of inducible diabetes mellitus, we explored the impact of hyperglycemia and/or hyperinsulinemia on development and severity of PH. We assessed insulin signaling and insulin-mediated glucose uptake in human endothelial cells with and without mutations in BMPR2. PH developed in control mice fed a Western diet and PH in BMPR2 mutant mice was increased by Western diet. Pulmonary artery pressure correlated strongly with fasting plasma insulin but not glucose. Reactive oxygen species were increased in lungs of insulin-resistant animals. BMPR2 mutation impaired insulin-mediated endothelial glucose uptake via reduced glucose transporter translocation despite intact insulin signaling. Experimental hyperinsulinemia is strongly associated with PH in both control and BMPR2-mutant mice, though to a greater degree in those with BMPR2 mutation. Human pulmonary endothelial cells with BMPR2 mutation have evidence of reduced glucose uptake due to impaired glucose transporter translocation. These experiments support a role for hyperinsulinemia in pulmonary vascular disease.

A systems biology analysis connects insulin receptor signaling with glucose transporter translocation in rat adipocytes

Type 2 diabetes is characterized by insulin resistance, which arises from malfunctions in the intracellular insulin signaling network. Knowledge of the insulin signaling network is fragmented, and because of the complexity of this network, little consensus has emerged for the structure and importance of the different branches of the network. To help overcome this complexity, systems biology mathematical models have been generated for predicting both the activation of the insulin receptor (IR) and the redistribution of glucose transporter 4 (GLUT4) to the plasma membrane. Although the insulin signal transduction between IR and GLUT4 has been thoroughly studied with modeling and time-resolved data in human cells, comparable analyses in cells from commonly used model organisms such as rats and mice are lacking. Here, we combined existing data and models for rat adipocytes with new data collected for the signaling network between IR and GLUT4 to create a model also for their interconnections. To describe all data (>140 data points), the model needed three distinct pathways from IR to GLUT4: (i) via protein kinase B (PKB) and Akt substrate of 160 kDa (AS160), (ii) via an AS160-independent pathway from PKB, and (iii) via an additional pathway from IR, e.g. affecting the membrane constitution. The developed combined model could describe data not used for training the model and was used to generate predictions of the relative contributions of the pathways from IR to translocation of GLUT4. The combined model provides a systems-level understanding of insulin signaling in rat adipocytes, which, when combined with corresponding models for human adipocytes, may contribute to model-based drug development for diabetes.

Metabolism and acetylation contribute to leucine-mediated inhibition of cardiac glucose uptake

High plasma leucine levels strongly correlate with type 2 diabetes. Studies of muscle cells have suggested that leucine alters the insulin response for glucose transport by activating an insulin-negative feedback loop driven by the mammalian target of rapamycin/p70 ribosomal S6 kinase (mTOR/p70S6K) pathway. Here, we examined the molecular mechanism involved in leucine's action on cardiac glucose uptake. Leucine was indeed able to curb glucose uptake after insulin stimulation in both cultured cardiomyocytes and perfused hearts. Although leucine activated mTOR/p70S6K, the mTOR inhibitor rapamycin did not prevent leucine's inhibitory action on glucose uptake, ruling out the contribution of the insulin-negative feedback loop. α-Ketoisocaproate, the first metabolite of leucine catabolism, mimicked leucine's effect on glucose uptake. Incubation of cardiomyocytes with [13C]leucine ascertained its metabolism to ketone bodies (KBs), which had a similar negative impact on insulin-stimulated glucose transport. Both leucine and KBs reduced glucose uptake by affecting translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Finally, we found that leucine elevated the global protein acetylation level. Pharmacological inhibition of lysine acetyltransferases counteracted this increase in protein acetylation and prevented leucine's inhibitory action on both glucose uptake and GLUT4 translocation. Taken together, these results indicate that leucine metabolism into KBs contributes to inhibition of cardiac glucose uptake by hampering the translocation of GLUT4-containing vesicles via acetylation. They offer new insights into the establishment of insulin resistance in the heart.NEW & NOTEWORTHY Catabolism of the branched-chain amino acid leucine into ketone bodies efficiently inhibits cardiac glucose uptake through decreased translocation of glucose transporter 4 to the plasma membrane. Leucine increases protein acetylation. Pharmacological inhibition of acetylation reverses leucine's action, suggesting acetylation involvement in this phenomenon.Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/leucine-metabolism-inhibits-cardiac-glucose-uptake/.

Novel therapies in type 2 diabetes: insulin resistance

AbstractInsulin resistance plays an important role in the pathogenesis of type 2 diabetes and cardiovascular disease. Obesity is a major risk factor for the development of insulin resistance. Hence, treating obesity with lifestyle interventions is the cornerstone of managing insulin resistance. However, when lifestyle interventions fail to produce sustainable impact, then pharmacotherapy and/or bariatric surgery can produce significant improvements in weight, insulin resistance and glycaemic measures. Ectopic fat in the liver and muscle is one of the main mechanisms via which obesity impacts on insulin sensitivity. Hence, there are currently multiple therapeutic interventions in development that aim to improve ectopic fat. With better understanding of the insulin signalling pathways, multiple agents are under development targeting different components of this pathway from the level of insulin receptor to the level of protein kinase B activation and the translocation of glucose transporters. The development of these agents has been slow due to the complexity of the insulin signalling pathway, the multiple negative feedback signals and the fact that the molecules involved in insulin signalling are also involved in other pathways such as cell survival and apoptosis. Sleep‐related disorders are increasingly recognised as independent risk factors for the development of insulin resistance and type 2 diabetes; targeting sleep‐related disorders as a treatment strategy for insulin resistance is under evaluation.In this article, I will briefly review the future treatments of insulin resistance that are in the pipeline, and I will also briefly review the role of bariatric surgery and sleep‐related disorders in the treatment of insulin resistance. Copyright © 2017 John Wiley & Sons.

Acute bout of exercise induced prolonged muscle glucose transporter-4 translocation and delayed counter-regulatory hormone response in type 1 diabetes.

Previous studies have demonstrated that an acute bout of aerobic exercise induces a subsequent delayed onset of hypoglycemia among patients with type 1 diabetes. However, the mechanisms of exercise-induced hypoglycemia in type 1 diabetes are still unclear. Streptozotocin (STZ) was injected to 6-week-old male Wistar rats, and three days after STZ injection, animals were randomly assigned into 2 groups: STZ with insulin only (STZ) and STZ with insulin and exercise (STZ+EX). Normal Wistar rats with exercise were used as control (CON+EX). Insulin was intraperitoneally injected (0.5 U/kg) to both STZ groups (−0.5 h), and a bout of aerobic exercise (15 m/min for 30 min) was conducted at euglycemic conditions (0 h). Blood was collected at 0, 1, 3, and 5 h after exercise from the carotid artery. While the blood glucose level was stable during the post-exercise period (0–5 h) in the STZ and CON+EX groups, it decreased significantly only in the STZ+EX group at 3 h. Plasma glucagon, adrenalin, and noradrenalin levels significantly increased at 1 h in the STZ group, whereas significant hormonal responses were observed at 5 h in the STZ+EX group. In skeletal muscle glucose metabolism-related pathway, the level of glucose transporter-4 (GLUT-4) translocation was significantly higher at 1 h in the CON and STZ groups. However, in the STZ+EX group, these activations were maintained by 5 h, indicating a sustained glucose metabolism in the STZ+EX group. A single bout of aerobic exercise induced a delayed onset of hypoglycemia in STZ-treated rats. A prolonged enhancement of GLUT-4 translocation and delayed counter-regulatory hormone responses may have contributed to the induction of hypoglycemia.

Metabolic Investigations of the Molecular Mechanisms Associated with Parkinson's Disease.

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by fibrillar cytoplasmic aggregates of α-synuclein (i.e., Lewy bodies) and the associated loss of dopaminergic cells in the substantia nigra. Mutations in genes such as α-synuclein (SNCA) account for only 10% of PD occurrences. Exposure to environmental toxicants including pesticides and metals (e.g., paraquat (PQ) and manganese (Mn)) is also recognized as an important PD risk factor. Thus, aging, genetic alterations, and environmental factors all contribute to the etiology of PD. In fact, both genetic and environmental factors are thought to interact in the promotion of idiopathic PD, but the mechanisms involved are still unclear. In this study, we summarize our findings to date regarding the toxic synergistic effect between α-synuclein and paraquat treatment. We identified an essential role for central carbon (glucose) metabolism in dopaminergic cell death induced by paraquat treatment that is enhanced by the overexpression of α-synuclein. PQ “hijacks” the pentose phosphate pathway (PPP) to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. PQ also stimulated an increase in glucose uptake, the translocation of glucose transporters to the plasma membrane, and AMP-activated protein kinase (AMPK) activation. The overexpression of α-synuclein further stimulated an increase in glucose uptake and AMPK activity, but impaired glucose metabolism, likely directing additional carbon to the PPP to supply paraquat redox cycling.

The carboxy-terminal region of the TBC1D4 (AS160) RabGAP mediates protein homodimerization

TBC1D4 (also known as AS160) is a Rab·GTPase-activating protein (RabGAP) which functions in insulin signaling. TBC1D4 is critical for translocation of glucose transporter 4 (GLUT4), from an inactive, intracellular, vesicle-bound site to the plasma membrane, where it promotes glucose entry into cells. The TBC1D4 protein is structurally subdivided into two N-terminal phosphotyrosine-binding (PTB) domains, a C-terminal catalytic RabGAP domain, and a disordered segment in between containing potential Akt phosphorylation sites. Structural predictions further suggest that a region C-terminal to the RabGAP domain adopts a coiled-coil motif. We show that C-terminal region (CTR) region is largely α-helical and mediates TBC1D4 RabGAP dimerization. RabGAP catalytic activity and thermal stability appear to be independent of CTR-mediated dimerization.

Effects of DEHP and its metabolite MEHP on insulin signalling and proteins involved in GLUT4 translocation in cultured L6 myotubes.

Di-(2-ethyl hexyl) phthalate (DEHP) is the plasticizer used in variety of medical and consumer products to impart structural flexibility. DEHP and its primary metabolite mono-(2-ethyl hexyl)phthalate (MEHP) posed a considerable interest because of their contribution to insulin resistance, type-2 diabetes and obesity. Experimental and epidemiological data have shown that DEHP affects blood glucose homeostasis. However, direct effect of DEHP and its metabolite MEHP on insulin signal transduction and glucose transporter 4 (GLUT4) translocation remain obscure. The present study was delineated to decipher the direct effects of DEHP and MEHP on insulin signal transduction and proteins involved in GLUT4 translocation in cultured L6 myotubes, the rat skeletal muscle model. For this study we have exposed cells with 50 and 100μM DEHP and MEHP for 24h followed by insulin stimulation for 20min. GLUT4 level in both cytosol and plasma membrane fractions were analysed by western blot analysis and found to be significantly decreased. Further, DEHP and MEHP treatment significantly altered the insulin signalling molecules and proteins involved in GLUT4 translocation (Rab8A (Ras related proteins in skeletal muscle), insulin - regulated amino peptidase (IRAP), synaptosomal - associated protein 23 (SNAP23), Syntaxin4, Munc18c) from cytosol to plasma membrane. Impaired GLUT4 in the plasma membrane resulted in decreased 14C-deoxy glucose uptake. 14C-glucose oxidation and glycogen content were also significantly decreased in treated groups. In essence, the present study is first of its kind to show the direct adverse effects of DEHP and MEHP on insulin signal transduction and GLUT4 translocation in cultured L6 myotubes. Further, MEHP is found to be more effective than DEHP as a result of its differential structure and physico-chemical properties.

Early energy metabolism-related molecular events in skeletal muscle of diabetic rats: The effects of l-arginine and SOD mimic

Considering the vital role of skeletal muscle in control of whole-body metabolism and the severity of long-term diabetic complications, we aimed to reveal the molecular pattern of early diabetes-related skeletal muscle phenotype in terms of energy metabolism, focusing on regulatory mechanisms, and the possibility to improve it using two redox modulators, l-arginine and superoxide dismutase (SOD) mimic. Alloxan-induced diabetic rats (120 mg/kg) were treated with l-arginine or the highly specific SOD mimic, M40403, for 7 days. As appropriate controls, non-diabetic rats received the same treatments. We found that l-arginine and M40403 restored diabetes-induced impairment of phospho-5′-AMP-activated protein kinase α (AMPKα) signaling by upregulating AMPKα protein itself and its downstream effectors, peroxisome proliferator-activated receptor-γ coactivator-1α and nuclear respiratory factor 1. Also, there was a restitution of the protein levels of oxidative phosphorylation components (complex I, complex II and complex IV) and mitofusin 2. Furthermore, l-arginine and M40403 induced translocation of glucose transporter 4 to the membrane and upregulation of protein of phosphofructokinase and acyl coenzyme A dehydrogenase, diminishing negative diabetic effects on limiting factors of glucose and lipid metabolism. Both treatments abolished diabetes-induced downregulation of sarcoplasmic reticulum calcium-ATPase proteins (SERCA 1 and 2). Similar effects of l-arginine and SOD mimic treatments suggest that disturbances in the superoxide/nitric oxide ratio may be responsible for skeletal muscle mitochondrial and metabolic impairment in early diabetes. Our results provide evidence that l-arginine and SOD mimics have potential in preventing and treating metabolic disturbances accompanying this widespread metabolic disease.

Insulin signaling via the PI3-kinase/Akt pathway regulates airway glucose uptake and barrier function in a CFTR-dependent manner.

Cystic fibrosis-related diabetes is the most common comorbidity associated with cystic fibrosis (CF) and correlates with increased rates of lung function decline. Because glucose is a nutrient present in the airways of patients with bacterial airway infections and because insulin controls glucose metabolism, the effect of insulin on CF airway epithelia was investigated to determine the role of insulin receptors and glucose transport in regulating glucose availability in the airway. The response to insulin by human airway epithelial cells was characterized by quantitative PCR, immunoblot, immunofluorescence, and glucose uptake assays. Phosphatidylinositol 3-kinase/protein kinase B (Akt) signaling and cystic fibrosis transmembrane conductance regulator (CFTR) activity were analyzed by pharmacological and immunoblot assays. We found that normal human primary airway epithelial cells expressed glucose transporter 4 and that application of insulin stimulated cytochalasin B-inhibitable glucose uptake, consistent with a requirement for glucose transporter translocation. Application of insulin to normal primary human airway epithelial cells promoted airway barrier function as demonstrated by increased transepithelial electrical resistance and decreased paracellular flux of small molecules. This provides the first demonstration that airway cells express insulin-regulated glucose transporters that act in concert with tight junctions to form an airway glucose barrier. However, insulin failed to increase glucose uptake or decrease paracellular flux of small molecules in human airway epithelia expressing F508del-CFTR. Insulin stimulation of Akt1 and Akt2 signaling in CF airway cells was diminished compared with that observed in airway cells expressing wild-type CFTR. These results indicate that the airway glucose barrier is regulated by insulin and is dysfunctional in CF.