Insulin-regulated Glucose Transporter Research Articles

The mechanisms of glucose transporter type 4 translocation regulated by insulin receptor signaling

Insulin-regulated glucose transport is dependent on glucose transporter GLUT4 translocation to the plasma membrane, which incorporates glucose into the cells, mainly in adipose tissue and skeletal muscle. Insulin receptor signaling can stimulate GLUT4 vesicle transport from perinuclear pool to the plasma membrane via the vesicle transport machinery. At first, insulin signaling is divided to the multiple pathways, such as Akt/PKB and PKC-zeta/lambda. Subsequently, PKC-zeta/lambda activates KIF3, motor protein based on microtubules, and sequentially Akt/PKB activates Myosin-Va, motor protein based on actin filaments. KIF3 motor moves GLUT4 vesicles from perinuclear pool to the end of microtubules, and Myosin-Va transports GLUT4 vesicles from the end of microtubules to the plasma membrane. Here we indicate the machinery of insulin-regulated GLUT4 vesicle translocation, showing that these motor proteins are the destinations of insulin receptor signaling to regulate glucose transport into the cells.

Reaction Network Analysis of Metabolic Insulin Signaling.

Absolute concentration robustness (ACR) and concordance are novel concepts in the theory of robustness and stability within Chemical Reaction Network Theory. In this paper, we have extended Shinar and Feinberg's reaction network analysis approach to the insulin signaling system based on recent advances in decomposing reaction networks. We have shown that the network with 20 species, 35 complexes, and 35 reactions is concordant, implying at most one positive equilibrium in each of its stoichiometric compatibility class. We have obtained the system's finest independent decomposition consisting of 10 subnetworks, a coarsening of which reveals three subnetworks which are not only functionally but also structurally important. Utilizing the network's deficiency-oriented coarsening, we have developed a method to determine positive equilibria for the entire network. Our analysis has also shown that the system has ACR in 8 species all coming from a deficiency zero subnetwork. Interestingly, we have shown that, for a set of rate constants, the insulin-regulated glucose transporter GLUT4 (important in glucose energy metabolism), has stable ACR.

Thyroid hormones and the potential for regulating glucose metabolism in cardiomyocytes during insulin resistance and T2DM.

In order for the heart to maintain its continuous mechanical work and provide the systolic movement to uphold coronary blood flow, substantial synthesis of adenosine triphosphate (ATP) is required. Under normal conditions cardiac tissue utilizes roughly 70% fatty acids (FA), and 30% glucose for the production of ATP; however, during impaired metabolic conditions like insulin resistance and diabetes glucose metabolism is dysregulated and FA account for 99% of energy production. One of the major consequences of a shift in FA metabolism in cardiac tissue is an increase in reactive oxygen species (ROS) and lipotoxicity, which ultimately lead to mitochondrial dysfunction. Thyroid hormones (TH) have direct effects on cardiac function and glucose metabolism during impaired metabolic conditions suggesting that TH may improve glucose metabolism in an insulin resistant condition. None‐classical TH signaling in the heart has shown to phosphorylate protein kinase B (Akt) and increase activity of phosphoinositide‐3‐kinase (PI3K), which are critical mediators in the insulin‐stimulated glucose uptake pathway. Studies on peripheral tissues such as skeletal muscle and adipocytes have demonstrated TH treatment improved glucose intolerance in a diabetic model and increased insulin‐regulated glucose transporter (GLUT4) mRNA levels. GLUT4 is a downstream target of thyroid response element (TRE), which demonstrates that THs regulate glucose via GLUT4. Elevated 3,5,3′‐triiodothyronine (T3) increased glucose oxidation rate and decreased the glycolytic intermediate, fructose 6‐phosphate (F6P) in cardiomyocytes, in addition to increasing mitochondrial biogenesis and pyruvate transport across the mitochondrial membrane. These findings along with a few other studies on T3 treatment in cardiac tissue suggest TH may improve glucose metabolism in an insulin resistant model and ameliorate the effects of diabetes and metabolic syndrome. This review highlights the potential benefits of exogenous TH on ameliorating metabolic dysfunction in the heart.

Defective insulin-stimulated GLUT4 translocation in brown adipocytes induces systemic glucose homeostasis dysregulation independent of thermogenesis in female mice

ObjectiveRecent studies indicate that brown adipose tissue, in addition to its role in thermogenesis, has a role in the regulation of whole-body metabolism. Here we characterize the metabolic effects of deleting Rab10, a protein key for insulin stimulation of glucose uptake into white adipocytes, solely from brown adipocytes. MethodsWe used a murine brown adipocyte cell line and stromal vascular fraction-derived in vitro differentiated brown adipocytes to study the role of Rab10 in insulin-stimulated GLUT4 translocation to the plasma membrane and insulin-stimulated glucose uptake. We generated a brown adipocyte-specific Rab10 knockout for in vivo studies of metabolism and thermoregulation. ResultsWe demonstrate that deletion of Rab10 from brown adipocytes results in a two-fold reduction of insulin-stimulated glucose transport by reducing translocation of the GLUT4 glucose transporter to the plasma membrane, an effect linked to whole-body glucose intolerance and insulin resistance in female mice. This effect on metabolism is independent of the thermogenic function of brown adipocytes, thereby revealing a metabolism-specific role for brown adipocytes in female mice. The reduced glucose uptake induced by Rab10 deletion disrupts ChREBP regulation of de novo lipogenesis (DNL) genes, providing a potential link between DNL in brown adipocytes and whole-body metabolic regulation in female mice. However, deletion of Rab10 from male mice does not induce systemic insulin resistance, although ChREBP regulation is disrupted. ConclusionsOur studies of Rab10 reveal the role of insulin-regulated glucose transport into brown adipocytes in whole-body metabolic homeostasis of female mice. Importantly, the contribution of brown adipocytes to whole-body metabolic regulation is independent of its role in thermogenesis. It is unclear whether the whole-body metabolic sexual dimorphism is because female mice are permissive to the effects of Rab10 deletion from brown adipocytes or because male mice are resistant to the effect.

A conserved haplotype in Wagyu cattle contains RAB4A whose encoded protein regulates glucose trafficking in muscle and fat cells

The Wagyu breed of taurine cattle possess favourable genetics for intramuscular fat (IMF) but genomic loci associated with the trait remain under characterised. Here, we report the identification of a previously unidentified genomic region possessing a particular haplotype structure in Wagyu. Through deployment of a genome-wide haplotype detection analysis that captures regions conserved in a target population but not other populations we screened 100 individual Wagyu and contrasted them with 100 individuals from two independent comparison breeds, Charolais and Angus, using high-density SNPs. An extreme level of Wagyu conservation was assigned to a single genomic window (spanning genomic coordinates BTA28:41088-300265bp). In fact, a five-SNP region spanning 27096bp is almost perfectly conserved among the 100 Wagyu individuals assayed and partially overlaps RAB4A. Focussing in, two consecutive SNPs (genomic coordinates 236949 and 239950) are apparently fixed within the Wagyu (BB and AA respectively), but at mixed frequencies in the other two breeds. These SNPs are located in the two introns straddling exon 7. In a separate analysis using the 1000 Bulls database, we found that, coincident with exon 7 of RAB4A first allele frequencies were highest in the high IMF Japanese Native (Wagyu) breeds (0.78) and lowest in the low IMF indicine breeds (Nelore and Brahman), with intermediate marbling breeds (Angus and Charolais) assigned intermediate rankings (0.42). RAB4A is known to encode a protein that regulates intracellular trafficking of the insulin-regulated glucose transporter GLUT4. RAB4A can be considered an attractive new positional candidate for IMF development.

Lack of collagen XVIII leads to lipodystrophy and perturbs hepatic glucose and lipid homeostasis.

Lack of collagen XVIII leads to lipodystrophy and perturbs hepatic glucose and lipid homeostasis.

A Detection Method for GLUT4 Exocytosis Based on Spontaneous Split Luciferase Complementation.

Glucose transporter 4 (GLUT4) is an insulin-regulated glucose transporter, which is vital for blood glucose homeostasis. To clarify the physiological roles of GLUT4, quantitative measurement of GLUT4 exocytosis is indispensable. Herein, we show a rapid detection system for GLUT4 on the cell surface using spontaneous split-luciferase reconstitution. Upon insulin-induced GLUT4 exocytosis, GLUT4 was exposed outside, where luciferase is reconstituted and emitted luminescence. Pretreatment with inhibitors reduced the insulin-induced signal elevation. The results indicate that the developed method is applicable to high-throughput analysis on GLUT4 trafficking, which will greatly accelerate comprehensive research on the physiological roles of GLUT4.

Molecular and cellular adaptations to exercise training in skeletal muscle from cancer patients treated with chemotherapy.

A growing body of evidence suggests that exercise training has beneficial effects in cancer patients. The aim of the present study was to investigate the molecular basis underlying these beneficial effects in skeletal muscle from cancer patients. We investigated expression of selected proteins involved in cellular processes known to orchestrate adaptation to exercise training by western blot. Skeletal muscle biopsies were sampled from ten cancer patients before and after 4-7weeks of ongoing chemotherapy, and subsequently after 10weeks of continued chemotherapy in combination with exercise training. Biopsies from ten healthy matched subjects served as reference. The expression of the insulin-regulated glucose transporter, GLUT4, increased during chemotherapy and continued to increase during exercise training. A similar trend was observed for ACC, a key enzyme in the biosynthesis and oxidation of fatty acids, but we did not observe any changes in other regulators of substrate metabolism (AMPK and PDH) or mitochondrial proteins (Cyt-C, COX-IV, SDHA, and VDAC). Markers of proteasomal proteolysis (MURF1 and ATROGIN-1) decreased during chemotherapy, but did not change further during chemotherapy combined with exercise training. A similar pattern was observed for autophagy-related proteins such as ATG5, p62, and pULK1 Ser757, but not ULK1 and LC3BII/LC3BI. Phosphorylation of FOXO3a at Ser318/321 did not change during chemotherapy, but decreased during exercise training. This could suggest that FOXO3a-mediated transcriptional regulation of MURF1 and ATROGIN-1 serves as a mechanism by which exercise training maintains proteolytic systems in skeletal muscle in cancer patients. Phosphorylation of proteins that regulate protein synthesis (mTOR at Ser2448 and 4EBP1 at Thr37/46) increased during chemotherapy and leveled off during exercise training. Finally, chemotherapy tended to increase the number of satellite cells in type 1 fibers, without any further change during chemotherapy and exercise training. Conversely, the number of satellite cells in type 2 fibers did not change during chemotherapy, but increased during chemotherapy combined with exercise training. Molecular signaling cascades involved in exercise training are disturbed during cancer and chemotherapy, and exercise training may prevent further disruption of these pathways. The study was approved by the local Scientific Ethics Committee of the Central Denmark Region (Project ID: M-2014-15-14; date of approval: 01/27/2014) and the Danish Data Protection Agency (case number 2007-58-0010; date of approval: 01/28/2015). The trial was registered at http//www.clinicaltrials.gov (registration number: NCT02192216; date of registration 07/17-2014).

Adipose Tissue, Inter-Organ Communication, and the Path to Type 2 Diabetes: The 2016 Banting Medal for Scientific Achievement Lecture.

My scientific career has focused on understanding the mechanisms underlying insulin resistance with the goal of developing new strategies to prevent and treat type 2 diabetes. My early studies focused on understanding how insulin promotes glucose transport into adipocytes, a classic model of highly insulin-responsive target cells. When we found changes in adipocyte glucose transport in altered metabolic states, we were highly motivated to understand the consequences of this on whole-body glucose homeostasis. In the late 1980s, when GLUT4, the major insulin-regulated glucose transporter, was identified, my lab observed that it was downregulated in adipocytes but not in skeletal muscle in insulin-resistant states, such as obesity and type 2 diabetes, in humans and rodents. We investigated the role of GLUT4 in adipose tissue and muscle in whole-body insulin sensitivity, making tissue-specific GLUT4-overexpressing and GLUT4 knockout mice. These studies led to the discovery that adipocytes, and specifically glucose transport into adipocytes, regulate whole-body glucose homeostasis. As adipocytes take up relatively little glucose, we investigated the underlying mechanisms. In the 1990s, we performed DNA microarrays on adipose tissue from adipose-specific GLUT4-overexpressing and GLUT4 knockout mice to find reciprocally regulated genes, and we identified several molecules that were not previously known to regulate systemic insulin sensitivity and/or energy balance. More recently, with Alan Saghatelian’s lab, we discovered a novel class of lipids with antidiabetes and anti-inflammatory effects. We also investigated the effects of the adipose-secreted hormone, leptin, on insulin sensitivity. We found that the AMP-activated protein kinase (AMPK) pathway mediates leptin’s effects on fatty acid oxidation in muscle and also plays a role in leptin’s anorexigenic effects in the hypothalamus. These studies transformed AMPK from a “fuel gauge” that regulates energy supply at the cellular level to a sensing and signaling pathway that regulates organismal energy balance. Overall, these studies have expanded our understanding of the multifaceted role of adipose tissue in metabolic health and how adipose dysfunction increases the risk for type 2 diabetes.

Constitutively Active Grp1 Promotes Basal Glucose Transport into White Adipose and Enhances Insulin Sensitivity in High-Fat Fed Mice

Insulin-stimulated phosphorylation of Grp1 (general receptor for phosphoinositides; a.k.a Cyth3, cytohesin3) in cultured adipocytes is known to promote recycling of insulin-regulated glucose transporter (Glut4)-containing vesicles from the endosome to plasma membrane, and to enhance glucose uptake thereafter. We found that Cre-dependent expression of a constitutively active form of Grp1 (caGrp1) specifically increased basal glucose uptake into WAT of young chow-fed mice, without altering Akt phosphorylation or insulin-stimulated uptake. Although early growth and glucose homeostasis in chow-fed caGrp1 mice were normal, caGrp1 mice fed a high fat diet (HFD) became markedly less adipose, and showed improved glucose tolerance and insulin sensitivity vs. Cre-negative controls. Fasting insulinemia in HFD-fed caGrp1 mice was lower, whereas circulating adiponectin was higher, and FGF-21 and leptin were unchanged. Hepatic lipid disposition in HFD-fed caGrp1 mice was comparable to that in controls, though circulating and liver free fatty acids were slightly lower. However, epigonadal WAT from caGrp1 mice showed markedly reduced staining for inflammatory marker CD68, and adipocyte size was also significantly greater. Collectively, these features distinguish caGrp1 mice from those with adipose-specific over-expression of Glut4 described by others, but remain compatible with an adipose-specific basis of improved systemic insulin sensitivity. Given recently improved understanding of the structural/functional changes induced in Grp1 via Akt phosphorylation, drug-based activation of Grp1 in WAT might prove an attractive route by which to favorably alter systemic insulin sensitivity and glycemia. Disclosure K.D. Copps: None. J. Li: None.

The significance of anthocyanins in the prevention and treatment of type 2 diabetes.

Anthocyanins are food compounds which belong to polyphenols and can mainly be found in dark fruits (e.g., blueberries, black currants, cranberries) and vegetables (e.g., red cabbage, radish, eggplant). The results of large research have shown that these compounds play an important role in the prevention of type 2 diabetes (T2D). In rodent studies and in studies with isolated omental adipocytes, it was observed that anthocyanins regulated the carbohydrate metabolism in the body due to the upregulation of GLUT4 (insulinregulated glucose transporter) translocation, increased activation of PPARγ (peroxisome proliferator-activated receptor-γ) in adipose tissue and skeletal muscles as well as increased secretion of adiponectin and leptin. Moreover, these compounds reduced the inflammation status in the body. Studies conducted on humans and experimental animals showed that anthocyanins decrease insulin resistance. This effect may be achieved by the upregulation of GLUT4 gene expression, activation of AMP-activated protein kinase and downregulation of retinol binding protein 4 (RBP4) expression. Anthocyanins also increased the uptake and utilization of glucose by tissues in streptozotocin-induced diabetic rats and mice, and they also protected pancreatic cells against necrosis induced by streptozotocin. Another mechanism that might explain the lower glucose level in the blood after a meal with anthocyanins compared to a meal without them is the inhibition of intestinal α-glucosidase and pancreatic α-amylase by these compounds. Moreover, anthocyanins improve insulin secretion, which can have a special meaning for people with T2D. The evidence from the presented studies suggests that foods rich in anthocyanins may be one of the diet elements supporting the prevention and treatment of T2D.

CHC22 and CHC17 clathrins have distinct biochemical properties and display differential regulation and function

Clathrins are cytoplasmic proteins that play essential roles in endocytosis and other membrane traffic pathways. Upon recruitment to intracellular membranes, the canonical clathrin triskelion assembles into a polyhedral protein coat that facilitates vesicle formation and captures cargo molecules for transport. The triskelion is formed by trimerization of three clathrin heavy-chain subunits. Most vertebrates have two isoforms of clathrin heavy chains, CHC17 and CHC22, generating two clathrins with distinct cellular functions. CHC17 forms vesicles at the plasma membrane for receptor-mediated endocytosis and at the trans-Golgi network for organelle biogenesis. CHC22 plays a key role in intracellular targeting of the insulin-regulated glucose transporter 4 (GLUT4), accumulates at the site of GLUT4 sequestration during insulin resistance, and has also been implicated in neuronal development. Here, we demonstrate that CHC22 and CHC17 share morphological features, in that CHC22 forms a triskelion and latticed vesicle coats. However, cellular CHC22-coated vesicles were distinct from those formed by CHC17. The CHC22 coat was more stable to pH change and was not removed by the enzyme complex that disassembles the CHC17 coat. Moreover, the two clathrins were differentially recruited to membranes by adaptors, and CHC22 did not support vesicle formation or transferrin endocytosis at the plasma membrane in the presence or absence of CHC17. Our findings provide biochemical evidence for separate regulation and distinct functional niches for CHC17 and CHC22 in human cells. Furthermore, the greater stability of the CHC22 coat relative to the CHC17 coat may be relevant to its excessive accumulation with GLUT4 during insulin resistance.

Insulin modulates hippocampally-mediated spatial working memory via glucose transporter-4

The insulin-regulated glucose transporter, GluT4, is a key molecule in peripheral insulin signaling. Although GluT4 is abundantly expressed in neurons of specific brain regions such as the hippocampus, the functional role of neuronal GluT4 is unclear. Here, we used pharmacological inhibition of GluT4-mediated glucose uptake to determine whether GluT4 mediates insulin-mediated glucose uptake in the hippocampus. Consistent with previous reports, we found that glucose utilization increased in the dorsal hippocampus of male rats during spontaneous alternation (SA), a hippocampally-mediated spatial working memory task. We previously showed that insulin signaling within the hippocampus is required for processing this task, and that administration of exogenous insulin enhances performance. At baseline levels of hippocampal insulin, inhibition of GluT4-mediated glucose uptake did not affect SA performance. However, inhibition of an upstream regulator of GluT4, Akt, did impair SA performance. Conversely, when a memory-enhancing dose of insulin was delivered to the hippocampus prior to SA-testing, inhibition of GluT4-mediated glucose transport prevented cognitive enhancement. These data suggest that baseline hippocampal cognitive processing does not require functional hippocampal GluT4, but that cognitive enhancement by supra-baseline insulin does. Consistent with these findings, we found that in neuronal cell culture, insulin increases glucose utilization in a GluT4-dependent manner. Collectively, these data demonstrate a key role for GluT4 in transducing the procognitive effects of elevated hippocampal insulin.

Pu’erh tea extract-mediated protection against hepatosteatosis and insulin resistance in mice with diet-induced obesity is associated with the induction of de novo lipogenesis in visceral adipose tissue

White adipose tissue (WAT) is important for the maintenance of metabolic homeostasis, and metabolic syndrome is sometimes associated with WAT dysfunction in humans and animals. WAT reportedly plays a key, beneficial role in the maintenance of glucose and lipid homeostasis during de novo lipogenesis (DNL). Pu'erh tea extract (PTE) can inhibit harmful, ectopic DNL in the liver, thus protecting against hepatosteatosis, in mice with diet-induced obesity. We examined whether PTE could induce DNL in WAT and consequently protect against hepatosteatosis. C57BL/6 male mice were fed a high-fat diet (HFD) with/without PTE for 16weeks. Systemic insulin sensitivity was determined using HOMA-IR, insulin- and glucose-tolerance tests, and WAT adipogenesis was evaluated by histological analysis. Adipogenesis-, inflammation-, and DNL-related gene expression in visceral AT (VAT) and subcutaneous AT (SAT) was measured using quantitative reverse transcription-PCR. Regression analysis was used to investigate the association between DNL in WAT and systemic insulin resistance or hepatosteatosis. Pu'erh tea extract significantly reduced the gain of body weight and SAT, but not VAT adiposity, in mice fed the high-fat diet and induced adipogenesis in VAT. The expression of DNL-related genes, including Glut4, encoding an important insulin-regulated glucose transporter (GLUT4), were highly elevated in VAT. Moreover, PTE inhibited VAT inflammation by simultaneously downregulating inflammatory molecules and inducing expression of Gpr120 that encodes an anti-inflammatory and pro-adipogenesis receptor (GPR-120) that recognizes unsaturated long-chain fatty acids, including DNL products. The expression of DNL-related genes in VAT was inversely correlated with hepatosteatosis and systemic insulin resistance. Activation of DNL in VAT may explain PTE-mediated alleviation of hepatosteatosis symptoms and systemic insulin resistance.

One Cycle Fuels Another: The Energetics of Neurotransmitter Release

In this issue of Neuron, Ashrafi etal. (2017) show that activity induces translocation of the insulin-regulated glucose transporter GLUT4 to the plasma membrane, where it sustains the ATP production required for synaptic vesicle cycling. However, translocation occurs from presynaptic membranes other than synaptic vesicles and involves a distinct molecular mechanism.

Adipose tissue regulates insulin sensitivity: role of adipogenesis, de novo lipogenesis and novel lipids.

Obesity, the major cause of the current global epidemic of type 2 diabetes (T2D), induces insulin resistance in peripheral insulin target tissues. Several mechanisms have been identified related to cross-talk between adipose tissue, skeletal muscle and liver. These mechanisms involve both increased free fatty acid release and altered secretion of adipokines from adipose tissue. A major determinant of metabolic health is the ability of subcutaneous adipose tissue (SAT) to store excess fat rather than allowing it to accumulate in ectopic depots including liver (i.e. in nonalcoholic fatty liver disease), muscle and heart, or in epicardial/pericardial and visceral fat depots which promote the metabolic complications of obesity. The ability to recruit and differentiate precursor cells into adipose cells (adipogenesis) in SAT is under genetic regulation and is reduced in high-risk individuals who have first-degree relatives with T2D. Early recruitment of new adipose cells is dependent on the cross-talk between canonical WNT and BMP4 signalling; WNT enhances their undifferentiated and proliferative state whereas BMP4 induces their commitment to the adipogenic lineage. Dysregulation of these signalling pathways is associated with impaired adipogenesis and impaired ability to respond to the need to store excess lipids in SAT. This leads to hypertrophic, dysfunctional and insulin-resistant adipose cells with a reduced content of GLUT4, the major insulin-regulated glucose transporter, which in turn reduces adipose tissue glucose uptake and de novo lipogenesis. We recently identified that reduced GLUT4 and lipogenesis in adipocytes impairs the synthesis of a novel family of lipids secreted by adipose tissue (and potentially other tissues), branched fatty acid esters of hydroxy fatty acids (FAHFAs). FAHFAs have beneficial metabolic effects, including enhancing insulin-stimulated glucose transport and glucose-stimulated GLP1 and insulin secretion, as well as powerful anti-inflammatory effects. FAHFA levels are reduced in subcutaneous adipose tissue in insulin-resistant individuals, and this novel family of lipids may become of future therapeutic use.

The Interacting Domains of PREP1 and p160 are Endowed with a Remarkable Structural Stability.

PREP1/p160 is a protein complex with relevant physiopathological roles in vivo. p160 regulates PREP1 transcriptional activity by preventing the formation of other PREP1-containing complexes, whereas PREP1 regulates p160 activity by increasing its stability. This induces the repression of the insulin-regulated glucose transporter GLUT4 dampening insulin sensitivity. In spite of the considerable amount of functional studies performed on the PREP1/p160 complex in vivo, a biochemical and structural characterization of this complex has not been so far undertaken, given the poor stability of the recombinant full-length proteins. Here, we report the design and preparation of PREP1 and p160 domains together with preliminary structural and binding studies. PREP1, residues 45-155, and p160, residues 20-160, have been expressed and purified as folded, monomeric domains. The two domains show both all-alpha secondary structures, as demonstrated by CD studies and are endowed with unusually high thermal stabilities. We have also estimated for the first time the PREP1-p160 interaction strength finding that the two recombinant domains interact with a KD ranging between about 0.3 and 1μM. Altogether, data suggest that the selected PREP1 and p160 domains are structurally independent and that their structure is underlined by high stability and a prevailing alpha-helical organization.

Arsenic Induces Insulin Resistance in Mouse Adipocytes and Myotubes Via Oxidative Stress-Regulated Mitochondrial Sirt3-FOXO3a Signaling Pathway.

Chronic exposure to arsenic via drinking water is associated with an increased risk for development of type 2 diabetes mellitus (T2DM). This study investigates the role of mitochondrial oxidative stress protein Sirtuin 3 (Sirt3) and its targeting proteins in chronic arsenic-induced T2DM in mouse adipocytes and myotubes. The results show that chronic arsenic exposure significantly decreased insulin-stimulated glucose uptake (ISGU) in correlation with reduced expression of insulin-regulated glucose transporter type 4 (Glut4). Expression of Sirt3, a mitochondrial deacetylase, was dramatically decreased along with its associated transcription factor, forkhead box O3 (FOXO3a) upon arsenic exposure. A decrease in mitochondrial membrane potential (Δψm) was observed in both 3T3L1 adipocytes and C2C12 myotubes treated by arsenic. Reduced FOXO3a activity by arsenic exhibited a decreased binding affinity to the promoters of both manganese superoxide dismutase (MnSOD) and peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α, a broad and powerful regulator of reactive oxygen species (ROS) metabolism. Forced expression of Sirt3 or MnSOD in mouse myotubes elevated Δψm and restored ISGU inhibited by arsenic exposure. Our results suggest that Sirt3/FOXO3a/MnSOD signaling plays a significant role in the inhibition of ISGU induced by chronic arsenic exposure.

Gestational protein restriction impairs insulin-regulated glucose transport mechanisms in gastrocnemius muscles of adult male offspring.

Type II diabetes originates from various genetic and environmental factors. Recent studies showed that an adverse uterine environment such as that caused by a gestational low-protein (LP) diet can cause insulin resistance in adult offspring. The mechanism of insulin resistance induced by gestational protein restriction is not clearly understood. Our aim was to investigate the role of insulin signaling molecules in gastrocnemius muscles of gestational LP diet-exposed male offspring to understand their role in LP-induced insulin resistance. Pregnant Wistar rats were fed a control (20% protein) or isocaloric LP (6%) diet from gestational day 4 until delivery and a normal diet after weaning. Only male offspring were used in this study. Glucose and insulin responses were assessed after a glucose tolerance test. mRNA and protein levels of molecules involved in insulin signaling were assessed at 4 months in gastrocnemius muscles. Muscles were incubated ex vivo with insulin to evaluate insulin-induced phosphorylation of insulin receptor (IR), Insulin receptor substrate-1, Akt, and AS160. LP diet-fed rats gained less weight than controls during pregnancy. Male pups from LP diet-fed mothers were smaller but exhibited catch-up growth. Plasma glucose and insulin levels were elevated in LP offspring when subjected to a glucose tolerance test; however, fasting levels were comparable. LP offspring showed increased expression of IR and AS160 in gastrocnemius muscles. Ex vivo treatment of muscles with insulin showed increased phosphorylation of IR (Tyr972) in controls, but LP rats showed higher basal phosphorylation. Phosphorylation of Insulin receptor substrate-1 (Tyr608, Tyr895, Ser307, and Ser318) and AS160 (Thr642) were defective in LP offspring. Further, glucose transporter type 4 translocation in LP offspring was also impaired. A gestational LP diet leads to insulin resistance in adult offspring by a mechanism involving inefficient insulin-induced IR, Insulin receptor substrate-1, and AS160 phosphorylation and impaired glucose transporter type 4 translocation.

Insulin stimulates glucose transport via protein kinase G type I alpha-dependent pathway in podocytes

Podocyte resistance to the actions of insulin on glucose transport could contribute to the pathogenesis of diabetic podocytopathy (DP) via disturbances in cyclic-dependent protein kinase signaling. To determine whether cGMP-dependent protein kinase (PKG) is involved in the insulin regulation of glucose transport, we measured insulin-dependent glucose uptake into cultured rat podocytes under conditions of modified PKG activity using pharmacological (PKG activator or inhibitor) and biochemical (siRNA PKGIα, siRNA insulin receptor β) means. Our findings indicate the participation of PKG in insulin-stimulated transport and provide new insights into how PKG may trigger the resistance of glucose transport to insulin in DP.