Stimulation Of Glucose Transport Research Articles

Characterization of SGLT1-mediated glucose transport in Caco-2 cell monolayers, and absence of its regulation by sugar or epinephrine

Caco-2 cells are increasingly used to study the absorption of drugs and nutrients, including D-glucose, an important nutrient that mainly gets absorbed from the intestine by the sodium/glucose cotransporter 1 (SGLT1). However, disadvantages of Caco-2 cells for such studies have been reported, e.g., D-glucose cannot elicit translocation of the intracellular pool of SGLT1 to the apical membrane, the origin of the cells affects glucose uptake, and Caco-2 cells exhibit heterogeneity. This study aimed to characterize SGLT1-mediated glucose transport across Caco-2 cell monolayers. We found that at lower glucose concentrations (5 mM) SGLT1 contributes more to total glucose transport than at higher (10 mM) glucose concentrations, suggesting contributions by another transporter at higher glucose concentrations. This contrasts with the in vivo situation, where SGLT1 dominant glucose transporter at all glucose concentrations. We also tested whether known regulators like sugars or catecholamines can stimulate glucose transport across Caco-2 cell monolayers. Neither epinephrine nor 2-deoxy-D-glucose could stimulate glucose transport. Moreover, the epinephrine could not induce accumulation of cyclic adenosine monophosphate (cAMP) in Caco-2 cells, indicating the absence of a functional β2-adrenoceptor in Caco-2 cells, which could explain the lack of epinephrine effect on glucose transport. Also, Caco-2 cells may lack some kinases required for increased SGLT1 transport. Overall, SGLT1-mediated glucose transport and its regulation in Caco-2 cells differ from that in vivo, and caution is advised when extrapolating glucose transport results obtained with this model to the in vivo situation.

Abstract 14844: Ampk is Required for Maintaining Atrial Metabolism and Oxidative Stress

Background: AMP-activated kinase (AMPK) has a critical role in cellular substrate and energy metabolism, regulating fatty acid oxidation, stimulating glucose transport and glycolysis. AMPK is crucial in the LV, preventing ischemic injury and heart failure. Atrial AMPK depletion induces atrial fibrillation in mice, but the role of AMPK in regulating atrial metabolism and oxidative stress is unknown. Methods: Atrial AMPK was selectively depleted in mice, utilizing sarcolipin-Cre mediated deletion of floxed α1 and α2 catalytic subunits (AMPKdKO). Floxed littermate mice were controls (CON). Microarray, immunoblotting, liquid chromatography-mass spectrometry (LC-MS), and electron microscope (EM) were used to access the gene, protein, metabolism, and mitochondria changes. Results: Pathway analysis of microarray data showed that fatty acid metabolism was downregulated in the AMPKdKO vs. CON atria (n=4 per group, p<0.0001). PGC1-α and downstream genes regulating fatty acid metabolism, including acyl-CoA thioesterase (ACOT), long-chain fatty acid-CoA ligase (ACSL), carnitine palmitoyltransferase 2 (CPT2), and fatty acid binding protein (FABP) were reduced in the AMPKdKO vs. CON atria (at 1 week of age). Atrial long-chain fatty acyl-CoA and acyl-carnitine levels were decreased (by LC-MS) in the AMPKdKO vs. CON atria (at 4 and 8 weeks of age) (n=3-4 per group, p<0.05). EM images showed evidence of swollen, broken and degraded mitochondrial in AMPKdKO atria (at 8 weeks age). Atrial expression of antioxidant enzymes, including SOD2 and PRDX3, was reduced (by immunoblotting) in AMPKdKO vs. CON atria (n=3-4, p<0.05). Conclusion: AMPK regulates critical mechanisms regulating atrial fatty acid metabolism and oxidative stress. Loss of atrial AMPK reduces the concentration of critical fatty acid intermediates for oxidative mitochondrial metabolism. These metabolic alterations may contribute to structural and electrical remodeling, and ensuing atrial fibrillation, that results from the loss of AMPK in the atria.

Building GLUT4 Vesicles: CHC22 Clathrin's Human Touch.

Insulin stimulates glucose transport by triggering regulated delivery of intracellular vesicles containing the GLUT4 glucose transporter to the plasma membrane. This process is defective in diseases such as type 2 diabetes (T2DM). While studies in rodent cells have been invaluable in understanding GLUT4 traffic, evolutionary plasticity must be considered when extrapolating these findings to humans. Recent work has identified species-specific distinctions in GLUT4 traffic, notably the participation of a novel clathrin isoform, CHC22, in humans but not rodents. Here, we discuss GLUT4 sorting in different species and how studies of CHC22 have identified new routes for GLUT4 trafficking. We further consider how different sorting-protein complexes relate to these routes and discuss other implications of these pathways in cell biology and disease.

The p21-activated kinase 2 (PAK2), but not PAK1, regulates contraction-stimulated skeletal muscle glucose transport.

AimMuscle contraction stimulates skeletal muscle glucose transport. Since it occurs independently of insulin, it is an important alternative pathway to increase glucose transport in insulin‐resistant states, but the intracellular signaling mechanisms are not fully understood. Muscle contraction activates group I p21‐activated kinases (PAKs) in mouse and human skeletal muscle. PAK1 and PAK2 are downstream targets of Rac1, which is a key regulator of contraction‐stimulated glucose transport. Thus, PAK1 and PAK2 could be downstream effectors of Rac1 in contraction‐stimulated glucose transport. The current study aimed to test the hypothesis that PAK1 and/or PAK2 regulate contraction‐induced glucose transport.MethodsGlucose transport was measured in isolated soleus and extensor digitorum longus (EDL) mouse skeletal muscle incubated either in the presence or absence of a pharmacological inhibitor (IPA‐3) of group I PAKs or originating from whole‐body PAK1 knockout, muscle‐specific PAK2 knockout or double whole‐body PAK1 and muscle‐specific PAK2 knockout mice.ResultsIPA‐3 attenuated (−22%) the increase in glucose transport in response to electrically stimulated contractions in soleus and EDL muscle. PAK1 was dispensable for contraction‐stimulated glucose transport in both soleus and EDL muscle. Lack of PAK2, either alone (−13%) or in combination with PAK1 (−14%), partly reduced contraction‐stimulated glucose transport compared to control littermates in EDL, but not soleus muscle.ConclusionContraction‐stimulated glucose transport in isolated glycolytic mouse EDL muscle is partly dependent on PAK2, but not PAK1.

A peroxidase mimetic protects skeletal muscle cells from peroxide challenge and stimulates insulin signaling.

Reactive oxygen species such as hydrogen peroxide have been implicated in causing metabolic dysfunction such as insulin resistance. Heme groups, either by themselves or when incorporated into proteins, have been shown to scavenge peroxide and demonstrate protective effects in various cell types. Thus, we hypothesized that a metalloporphyrin similar in structure to heme, Fe(III)tetrakis(4-benzoic acid)porphyrin (FeTBAP), would be a peroxidase mimetic that could defend cells against oxidative stress. After demonstrating that FeTBAP has peroxidase activity with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and NADH as reducing substrates, we determined that FeTBAP partially rescued C2C12 myotubes from peroxide-induced insulin resistance as measured by phosphorylation of AKT (S473) and insulin receptor substrate 1 (IRS-1, Y612). Furthermore, we found that FeTBAP stimulates insulin signaling in myotubes and mouse soleus skeletal muscle to about the same level as insulin for phosphorylation of AKT, IRS-1, and glycogen synthase kinase 3β (S9). We found that FeTBAP lowers intracellular peroxide levels and protects against carbonyl formation in myotubes exposed to peroxide. Additionally, we found that FeTBAP stimulates glucose transport in myotubes and skeletal muscle to about the same level as insulin. We conclude that a peroxidase mimetic can blunt peroxide-induced insulin resistance and also stimulate insulin signaling and glucose transport, suggesting a possible role of peroxidase activity in regulation of insulin signaling.

The ULK1/2 and AMPK Inhibitor SBI-0206965 Blocks AICAR and Insulin-Stimulated Glucose Transport.

The small molecule kinase inhibitor SBI-0206965 was originally described as a specific inhibitor of ULK1/2. More recently, it was reported to effectively inhibit AMPK and several studies now report its use as an AMPK inhibitor. Currently, we investigated the specificity of SBI-0206965 in incubated mouse skeletal muscle, measuring the effect on analog 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)-stimulated AMPK-dependent glucose transport and insulin-stimulated AMPK-independent glucose uptake. Pre-treatment with 10 µM SBI-0206965 for 50 min potently suppressed AICAR-stimulated glucose transport in both the extensor digitorum longus (EDL) and soleus muscle. This was despite only a modest lowering of AICAR-stimulated AMPK activation measured as ACC2 Ser212, while ULK1/2 Ser555 phosphorylation was prevented. Insulin-stimulated glucose transport was also potently inhibited by SBI-0206965 in soleus. No major changes were observed on insulin-stimulated cell signaling. No general effect of SBI-0206965 on intracellular membrane morphology was observed by transmission electron microscopy. As insulin is known to neither activate AMPK nor require AMPK to stimulate glucose transport, and insulin inhibits ULK1/2 activity, these data strongly suggest that SBI-0206965 has a non-specific off-target inhibitory effect on muscle glucose transport. Thus, SBI-0206965 is not a specific inhibitor of the AMPK/ULK-signaling axis in skeletal muscle, and data generated with this inhibitor must be interpreted with caution.

Hypoxia preconditioning protects neuronal cells against traumatic brain injury through stimulation of glucose transport mediated by HIF-1\u03b1/GLUTs signaling pathway in rat

Hypoxia preconditioning (HPC), a well-established preconditioning model, has been shown to protect the brain against severe hypoxia or ischemia caused by traumatic brain injury (TBI), but the mechanism has not been well elucidated. Anaerobic glycolysis is the major way for neurons to produce energy under cerebral ischemia and hypoxia after TBI, and it requires large amounts of glucose. We hypothesized that glucose transport, as a rate-limiting step of glucose metabolism, may play key roles in the neuroprotective effects of HPC on cerebral cortex tissue against TBI. The aim of this study was to investigate the effect of HPC on glucose transport activity of rat cerebral cortex tissue after TBI through examining the gene expression of two major glucose transporters (GLUT1 and GLUT3) and their upstream target gene hypoxia-inducible factor-1α (HIF-1α). Sprague-Dawley rats were treated with HPC (50.47 kPa, 3 h/d, 3d). Twenty-four hours after the last treatment, the rats were injured using the Feeney free falling model. Cortex tissues of injured rats were removed at 1 h, 4 h, 8 h, 12 h, 1 day, 3 days, 7 d, and 14 days post-injury for histological analysis. Compared with TBI alone, HPC before TBI resulted in the expression of HIF-1α, GLUT1, and GLUT3 to increase at 1 h; they were markedly increased at 4 h, 8 h, 12 h, 1 day, and 3 days and decreased thereafter (p < 0.05). HPC before TBI could improve neuronal survival in rats by examining NeuN staining and observing reduced apoptosis by examining TUNEL staining. The result showed that HPC before TBI could increase the expression of GLUT1 and GLUT3. And through double immunofluorescence staining for GLUT3 and NeuN, the results strongly suggest that HPC improved glucose transport activity of neurons in rats with TBI. In summary, our results further support that HPC can improve hypoxia tolerance and attenuate neuronal loss of cerebral cortex in rats after TBI. The mechanism is mainly related to the increase of glucose transport activity through inducing GLUT1 and GLUT3 expression through upregulating HIF-1α expression.

Cytosolic ROS production by NADPH oxidase 2 regulates muscle glucose uptake during exercise

Reactive oxygen species (ROS) act as intracellular compartmentalized second messengers, mediating metabolic stress-adaptation. In skeletal muscle fibers, ROS have been suggested to stimulate glucose transporter 4 (GLUT4)-dependent glucose transport during artificially evoked contraction ex vivo, but whether myocellular ROS production is stimulated by in vivo exercise to control metabolism is unclear. Here, we combined exercise in humans and mice with fluorescent dyes, genetically-encoded biosensors, and NADPH oxidase 2 (NOX2) loss-of-function models to demonstrate that NOX2 is the main source of cytosolic ROS during moderate-intensity exercise in skeletal muscle. Furthermore, two NOX2 loss-of-function mouse models lacking either p47phox or Rac1 presented striking phenotypic similarities, including greatly reduced exercise-stimulated glucose uptake and GLUT4 translocation. These findings indicate that NOX2 is a major myocellular ROS source, regulating glucose transport capacity during moderate-intensity exercise.

Perspectives on Insulin Stimulation of Glucose Transport

Perspectives on Insulin Stimulation of Glucose Transport

Chronic AICAR treatment prevents metabolic changes in cardiomyocytes exposed to free fatty acids.

The stimulation of glucose transport by metabolic stress is an important determinant of myocardial susceptibility to ischemia and reperfusion injury. Stimulation of glucose transport is markedly impaired in cardiomyocytes chronically exposed to excess free fatty acids (FFA), as occurs in vivo in type 2 diabetes. To determine whether chronic low-grade activation of AMP-activated kinase (AMPK) improves substrate metabolism in cardiomyocytes exposed to FFA, isolated cultured cardiomyocytes were exposed for 7days to FFA ± the AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). Glucose transport and glycolysis were then measured during acute metabolic stress provoked by oligomycin. Chronic treatment with AICAR improved basal and oligomycin-stimulated glucose transport in FFA-exposed but not in control cardiomyocytes. Similarly, basal and oligomycin-stimulated glycolysis was reduced in FFA-exposed cardiomyocytes but restored by chronic AICAR treatment. Conversely, fatty acid oxidation was increased in FFA-exposed cardiomyocytes and reduced by chronic AICAR treatment. Chronic AICAR treatment induced in FFA-exposed cardiomyocytes the biogenesis of numerous lipid droplets. Curiously, whereas acute treatment of cardiomyocytes with AICAR increased phosphorylation of the AMPKα subunit on T172, a classical marker of AMPK activation, chronic AICAR treatment almost completely obliterated T172 phosphorylation. However, phosphorylation of the AMPK target protein raptor on S792 was reduced in FFA-exposed cardiomyocytes but restored by AICAR treatment. In conclusion, chronic AICAR treatment induces a metabolic shift in FFA-exposed cardiomyocytes, characterized by improved glucose transport and glycolysis and redirection of fatty acids towards neutral storage. Such metabolic changes in vivo could protect the hearts of patients with type 2 diabetes against ischemia-reperfusion injury.

Long term treatment with quercetin in contrast to the sulfate and glucuronide conjugates affects HIF1α stability and Nrf2 signaling in endothelial cells and leads to changes in glucose metabolism

Endothelial functionality profoundly contributes to cardiovascular health. The effects of flavonoids shown to improve endothelial performance include regulating blood pressure by modulating endothelial nitric oxide synthase and NADPH oxidases, but their impact on glucose uptake and metabolism has not been explored. We treated human umbilical vein endothelial cells (HUVEC) with the flavonoid quercetin and its circulating metabolites acutely and chronically, then assessed glucose uptake, glucose metabolism, gene transcription and protein expression. Acute treatment had no effect on glucose uptake, ruling out any direct interaction with sugar transporters. Long term treatment with quercetin, but not quercetin 3-O-glucuronide or 3′-O-sulfate, significantly increased glucose uptake. Heme oxygenase-1 (HO-1) was induced by quercetin but not its conjugates, but was not implicated in the glucose uptake stimulation since hemin, a classical inducer of HO-1, did not affect glucose metabolism. Quercetin increased stability of the transcription factor hypoxia induced factor 1α (HIF1α), a powerful stimulant of glucose metabolism, which was also paralleled by treatment with a prolyl-4-hydroxylase inhibitor dimethyloxalylglycine (DMOG). 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), which regulates the rate of glycolysis, was upregulated by both quercetin and DMOG. Pyruvate dehydrogenase kinase (PDK) isoforms regulate pyruvate dehydrogenase; PDK2 and PDK4 were down-regulated by both effectors, but only DMOG also upregulated PDK1 and PDK3. Quercetin, but not DMOG, increased glucose-6-phosphate dehydrogenase. Chronic quercetin treatment also stimulated glucose transport across the HUVEC monolyer in a 3D culture model. Gene expression of several flavonoid transporters was repressed by quercetin, but this was either abolished (Organic anion transporter polypeptide 4C1) or reversed (Multidrug resistance gene 1) by both conjugates. We conclude that quercetin and its circulating metabolites differentially modulate glucose uptake/metabolism in endothelial cells, through effects on HIF1α and transcriptional regulation of energy metabolism.

OR05-5 AZP-3404, a 9-Amino Acid Peptide Analog of Insulin-Like Growth Factor-Binding Protein 2, Reverses Insulin Resistance in Leptin-Deficient ob/ob Mice

Insulin-like growth factor-binding protein-2 (IGFBP-2), one of six highly-conserved binding proteins that modulate circulating IGF1, has significant regulatory actions on carbohydrate, lipid and bone metabolism that are independent of its IGF-1 binding role. The metabolic activity of IGFBP-2 has been localized to a short peptide sequence within its unique heparin-binding domain, HBD-1, that is able to reproduce the metabolic actions of the full IGFBP-2 on bone and fat. Through structure-activity studies of this sequence, an acylated 9-amino acid peptide, AZP-3404, with enhanced stability and pharmacokinetics, has been developed that retains the activity of IGFBP-2 on fat and bone metabolism. As IGFBP-2 treatment has also been demonstrated to improve glucose homeostasis in animal models of severe insulin resistance, including the leptin-deficient ob/ob mouse, the present study was undertaken to determine whether AZP-3404 is able to reverse insulin resistance and improve glucose homeostasis in the ob/ob mouse. Male B6.Cg-Lepob/J mice were implanted with HD-XG telemetric devices (Data Sciences International), which allows continuous, wireless monitoring of carotid blood glucose under awake, unrestrained conditions. Animals were randomly assigned to groups (n=8) receiving either saline or AZP-3404 at 1 or 3 mg/kg, b.i.d. for four weeks. The mean daily blood glucose level prior to study initiation was 507 ± 102 mg/dL. Treatment with AZP-3404 dose-dependently decreased the average daily glucose concentration, such that by day 27, glucose levels were reduced by 64 ± 29 and 100 ± 16 mg/dL in the 1 and 3 mg/kg AZP-3404-treated groups, respectively, while slightly increased by 14 ± 29 mg/dL in the vehicle-treated group. On days 7, 14, 21 and 28, following a 24-hour fast, the mice were given an intraperitoneal glucose tolerance test (IPGTT: 1g glucose/kg) to evaluate the impact of AZP-3404 on glucose disposal which is impaired in ob/ob mice. By day 28, the fasting glucose level was reduced by 8% and 20% in the 1 and 3 mg/kg AZP-3404-treated groups, respectively, as compared with fasting levels on day 7. The IPGTTs revealed a progressive, dose-dependent reduction in glucose excursion that reached a decrease of 40% after 4 weeks of treatment with 3mg/kg AZP-3404. In related studies, we have observed that AZP-3404, both alone and in an additive manner with insulin, stimulates glucose transport in mouse C2C12 differentiated myotubes through activation of AMP-activated protein kinase (AMPK). These findings suggest that AZP- 3404 stimulates glucose uptake by muscle tissue in ob/ob mice thereby improving glucose homeostasis. The results from these studies confirm that AZP-3404 retains the ability of IGFBP-2 to restore glucose homeostasis and support the development of AZP-3404 as a novel therapeutic approach for syndromes of severe insulin resistance.

A Metalloporphyrin that Serves as a Peroxidase and Stimulates Insulin Signaling in Skeletal Muscle

Hydrogen peroxide is a form of reactive oxygen species (ROS) that has been associated with metabolic dysfunction such as insulin resistance. We hypothesized that the metalloporphyrin Fe(III)tetrakis(4‐benzoic acid)porphyrin (FeTBAP), which is a peroxidase mimetic, would protect skeletal muscle cells from the damaging effects of peroxide. To determine whether FeTBAP could be a means to deliver peroxidase activity to living cells, we incubated C2C12 myotubes with FeTBAP. We then confirmed in SDS polyacrylamide gels that cell lysates contained FeTBAP's peroxidase activity by the appearance of blue bands corresponding to oxidized tetramethylbenzidine (TMB) when incubated in TMB and peroxide. In order to determine whether FeTBAP could lower peroxide levels in cells challenged with peroxide, we transfected C2C12 myoblasts with the cytosolic, ratiometric fluorescent peroxide sensor, HyPer3. Cells were then pretreated with FeTBAP and finally exposed to glucose oxidase (GO), an enzyme that utilizes glucose to produce hydrogen peroxide. Exposure to GO increased the Hyper3 fluorescence ratio, indicating higher cytosolic peroxide concentration. This increased intracellular peroxide was prevented in cells that were pretreated with FeTBAP. Carbonyl formation, a marker of oxidative protein modification, was also monitored in myotubes pretreated with FeTBAP and exposed to glucose oxidase. We found that there was an increase in carbonyl content in cells exposed to GO, while prior treatment with FeTBAP prevented an increase in carbonyl formation. To assess the ability of FeTBAP to protect myotubes from peroxide induced insulin resistance, cells were pretreated with FeTBAP followed by incubation in GO and then insulin. Exposure to GO decreased insulin‐stimulated phosphorylation of insulin receptor substrate 1 (IRS‐1, Y612). Prior treatment of cells to FeTBAP rescued myotubes from this peroxide induced insulin resistance. Next, we asked whether FeTBAP could impact insulin signaling in the absence of insulin. C2C12 myotubes and mouse soleus muscle were pretreated with FeTBAP followed by an incubation in insulin. We found that FeTBAP increases insulin signaling in myotubes and soleus to about the same level as insulin for phosphorylation of AKT (S473), IRS‐1 (Y612), and glycogen synthase kinase 3 beta (S9). Further, we found that FeTBAP stimulates glucose transport in myotubes. We conclude that FeTBAP can blunt peroxide induced insulin resistance and also stimulate insulin signaling and glucose transport, suggesting that it could be a lead compound for new approaches to prevention of insulin resistance.Support or Funding InformationThis project was supported by United States Public Health Service award R15DK102122 from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Efektivitas antara Brisk Walk Exercise dan Relaksasi Otot Progresif terhadap Penurunan Kadar Gula Darah pada Diabetes Mellitus tipe 2

Kadar gula darah pada pasien diabetes mellitus tipe 2 dapat diturunkan dengan terapi brisk walk exercise dan relaksasi otot progresif. Hal ini dikarenakan bila latihan jalan kaki dilakukan secara teratur dapat merangsang sintesis glikogen melalui peningkatan aksi insulin dan merangsang transportasi glukosa oleh tranporter glukosa GLUT4. Sedangkan ketika relaksasi otot progresif dilakukan secara teratur mampu menghambat jalur stressor sehingga memperkuat sikap positif terhadap rangsangan stress dan mengakibatkan hipotalamus berkurang. Penelitian ini bertujuan untuk mengetahui efektivitas antara Brisk Walk Exercise dan Relaksasi Otot Progresif terhadap penurunan kadar gua darah pada Diabetes Mellitus Tipe 2. Desain penelitian ini menggunakan Quasy Eksperimental dengan jenis rancangan Pre-test and Post-test Group Design dengan teknik pengambilan sample consecutive sampling. Besar Sample adalah 30 responden, dibagi menjadi 2 kelompok yaitu kelompok Brisk Walk Exercise dan kelompok RelaksasiEfektivitas Antara Brisk Walk Exercise Dan Relaksasi Otot...60 pISSN 2301-4024 eISSN 2442-6873Otot Progresif. Hasil uji Paired T-test kelompok Brisk Walk Exercise didapatkan nilai Pvalue =0,000&lt;α=0.05 hal ini berarti ada perbedaan yang signifikan. Sedangkan pada kelompok Relaksasi Otot Progresif nilai Pvalue=0,000&lt;α=0.05 berarti ada perbedaan yang signifikan. Pada uji Paired T-test didapatkan nilai Pvalue =0,000&lt;α=0.05 artinya ada perbedaan bermakna antara Brisk Walk Exercise dan Relaksasi Otot Progresif. Sebagai perawat diharapkan dapat menerapkan terapi non farmakologis seperti Brisk Walk Exercise dan Relaksasi Otot Progresif sebagai terapi alternatif pilihan pasien yang mudah untuk dilakukan untuk menurunkan kadar gula darah.

The deubiquitinating enzyme USP25 binds tankyrase and regulates trafficking of the facilitative glucose transporter GLUT4 in adipocytes

Key to whole body glucose homeostasis is the ability of fat and muscle cells to sequester the facilitative glucose transporter GLUT4 in an intracellular compartment from where it can be mobilized in response to insulin. We have previously demonstrated that this process requires ubiquitination of GLUT4 while numerous other studies have identified several molecules that are also required, including the insulin-responsive aminopeptidase IRAP and its binding partner, the scaffolding protein tankyrase. In addition to binding IRAP, Tankyrase has also been shown to bind the deubiquinating enzyme USP25. Here we demonstrate that USP25 and Tankyrase interact, and colocalise with GLUT4 in insulin-sensitive cells. Furthermore depletion of USP25 from adipocytes reduces cellular levels of GLUT4 and concomitantly blunts the ability of insulin to stimulate glucose transport. Collectively, these data support our model that sorting of GLUT4 into its insulin-sensitive store involves a cycle of ubiquitination and subsequent deubiquitination.

EGCG regulation of non-insulin-responsive endosomal compartments in insulin-resistant skeletal muscle

Tea consumption reduces the risk of type 2 diabetes mellitus, but how (-)-epigallocatechin-3-gallate (EGCG), the most abundant catechin in tea, mediates glucose transporter (GLUT)-containing vesicles in muscle cells is still unknown. This study investigated the hypothesis that EGCG can stimulate glucose transport by regulating endosome-specific signaling control systems, thus enhancing glucose uptake by skeletal muscle cells. To understand the relationship between the endosomal system and the EGCG-regulated pathway in skeletal muscle, the insulin-resistance L6 myotubes, induced by palmitate treatment, were treated with vesicle ablation, actin filament disruption and signaling inhibition and results were analyzed. The horseradish peroxidase-conjugated human transferrin was localized to endosomes in the cells, followed by incubation with diaminobenzidine/H2O2. The unablated cell-associated glucose uptake was determined using quantitative fluorescence assay. Ablation of endosomal compartments showed that insulin had little effect on the endosomal recycling pathway, and it mainly affected GLUT4 storage vesicles. Conversely, EGCG stimulated endosomal compartments to facilitate glucose transport using a mechanism independent of the actin-based cytoskeletal transit system. Both Akt and atypical protein kinase C, the downstream effectors of phosphatidylinositol-3-kinase (PI3K), participated in the development of palmitate-induced muscle cells insulin resistance. However, EGCG-stimulated glucose uptake is mediated using a PI3K-independent mechanism that involves silent information regulator 1. Thus, glucose transport is regulated by at least two signal cascades and by two types of vesicles, and EGCG can improve glucose uptake in insulin-resistant skeletal muscle through the non-insulin-responsive endosomal pathway.

Angiotensin II Modulates Podocyte Glucose Transport.

Podocytes play a central role in the maintenance of the glomerular filtration barrier and are cellular targets of angiotensin II (AngII). Non-hemodynamic pathways of AngII signaling regulate cellular function and mediate podocyte abnormalities that are associated with various glomerulopathies, including diabetic kidney disease. In this study we investigated the capacity of AngII to modulate glucose uptake in mouse podocytes expressing the human AT1 receptor (AT1R+) after 5 days of exposure to normal (NG, 5.6 mmol/L) or to high (HG, 30 mmol/L) glucose. Short (30 min) as well as long-term (24 h) incubations with AngII markedly enhanced glucose transport in both NG and HG cells. In podocytes cultured under NG conditions, AngII inhibited insulin-stimulated glucose uptake. Regardless of the presence or absence of AngII, no effect of insulin on glucose uptake was observed in HG cells. Stimulation of glucose transport by AngII was mediated by protein kinase C and by phosphoinositide 3-kinase. Glucose dependent surface expression of the glucose transporters GLUT1, GLUT2, and GLUT4 was modulated by AngII in a time and glucose concentration dependent manner. Furthermore, despite its inhibitory effect on insulin's action, AngII elevated the number of podocyte insulin receptors in both NG and HG cultured cells. These findings demonstrate that AngII modulates podocyte basal, as well as insulin-dependent glucose uptake by regulating glucose transporters and insulin signaling.

High glucose induces Glucose transporter‐2 (GLUT2), but not Na‐glucose cotransporter‐1 (SGLT1) mediated glucose absorption

BackgroundGlucose dependent insulinotropic peptide (GIP) is a hormone that has a cyclic pathway in the stimulation of glucose transport. GIP increases transport of glucose, which in turn stimulates GIP secretion. This cycle increases the jejunal absorption of glucose, which stimulates insulin release. High glucose results in excess GIP secretion that causes unusually high glucose absorption and high blood glucose that stimulates insulin release that causes insulin insensitivity. Presence of high blood glucose and insulin lead to obesity and Type‐2 diabetes. Although SGLT1 is the primary apical glucose absorptive process, GLUT2 has been localized on the intestinal apical membranes of obese human and mouse models. However, signaling mechanisms of how GIP regulates glucose transporters in the presence of normal versus high levels of extracellular glucose concentrations is not known.HypothesisExtracellular glucose concentration that alters cellular GIP level would regulate glucose transporters on to the apical membrane of differentiated cells.MethodsIntestinal epithelial crypt cell‐6 (IEC6 cells) where grown 5‐days post confluent on transwell snapwells using standard DMEM media supplemented with 5% FBS and either 4.5 or 0.45 g/liter of glucose for high and low glucose, respectively. Mucosal to serosal (m‐s) glucose fluxes were measured across IEC6 cell layers that were mounted under voltage clamp conditions in Ussing chambers. Short circuit currents were measured to distinguish the SGLT1 (i.e. electrogenic) and GLUT2 (i.e. electroneutral) mediated glucose absorption. Apical membranes were isolated from IEC6 cell monolayers using a biotinylation technique. Western blot analyses were performed using SGLT1 and GLUT2 antibodies.ResultsGlucose absorption is substantially higher in IEC6 cells that were grown in high glucose medium compared to that grown in low glucose medium. In contrast to increased rate of glucose absorption, Isc is not significantly altered as an indication that high glucose increased GLUT2 mediated, but not SGLT1 mediated glucose absorption in IEC6 cells. Western blot analyses revealed that GLUT2, but not SGLT1 specific protein expression is increased on the apical membrane of IEC6 cells grown in the high glucose medium.ConclusionsHigh extracellular glucose environment stimulates GLUT2, but not SGLT1 expression and GLUT2 mediated glucose absorption in intestinal epithelial cells.SpeculationWe speculate that GLUT2 might be regulated similarly in in the villus cells of mouse intestine.Support or Funding InformationIMBRE grant #G110‐18‐W6619This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation of bombesin receptor Subtype-3 by [D-Tyr6,β-Ala11,Phe13,Nle14]bombesin6-14 increased glucose uptake and lipogenesis in human and rat adipocytes

BRS-3 has an important role in glucose homeostasis. Its expression was reduced in skeletal muscle from obese and/or diabetic patients, and BRS-3 KO-mice developed obesity. In this work, focused on rat/human adipose tissue, BRS-3 gene-expression was lower than normal-levels in hyperlipidemic, type-2-diabetic (T2D), and type-1-diabetic rats and also in obese (OB) and T2D patients. Moreover, BRS-3 protein levels were decreased in diabetic rat and in obese and diabetic human fat pieces; but neither mutation nor even polymorphism in the BRS-3-gene was found in OB or T2D patients. Interestingly, in rat and human adipocytes, without metabolic alterations, [D-Tyr6,β-Ala11,Phe13,Nle14]bombesin6-14 -BRS-3-agonist-, as insulin, enhanced BRS-3 gene/protein expression, increased, PKB, p70s6K, MAPKs and p90RSK1 phosphorylation-levels, and induced a concentration-related stimulation of glucose transport, GLUT-4 membrane translocation and lipogenesis, exclusively mediated by BRS-3, and abolished by wortmannin, PD98059 or rapamacyn. These results confirm that BRS-3 and/or its agonist are a potential therapeutic tool for obesity/diabetes.

Mechanism of insulin-like effect of chromium(III) ions on glucose uptake in C2C12 mouse myotubes involves ROS formation

Chromium is considered a trace element which improves glucose tolerance, but mechanism accounting for this insulin-like action is not recognized. The main purpose of this study was to examine the role of reactive oxygen species (ROS) in chromium and insulin stimulated glucose transport using antioxidants. Effect of chromium ions on phosphatases, enzymes involved in inhibition of insulin signaling was also investigated. Experiments were performed in vitro on C2C12 mouse myotubes. ROS level was measured with the use of confocal microscope and 2’,7’ dichlorodihydrofluorescein diacetate (DCFH-DA). Glucose metabolism was assayed by the measurement of 2-[3H]-deoxyglucose uptake. Cr3+ ions and insulin treatment caused significant increase of ROS formation and also stimulated glucose uptake in C2C12 cells in concentration dependent manner. Antioxidants (L-ascorbic acid and N-acetyl cysteine 100μM) and DPI (diphenyleneiodonium-NADPH oxidase inhibitor, 10μM) abolished insulin- and Cr-inducted glucose transport. Our results confirm the hypothesis that the ROS are integral part of insulin signaling pathway and that the insulin mimetic effect of Cr3+ ions depends on the antioxidant status of the cells. Surprisingly, chromium treatment resulted in increased activity of membrane phosphatases.