Contractile activity per se induces transcriptional activation of SLC2A4 gene in soleus muscle: involvement of MEF2D, HIF-1a, and TR alpha transcriptional factors

Lima GA, Anhe GF, Giannocco G, Nunes MT, Correa-Giannella ML, Machado UF. Contractile activity per se induces transcriptional activation of SLC2A4 gene in soleus muscle: involvement of MEF2D, HIF-1a, and TR alpha transcriptional factors. Am J Physiol Endocrinol Metab 296: E132-E138, 2009. First published October 28, 2008; doi: 10.1152/ajpendo.90548.2008.-Skeletal muscle is a target tissue for approaches that can improve insulin sensitivity in insulin-resistant states. In muscles, glucose uptake is performed by the GLUT-4 protein, which is encoded by the SLC2A4 gene. SLC2A4 gene expression increases in response to conditions that improve insulin sensitivity, including chronic exercise. However, since chronic exercise improves insulin sensitivity, the increased SLC2A4 gene expression could not be clearly attributed to the muscle contractile activity per se and/or to the improved insulin sensitivity. The present study was designed to investigate the role of contractile activity per se in the regulation of SLC2A4 gene expression as well as in the participation of the transcriptional factors myocyte enhancer factor 2D (MEF2D), hypoxia inducible factor 1a (HIF-1a), and thyroid hormone receptor-alpha (TR alpha). The performed in vitro protocol excluded the interference of metabolic, hormonal, and neural effects. The results showed that, in response to 10 min of electrically induced contraction of soleus muscle, an early 40% increase in GLUT-4 mRNA (30 min) occurred, with a subsequent 65% increase (120 min) in GLUT-4 protein content. EMSA and supershift assays revealed that the stimulus rapidly increased the binding activity of MEF2D, HIF-1a, and TR alpha into the SLC2A4 gene promoter. Furthermore, chromatin immunoprecipitation assay confirmed, in native nucleosome, that contraction induced an approximate fourfold (P < 0.01) increase in MEF2D and HIF-1a-binding activity. In conclusion, muscle contraction per se enhances SLC2A4 gene expression and that involves MEF2D, HIF-1a, and TR alpha transcription factor activation. This finding reinforces the importance of physical activity to improve glycemic homeostasis independently of other additional insulin sensitizer approaches.

Frequency distribution of XbaIG > T and HaeIIIT > C GLUT1 polymorphisms among different Brazilian ethnic groups

GLUT is the major glucose transporter in mammalian cells. Single nucleotide polymorphisms (SNP) at GLUT1 promoter and regulatory regions have been associated to the risk of developing nephropathy in different type 1 and type 2 diabetic populations. It has been demonstrated that differences in allelic and genotypic frequencies of GLUT1 gene (SLC2A1) polymorphisms occur among different populations. Therefore, ethnic differences in distribution of GLUT1 gene polymorphisms may be an important factor in determining gene-disease association. In this study, we investigated the XbaIG > T and HaeIIIT > C polymorphisms in six different Brazilian populations: 102 individuals from Salvador population (Northern Brazil), 56 European descendants from Joinville (South Brazil), 85 Indians from Tiryi tribe (North Brazil) and 127 samples from Southern Brazil: 44 from European descendants, 42 from African descendants and 41 from Japanese descendants. Genotype frequencies from both sites did not differ significantly from those expected under the Hardy-Weinberg equilibrium. We verified that the allele frequencies of both polymorphisms were heterogeneous in these six Brazilian ethnic groups.

Potential Role of Growth Hormone in Impairment of Insulin Signaling in Skeletal Muscle, Adipose Tissue, and Liver of Rats Chronically Treated with Arginine

We have shown that rats chronically treated with Arginine (Arg), although normoglycemic, exhibit hyperinsulinemia and decreased blood glucose disappearance rate after an insulin challenge. Attempting to investigate the processes underlying these alterations, male Wistar rats were treated with Arg (35 mg/d), in drinking water, for 4 wk. Rats were then acutely stimulated with insulin, and the soleus and extensorum digitalis longus muscles, white adipose tissue (WAT), and liver were excised for total and/or phosphorylated insulin receptor (IR), IR substrate 1/2, Akt, Janus kinase 2, signal transducer and activator of transcription (STAT) 1/3/5, and p85 alpha/55 alpha determination. Muscles and WAT were also used for plasma membrane (PM) and microsome evaluation of glucose transporter (GLUT) 4 content. Pituitary GH mRNA, GH, and liver IGF-I mRNA expression were estimated. It was shown that Arg treatment: 1) did not affect phosphotyrosine-IR, whereas it decreased phosphotyrosine-IR substrate 1/2 and phosphoserine-Akt content in all tissues studied, indicating that insulin signaling is impaired at post-receptor level; 2) decreased PM GLUT4 content in both muscles and WAT; 3) increased the pituitary GH mRNA, GH, and liver IGF-I mRNA expression, the levels of phosphotyrosine-STAT5 in both muscles, phosphotyrosine-Janus kinase 2 in extensorum digitalis longus, phosphotyrosine-STAT3 in liver, and WAT as well as total p85 alpha in soleus, indicating that GH signaling is enhanced in these tissues; and 4) increased p55 alpha total content in muscles, WAT, and liver. The present findings provide the molecular mechanisms by which insulin resistance and, by extension, reduced GLUT4 content in PM of muscles and WAT take place after chronic administration of Arg, and further suggest a putative role for GH in its genesis, considering its diabetogenic effect. (Endocrinology 150: 2080-2086, 2009)

Insulin resistance of pregnancy involves estrogen-induced repression of muscle GLUT4

Pregnancy is accompanied by hyperestrogenism, however, the role of estrogens in the gestational-induced insulin resistance is unknown. Skeletal muscle plays a fundamental role in this resistance, where GLUT4 regulates glucose uptake. We investigated: (1) effects of oophorectomy and estradiol (E2) on insulin sensitivity and GLUT4 expression. E2 (similar to 200 nM) for 7 days decreased sensitivity, reducing similar to 30% GLUT4 mRNA and protein (P< 0.05) and plasma membrane expression in muscle; (2) the expression of ER alpha and ER beta in L6 myotubes, showing that both coexpress in the same nucleus; (3) effects of E2 on GLUT4 in L6, showing a time- and dose-dependent response. High concentration (100 nM) for 6 days reduced similar to 25% GLUT4 mRNA and protein (P < 0.05). Concluding, E2 regulates GLUT4 in muscle, and at high concentrations, such as in pregnancy, reduces GLUT4 expression and, in vivo, decreases insulin sensitivity. Thus, hyperestrogenism may be involved in the pregnancy-induced insulin resistance and/or gestational diabetes. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Glucose-Induced Regulation of NHEs Activity and SGLTs Expression Involves the PKA Signaling Pathway

The effect of glucose on the intracellular pH (pH(i)) recovery rate (dpH(i)/dt) and Na(+)-glucose transporter (SGLT) localization was investigated in HEK-293 cells, a cell line that expresses endogenous NHE1, NHE3, SGLT1, and SGLT2 proteins. The activity of the Na(+)/H(+) exchangers (NHEs) was evaluated by using fluorescence microscopy. The total and membrane protein expression levels were analyzed by immunoblotting. In cells cultivated in 5 mM glucose, the pH(i) recovery rate was 0.169 +/- A 0.020 (n = 6). This value did not change in response to the acute presence of glucose at 2 or 10 mM, but decreased with 25 mM glucose, an effect that was not observed with 25 mM mannitol. Conversely, the chronic effect of high glucose (25 mM) increased the pH(i) recovery rate (similar to 40%, P < 0.05), without changes in the total levels of NHE1, NHE3, or SGLT1 expression, but increasing the total cellular (similar to 50%, P < 0.05) and the plasma membrane (similar to 100%, P < 0.01) content of SGLT2. Treatment with H-89 (10(-6) M) prevented the stimulatory effect of chronic glucose treatment on the pH(i) recovery rate and SGLT2 expression in the plasma membrane. Our results indicate that the effect of chronic treatment with a high glucose concentration is associated with increased NHEs activity and plasma membrane expression of SGLT2 in a protein kinase A-dependent way. The present results reveal mechanisms of glucotoxicity and may contribute to understanding the diabetes-induced damage of this renal epithelial cell.

Hyperglycaemia protects the heart after myocardial infarction: aspects of programmed cell survival and cell death

Exposure to a high glucose medium or diabetes has been found to protect the heart against ischaemia. The activation of antiapoptotic and proliferative factors seems to be involved in this cardioprotection. This study was designed to evaluate the role of hyperglycaemia in cardiac function, programmed cell survival, and cell death in diabetic rats after myocardial infarction (MI). Male Wistar rats were divided into four groups (n = 8): control (C), diabetic (D), myocardial infarcted (MI), and diabetic myocardial infarcted (DI). The following measures were assessed in the left ventricle: size of MI, systolic and diastolic function by echocardiography, cytokines by ELISA (TNF-alpha, IL-1 beta, IL-6, and IL-10), gene expression by real-time PCR (Bax, Fas, p53, Bcl-2, HIF1-alpha, VEGF, and IL8r), caspase-3 activity by spectrofluorometric assay, glucose transporter type 1 and 4 (GLUT-1 and GLUT-4) protein expression by western blotting, and capillary density and fibrosis by histological analysis. Systolic function was improved by hyperglycaemia in the DI group, and this was accompanied by no improvement in diastolic dysfunction, a reduction of 36% in MI size, reduced proinflammatory cytokines, apoptosis activation, and an increase in cell survival factors (HIF1-alpha, VEGFa and IL8r) assessed 15 days post-MI. Moreover, hyperglycaemia resulted in angiogenesis (increased capillary density) before and after MI, accompanied by a reduction in fibrosis. Together, these results suggest that greater plasticity and cellular resistance to ischaemic injury result from chronic diabetic hyperglycaemia in rat hearts.

beta-ADRENERGIC ACTIVITY PRESERVES GLUT4 PROTEIN IN GLYCOLYTIC FIBERS IN FASTING

Glucose transporter 4 (GLUT4) expression in adipose tissue decreases during fasting. In skeletal muscle, we hypothesized that GLUT4 expression might be maintained in a beta-adrenergic-dependent way to ensure energy disposal for contractile function. Herein we investigate beta-blockade or beta-stimulation effects on GLUT4 expression in oxidative (soleus) and glycolytic [extensor digitorum longus (EDL)] muscles of fasted rats. Fasting increased GLUT4 mRNA in soleus (24%) and EDL (40%) but the protein content increased only in soleus (30%). beta 1-beta 2-, and beta 1-beta 2-beta 3-blockade decreased (20-30%) GLUT4 mRNA content in both muscles, although GLUT4 protein decreased only in EDL. When mRNA and GLUT4 protein regulations were discrepant, changes in the mRNA poly(A) tail length were detected, indicating a posttranscriptional modulation of gene expression. These results show that beta-adrenergic activity regulates GLUT4 gene expression in skeletal muscle during fasting, highlighting its participation in preservation of GLUT4 protein in glycolytic muscle. Muscle Nerve 40: 847-854, 2009

Endurance training activates AMP-activated protein kinase, increases expression of uncoupling protein 2 and reduces insulin secretion from rat pancreatic islets

Endurance exercise is known to enhance peripheral insulin sensitivity and reduce insulin secretion. However, it is unknown whether the latter effect is due to the reduction in plasma substrate availability or alterations in beta-cell secretory machinery. Here, we tested the hypothesis that endurance exercise reduces insulin secretion by altering the intracellular energy-sensitive AMP-activated kinase (AMPK) signaling pathway. Male Wistar rats were submitted to endurance protocol training one, three, or five times per week, over 8 weeks. After that, pancreatic islets were isolated, and glucose-induced insulin secretion (GIIS), glucose transporter 2 (GLUT2) protein content, total and phosphorylated calmodulin kinase kinase (CaMKII), and AMPK levels as well as peroxisome proliferator-activated receptor-gamma coactivator-1-alpha (PGC-1 alpha) and uncoupling protein 2 (UCP2) content were measured. After 8 weeks, chronic endurance exercise reduced GIIS in a dose-response manner proportionally to weekly exercise frequency. Contrariwise, increases in GLUT2 protein content, CaMKII and AMPK phosphorylation levels were observed. These alterations were accompanied by an increase in UCP2 content, probably mediated by an enhancement in PGC-1 alpha protein expression. In conclusion, chronic endurance exercise induces adaptations in beta-cells leading to a reduction in GIIS, probably by activating the AMPK signaling pathway. Journal of Endocrinology (2011) 208, 257-264

Anti-inflammatory effect of atorvastatin ameliorates insulin resistance in monosodium glutamate-treated obese mice

Considering that inflammation contributes to obesity-induced insulin resistance and that statins have been reported to have other effects beyond cholesterol lowering, the present study aimed to it whether atorvastatin treatment has anti-inflammatory action in white adipose tissue of obese mice, consequently improving insulin sensitivity. Insulin sensitivity in vivo (by insulin tolerance test); metabolic-hormonal profile; plasma tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and adiponectin; adipose tissue immunohistochemistry; glucose transporter (GLUT) 4; adiponectin; INF-alpha; IL-1 beta; and IL-6 gene expression; and I kappa B kinase (IKK)-alpha/beta activity were assessed in 23-week-old monosodium glutamate induced obese mice untreated or treated with atorvastatin for 4 weeks. Insulin-resistant obese mice had increased plasma triglyceride, insulin, TNF-alpha, and IL-6 plasma levels. Adipose tissue of obese animals showed increased macrophage infiltration, IKK-alpha (42%, P < .05) and IKK-beta (73%, P < .05) phosphorylation, and INF-alpha and IL-6 messenger RNA (mRNA) (similar to 15%, P < .05) levels, and decreased GLUT4 mRNA and protein (30%, P < .05) levels. Atorvastatin treatment lowered cholesterol, triglyceride, insulin, INF-alpha, and IL-6 plasma levels, and restored whole-body insulin sensitivity. In adipose tissue, atorvastatin decreased macrophage in and normalized IKK-alpha/beta phosphorylation; INF-alpha, IL-6, and GLUT4 mRNA; and GLUT4 protein to control levels. The present findings demonstrate that atorvastatin has anti-inflammatory effects on adipose tissue of obese mice, which may be important to its local and whole-body insulin-sensitization effects. (C) 2010 Published by Elsevier Inc.

Treinamento resistido reduz inflamação em músculo esquelético e melhora a sensibilidade à insulina periférica em ratos obesos induzidos por dieta hiperlipídica

OBJETIVO: Investigar em ratos obesos o efeito da prática de exercício resistido sobre a sensibilidade à insulina e sobre a expressão de citocinas pró-inflamatórias e de transportador de glicose em músculo solear. MATERIAIS E MÉTODOS: Ratos Wistar alimentados com dieta hiperlipídica (grupos obesos) foram submetidos ao protocolo de exercício tipo jump squat. A sensibilidade à insulina e a expressão gênica de Tnf-α, SOCS3 e GLUT4 foram comparadas entre os grupos obesos sedentários (OS) e exercitados (OE) e controles sedentários (CS) e exercitados (CE). RESULTADOS: A sensibilidade à insulina estava reduzida no grupo OS e elevada no OE. Os conteúdos de RNAm de Tnf-α e de SOCS3 estavam aumentados no músculo esquelético do grupo OS e reduzidos no OE. O conteúdo proteico e de RNAm de GLUT4 não diferiu entre os grupos. CONCLUSÃO: O exercício resistido reverte o quadro de resistência à insulina periférica e de inflamação no músculo esquelético de obesos induzidos por dieta.

Performance evaluation and phylogenetic characterization of anaerobic fluidized bed reactors using ground tire and pet as support materials for biohydrogen production

This study evaluated two different support materials (ground tire and polyethylene terephthalate [PET]) for biohydrogen production in an anaerobic fluidized bed reactor (AFBR) treating synthetic wastewater containing glucose (4000 mg L(-1)). The AFBR, which contained either ground tire (R1) or PET (R2) as support materials, were inoculated with thermally pretreated anaerobic sludge and operated at a temperature of 30 degrees C. The AFBR were operated with a range of hydraulic retention times (HRT) between 1 and 8 h. The reactor R1 operating with a HRT of 2 h showed better performance than reactor R2, reaching a maximum hydrogen yield of 2.25 mol H(2) mol(-1) glucose with 1.3 mg of biomass (as the total volatile solids) attached to each gram of ground tire. Subsequent 16S rRNA gene sequencing and phylogenetic analysis of particle samples revealed that reactor R1 favored the presence of hydrogen-producing bacteria such as Clostridium, Bacillus, and Enterobacter. (C) 2010 Elsevier Ltd. All rights reserved.

Biohydrogen production in anaerobic fluidized bed reactors: Effect of support material and hydraulic retention time

This study evaluated two different support materials (polystyrene and expanded clay) for biohydrogen production in an anaerobic fluidized bed reactor (AFBR) treating synthetic wastewater containing glucose (4000 mg L(-1)). The AFBRs contained either polystyrene (R1) or expanded clay (R2) as support materials were inoculated with thermally pre-treated anaerobic sludge and operated at a temperature of 30 degrees C and a pH of approximately 5.5. The AFBRs were operated with a range of hydraulic retention times (HRTs) between 1 and 8 h. For R1 with an HRT of 2 h, the maximum hydrogen yield (HY) was 1.90 mol H(2) mol(-1) glucose, with 0.805 mg of biomass (as total volatile solids, or TVS) attached to each g of polystyrene. For R2 operated at an HRT of 2 h, the maximum HY was 2.59 mol H(2) moll glucose, with 1.100 mg of attached biomass (as TVS) g(-1) expanded clay. The highest hydrogen production rates (HPR) were 0.95 and 1.21 L h(-1) L(-1) for R1 and R2, respectively, using an HRT of 1 h. The H(2) content increased from 16-47% for R1 and from 22-51% for R2. No methane was detected in the biogas produced throughout the period of AFBR operation. These results show that the values of HY, HPR, H(2) content, and g of attached biomass g(-1) support material were all higher for AFBRs containing expanded clay than for reactors containing polystyrene. (C) 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

Absorption and metabolism of cyanidin-3-glucoside and cyanidin-3-rutinoside extracted from wild mulberry (Morus nigra L.) in rats

The mechanism of uptake of anthocyanins (as well as the type) from food in the intestine is not clear. Anthocyanin-rich extract from wild mulberry, composed of cyanidin-3-glucoside (79%) and cyanidin-3-rutino side (cy-3-rut) (19%), was orally administered to Wistar rats, and their concentrations were determined in plasma, kidney, and the gastrointestinal (GI) tract. The 2 glycosylated forms showed maximum concentration at 15 minutes after oral administration, both in plasma and kidney. The cyanidin-3-glucoside and cy-3-rut were found in plasma as glucuronides, as sulfates of cyanidin, and as unchanged forms. The area under the curve of concentration vs time (AUC(0-8h)) was 2.76 +/- 0.88 mu g hour/mL and 9.74 +/- 0.75 mu g hour/g for plasma and kidney, respectively. In spite of the low absorption, the increase in plasma anthocyanin level resulted in a significant increase in antioxidant capacity (P < .05). In the GI tract (stomach and small and large intestines), cyanidin glycosides were found unchanged, but a low amount of the aglycone form was present. Anthocyanin glycosides were no longer detected in the GI tract after 8 hours of administration. In vitro fermentation showed that the 2 cyanidin glycosides were totally metabolized by the rat colonic microflora, explaining their disappearance. In addition, the 2 products of their degradation, cyanidin and protocatechuic acid, were not detected in plasma and probably do not influence plasma antioxidant capacity. As found by the everted sac model, anthocyanins were transported across the enterocyte by the sodium-dependent glucose transporter. (c) 2008 Elsevier Inc. All rights reserved.

The expression of efflux and uptake transporters are regulated by statins in Caco-2 and HepG2 cells

Aim: Statin disposition and response are greatly determined by the activities of drug metabolizing enzymes and efflux/uptake transporters. there is little information on the regulation of these proteins in human cells after statin therapy. In this study, the effects of atorvastatin and simvastatin on mRNA expression of efflux (ABCB1, ABCG2 and ABCC2) and uptake (SLCO1B1, SLCO2B1 and SLC22A1) drug transporters in Caco-2 and HepG2 cells were investigated. Methods: Quantitative real-time PCR was used to measure mRNA levels after exposure of HepG2 and Caco-2 cells to statins. Results: Differences in mRnA basal levels of the transporters were as follows: ABCC2>ABCG2>ABCB1>SLCO1B1>>>SLC22A1>SLC O2B1 for HepG2 cells, and SLCO2B1>>ABCC2>ABCB1>ABCG2>>>SLC22A1 for Caco-2 cells. While for HepG2 cells, ABCC2, ABCG2 and SLCO2B1 mRnA levels were significantly up-regulated at 1, 10 and 20 mu mol/L after 12 or 24 h treatment, in Caco-2 cells, only the efflux transporter ABCB1 was significantly down-regulated by two-fold following a 12 h treatment with atorvastatin. Interestingly, whereas treatment with simvastatin had no effect on mRNA levels of the transporters in HepG2 cells, in Caco-2 cells the statin significantly down-regulated ABCB1, ABCC2, SLC22A1, and SLCO2B1 mRnA levels after 12 or 24 h treatment. Conclusion: These findings reveal that statins exhibits differential effects on mRNA expression of drug transporters, and this effect depends on the cell type. Furthermore, alterations in the expression levels of drug transporters in the liver and/or intestine may contribute to the variability in oral disposition of statins.

Exercise-stimulated GLUT4 Expression is Similar in Normotensive and Hypertensive Rats

The effects of exercise training on systolic blood pressure (BP), insulin sensitivity, and plasma membrane GLUT4 protein content in spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats were compared. 16 SHR and 16 WKY male rats, aged 6 months, were randomized into sedentary and trained (tread-mill running, 5 days/week, 60 min/day for 10 weeks) groups (n = 8/group). At baseline, SHR had lower insulin sensitivity than WKY rats, however, there were no differences between WKY and SHR GLUT4 expression. The 10-week training reduced BP by similar to 19% in SHR, improved insulin sensitivity by similar to 24% in SHR, but not in WKY, and increased GLUT4 expression in both animal models. Compared to the sedentary group, there was an increase of GLUT4 in WKY rats by similar to 25% in the heart, by similar to 23% in the gastrocnemius, and by similar to 15% in the fat tissue. Trained SHR presented an increase in GLUT4 of similar to 21%, similar to 20%, and similar to 14%, in the same tissues, respectively. There were no differences between SHR and WKY rats in post-training GLUT4 expression. We conclude that training determined BP and insulin resistance reduction in SHR, and increased GLUT4 expression in both normotensive and hypertensive rats. However, considering the similar rise in GLUT4-induced training in SHR and WKY, it is possible that GLUT4 levels in plasma membrane fraction do not have a pivotal role in the exercise-induced improvement of insulin sensitivity in SHR.