Sympathetic Regulation of Slc2a4 Gene Expression: Participation of a Putative cAMP Responsive Element (CRE) Site in the Slc2a4 Promoter.

BACKGROUND/AIMS: Studies have indicated that sympathetic activity enhances GLUT4 expression (Slc2a4 gene) by activating beta-adrenergic receptors. This could be mediated by a direct enhancer effect of cyclic AMP-responsive element binding protein (CREB) and family members upon Slc2a4 gene. However, a cAMP responsive element (CRE) in Slc2a4 promoter has never been demonstrated. METHODS: Slc2a4 CRE-site was searched by in silico analysis. In skeletal muscles from rats displaying high sympathetic activity (SHR), Slc2a4 CRE-site was investigated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay; and Slc2a4 expression was analyzed by RT-qPCR. Functional activity of the CRE-site was investigated by luciferase assay, 2 hours after 8-br-cAMP stimulation, in 3T3L1 adipocytes transientely transfected with native and mutated CRE-sites. RESULTS: In silico analysis indicated the -480/-473 segment as a putative CRE-site, with 62.5% of identity to CRE consensus sequence, and highly preserved in mouse, rat and human. CREB/CREM binding in this CRE-site was confirmed to occur in vitro (EMSA) and in vivo (ChIP assay). Enhancer activity of this segment in Slc2a4 transcription was confirmed in 3T3-L1 cells. Finally, in extensor digitorum longus muscle from SHR, 80% increase in Slc2a4 mRNA expression was observed to be accompanied by increased CREB/CREM binding into the CRE-site both in vitro and in vivo. CONCLUSION: This study demonstrates the presence of a functional CRE-site at -480/-473 sequence of the Slc2a4 gene. This CRE-site has an enhancing activity on Slc2a4 expression and participates in the Slc2a4 increased expression observed in glycolytic muscles of rats displaying high sympathetic activity.

Glucose transporter 1 expression accompanies hypoxia sensing in the cyclic canine corpus luteum.

The canine corpus luteum (CL) functions as a source of progesterone (P4) and 17ß-oestradiol (E2); however, the transport of energy substrates to maintain its high hormonal output has not yet been characterised. This study involved the localisation and temporal distribution of the facilitative glucose transporter 1 and the quantification of the corresponding protein (GLUT1) and gene (SLC2A1) expression. Some GLUT1/SLC2A1 regulatory proteins, such as hypoxia-inducible factor 1α (HIF1A) and fibroblast growth factor 2 (FGF2); mRNAs, such as HIF1A, FGF2 and vascular endothelial growth factor A (VEGFA); and VEGFA receptors 1 and 2 (FLT1 and KDR) were also analysed from days 10 to 70 after ovulation. Additionally, plasma P4 and E2 levels were assessed via chemiluminescence. Moreover, the canine KDR sequence has been cloned, thereby enabling subsequent semi-quantitative PCR analysis. Our results demonstrate time-dependent variations in the expression profile of SLC2A1 during dioestrus, which were accompanied by highly correlated changes (0.84

Carbohydrate- and lipid-enriched meals acutely disrupt glycemic homeostasis by inducing transient insulin resistance in rats.

Chronic intake of high-carbohydrate or high-lipid diets is a well-known insulin resistance inducer. This study investigates the immediate effect (1-6 h) of a carbohydrate- or lipid-enriched meal on insulin sensitivity. Fasted rats were refed with standard, carbohydrate-enriched (C), or lipid-enriched (L) meal. Plasma insulin, glucose, and non-esterified fatty acids (NEFA) were measured at 1, 2, 4, and 6 h of refeeding. The glucose-insulin index showed that either carbohydrates or lipids decreased insulin sensitivity at 2 h of refeeding. At this time point, insulin tolerance tests (ITTs) and glucose tolerance tests (GTTs) detected insulin resistance in C rats, while GTT confirmed it in L rats. Reduced glycogen and phosphorylated AKT and GSK3 content revealed hepatic insulin resistance in C rats. Reduced glucose uptake in skeletal muscle subjected to the fatty acid concentration that mimics the high NEFA level of L rats suggests insulin resistance in these animals is mainly in muscle. In conclusion, carbohydrate- or lipid-enriched meals acutely disrupt glycemic homeostasis, inducing a transient insulin resistance, which seems to involve liver and skeletal muscle, respectively. Thus, the insulin resistance observed when those types of diets are chronically consumed may be an evolution of repeated episodes of this transient insulin resistance.

Maternal Exercise-Induced SOD3 Reverses the Deleterious Effects of Maternal High-Fat Diet on Offspring Metabolism Through Stabilization of H3K4me3 and Protection Against WDR82 Carbonylation.

Preclinical studies reveal maternal exercise as a promising intervention to reduce the transmission of multigenerational metabolic dysfunction caused by maternal obesity. The benefits of maternal exercise on offspring health may arise from multiple factors and have recently been shown to involve DNA demethylation of critical hepatic genes leading to enhanced glucose metabolism in offspring. Histone modification is another epigenetic regulator, yet the effects of maternal obesity and exercise on histone methylation in offspring are not known. Here, we find that maternal high-fat diet (HFD; 60% kcal from fat) induced dysregulation of offspring liver glucose metabolism in C57BL/6 mice through a mechanism involving increased reactive oxygen species, WD repeat-containing 82 (WDR82) carbonylation, and inactivation of histone H3 lysine 4 (H3K4) methyltransferase leading to decreased H3K4me3 at the promoters of glucose metabolic genes. Remarkably, the entire signal was restored if the HFD-fed dams had exercised during pregnancy. WDR82 overexpression in hepatoblasts mimicked the effects of maternal exercise on H3K4me3 levels. Placental superoxide dismutase 3 (SOD3), but not antioxidant treatment with N-acetylcysteine was necessary for the regulation of H3K4me3, gene expression, and glucose metabolism. Maternal exercise regulates a multicomponent epigenetic system in the fetal liver resulting in the transmission of the benefits of exercise to offspring.

Placental superoxide dismutase 3 mediates benefits of maternal exercise on offspring health.

Poor maternal diet increases the risk of obesity and type 2 diabetes in offspring, adding to the ever-increasing prevalence of these diseases. In contrast, we find that maternal exercise improves the metabolic health of offspring, and here, we demonstrate that this occurs through a vitamin D receptor-mediated increase in placental superoxide dismutase 3 (SOD3) expression and secretion. SOD3 activates an AMPK/TET signaling axis in fetal offspring liver, resulting in DNA demethylation at the promoters of glucose metabolic genes, enhancing liver function, and improving glucose tolerance. In humans, SOD3 is upregulated in serum and placenta from physically active pregnant women. The discovery of maternal exercise-induced cross talk between placenta-derived SOD3 and offspring liver provides a central mechanism for improved offspring metabolic health. These findings may lead to novel therapeutic approaches to limit the transmission of metabolic disease to the next generation.

Effects of maternal and paternal exercise on offspring metabolism.

Maternal and paternal obesity and type 2 diabetes are recognized risk factors for the development of metabolic dysfunction in offspring, even when the offspring follow a healthful lifestyle. Multiple studies have demonstrated that regular physical activity in mothers and fathers has striking beneficial effects on offspring health, including preventing the development of metabolic disease in rodent offspring as they age. Here, we review the benefits of maternal and paternal exercise in combating the development of metabolic dysfunction in adult offspring, focusing on offspring glucose homeostasis and adaptations to metabolic tissues. We discuss recent findings regarding the roles of the placenta and sperm in mediating the effects of parental exercise on offspring metabolic health, as well as the mechanisms hypothesized to underlie these beneficial changes. Given the worldwide epidemics of obesity and type 2 diabetes, if these findings translate to humans, regular exercise during the reproductive years might limit the vicious cycles in which increased metabolic risk propagates across generations.

Maternal and paternal exercise regulate offspring metabolic health and beta cell phenotype.

OBJECTIVE: Poor maternal and paternal environments increase the risk for obesity and diabetes in offspring, whereas maternal and paternal exercise in mice can improve offspring metabolic health. We determined the effects of combined maternal and paternal exercise on offspring health and the effects of parental exercise on offspring pancreas phenotype, a major tissue regulating glucose homeostasis. RESEARCH DESIGN AND METHODS: Breeders were high fat fed and housed±running wheels before breeding (males) and before and during gestation (females). Offspring groups were: both parents sedentary (Sed); maternal exercise only (Mat Ex); paternal exercise only (Pat Ex); and maternal+paternal exercise (Mat+Pat Ex). Offspring were sedentary, chow fed, and studied at weaning, 12, 20 and 52 weeks. RESULTS: While there was no effect of parental exercise on glucose tolerance at younger ages, at 52 weeks, offspring of Mat Ex, Pat Ex and Mat+Pat Ex displayed lower glycemia and improved glucose tolerance. The greatest effects were in offspring from parents that both exercised (Mat+Pat Ex). Offspring from Mat Ex, Pat Ex, and Mat+Pat Ex had decreased beta cell size, whereas islet size and beta cell mass only decreased in Mat+Pat Ex offspring. CONCLUSIONS: Maternal and paternal exercise have additive effects to improve glucose tolerance in offspring as they age, accompanied by changes in the offspring endocrine pancreas. These findings have important implications for the prevention and treatment of type 2 diabetes.

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%). beta1-beta2-, and beta1-beta2-beta3-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.

Maternal Exercise Improves Glucose Tolerance in Female Offspring.

Poor maternal diet can lead to metabolic disease in offspring, whereas maternal exercise may have beneficial effects on offspring health. In this study, we determined ifmaternal exercise could reverse the detrimental effects of maternal high-fat feeding on offspring metabolism of female mice. C57BL/6 female mice were fed a chow (21%) or high-fat (60%) diet and further divided by housing in static cages or cages with running wheels for 2 weeks prior to breeding and throughout gestation. Females were bred with chow-fed sedentary C57BL/6 males. High fat-fed sedentary dams produced female offspring with impaired glucose tolerance compared with offspring of chow-fed dams throughout their first year of life, an effect not present in the offspring from high fat-fed dams that had trained. Offspring from high fat-fed trained dams had normalized glucose tolerance, decreased fasting insulin, and decreased adiposity. Liver metabolic function, measured by hepatic glucose production in isolated hepatocytes, hyperinsulinemic-euglycemic clamps, liver triglyceride content, and liver enzyme expression, was enhanced in offspring from trained dams. In conclusion, maternal exercise negates the detrimental effects of a maternal high-fat diet on glucose tolerance and hepatocyte glucose metabolism in female offspring. The ability of maternal exercise to improve the metabolic health of female offspring is important, as this intervention could combat the transmission of obesity and diabetes to subsequent generations.

Is the canine corpus luteum an insulin-sensitive tissue?

This study aimed to determine in the canine corpus luteum throughout the dioestrus (1) the influence of insulin on glucose uptake; (2) the regulation of genes potentially involved; and (3) the influence of hypoxia on glucose transporter expression and steroidogenesis, after treatment with cobalt chloride (CoCl2). Glucose uptake by luteal cells increased 2.7 folds (P < 0.05) in response to insulin; a phenomenon related to increased expression of glucose transporter (GLUT) 4 and phosphorylation of protein kinase B (AKT). The gene expression of insulin receptor and SLC2A4 (codifier of GLUT4) genes after insulin stimulation increased on day 20 post ovulation (p.o.) and declined on day 40 p.o. (P < 0.05). Regarding potentially involved molecular mechanisms, the nuclear factor kappa B gene RELA was upregulated on days 30/40 p.o., when SLC2A4 mRNA was low, and the interleukin 6 (IL6) gene was upregulated in the first half of dioestrus, when SLC2A4 mRNA was high. CoCl2 in luteal cell cultures increased the hypoxia-inducible factor HIF1A/HIF1A and the SLC2A4/GLUT4 expression, and decreased progesterone (P4) production and hydroxyl-delta-5-steroid dehydrogenase 3 beta (HSD3B) mRNA expression (P < 0.05). This study shows that the canine luteal cells are responsive to insulin, which stimulates glucose uptake in AKT/GLUT4-mediated pathway; that may be related to local activity of RELA and IL6. Besides, the study reveals that luteal cells under hypoxia activate HIF1A-modulating luteal function and insulin-stimulated glucose uptake. These data indicate that insulin regulates luteal cells' glucose disposal, participating in the maintenance and functionality of the corpus luteum.

Beta-adrenergic blockade increases GLUT4 and improves glycemic control in insulin-treated diabetic Wistar rats.

OBJECTIVE: Unequivocal modulation of glycemic homeostasis by chronic beta-adrenergic blockade in diabetes has never been demonstrated. This study investigates the participation of beta-adrenergic system in glycemic control and muscle glucose transporter GLUT4 expression in insulin-treated diabetic rats. METHODS: Insulin-treated diabetic Wistar (W) or spontaneously hypertensive rats (SHR) were additionally treated with propranolol, and glycemic homeostasis and expression of some target mRNAs and proteins in soleus and extensor digitorum longus (EDL) muscles were analyzed. RESULTS: Insulin improved glycemic control in both strains. Importantly, in W, propranolol promoted a further improvement in glycemic control, which was accompanied by decreased PKA and Tnf expression, and increased Slc2a4 and GLUT4 in EDL. Those effects were not observed in diabetic-SHR. DISCUSSION: Propranolol-induced decrease in beta-adrenergic activity in skeletal muscles of insulin-treated diabetic Wistar rats increases GLUT4 expression in EDL, improving glycemic control. These outcomes represent a positive effect of nonselective beta-blockade, which might be extended to autonomic neuropathy.

Decreased diabetes-induced glycemic impairment in WKY and SHR involves enhanced skeletal muscle Slc2a4/GLUT4 expression.

BACKGROUND: Hypertension has been associated to diabetes, and participates in the development of diabetic complications. The spontaneously hypertensive rat (SHR) is the gold standard model for the study of hypertension, and experimental diabetes has been currently investigated in SHR. Wistar-Kyoto rat is usually taken as control for SHR, however, regarding the glycemic homeostasis, WKY may be similar to SHR, when compared to the standard Wistar rat, importantly affecting the interpretation of data. Slc2a4 gene, which encodes the GLUT4 protein, is expressed in insulin-sensitive tissues, such as muscle cells and adipocytes, and alteration in Slc2a4/GLUT4 expression is inversely related to glycemic levels. We investigated the effect of diabetes on the expression of Slc2a4/GLUT4 and glycemic control in Wistar-Kyoto and SHR. FINDINGS: Slc2a4 mRNA (Northern-blotting) and GLUT4 protein (Western-blotting) were investigated in skeletal muscles (soleus and extensor digitorum longus) of Wistar, Wistar-Kyoto and SHR, rendered or not diabetic for 1 month. Non-diabetic SHR shows hyperinsulinemia, and unaltered GLUT4 expression. The hyperglycemia was significantly attenuated in diabetic Wistar-Kyoto and SHR, compared to that observed in diabetic Wistar, although all of them presented the same hypoinsulinemic levels. Besides, diabetes significantly reduced Slc2a4/GLUT4 in Wistar, as expected; however, that was not observed in diabetic Wistar-Kyoto and SHR. CONCLUSIONS: Non-diabetic SHR is insulin resistant, despite unaltered GLUT4 expression. Diabetic Wistar-Kyoto and diabetic SHR presented high Slc2a4/GLUT4 expression in skeletal muscle, as compared to diabetic Wistar. This Slc2a4/GLUT4 regulation does not depend on insulin level and possibly protects the WKY and SHR from severe glycemic impairment.

Recovery of insulin sensitivity and Slc2a4 mRNA expression depend on T3 hormone during refeeding.

OBJECTIVE: GLUT4 protein, encoded by the Slc2a4 gene, plays a key role in muscle glucose uptake, and its expression decreases in muscles under insulin resistance. Slc2a4/GLUT4 decreases with fasting and rapidly increases with refeeding and the same occurs to plasma glucose, amino acids, insulin and T3. Thus, they might be potential regulators of the Slc2a4 gene, which makes them promising targets for strategies to improve GLUT4 expression. Herein, we investigate the role of metabolic-hormonal parameters triggered by refeeding upon the Slc2a4 expression. MATERIALS/METHODS: Plasma glucose/insulin/T3, and gastrocnemius Slc2a4 mRNA contents were measured in rats studied at the end of 48-h fasting, and subsequently at: i) 2-4h after spontaneous refeeding; ii) 2-4h after T3 injection, without refeeding; and iii) 0.5-2h after intravenous infusion of insulin, insulin+glucose and insulin+amino acids, without refeeding. RESULTS: Refeeding increased plasma glucose/insulin/T3 and muscle Slc2a4 mRNA, reverting insulin resistance. Post-fasting infusions surprisingly induced a further Slc2a4 mRNA decrease (~20%, P<0.05 vs. fasting), but T3 injection induced a ~2-fold increase in Slc2a4 mRNA, 2-4h later (P<0.001). Moreover, T3 increased glycemia and insulinemia to the 2h-refed rats levels, suggesting that T3 elevation is a key factor to the mechanisms of metabolic balance during refeeding. CONCLUSIONS: Refeeding induces a rapid increase in muscle Slc2a4 expression, not associated with increased plasma glucose, insulin or amino acids, but highly correlated to increased plasma T3 concentration. This result points out T3 hormone as a powerful Slc2a4 enhancer, an effect that may be acutely explored in situations of insulin resistance.