GLUT4 mRNA In Skeletal Muscle Research Articles

Effects of Resistance Exercise Training on Leptin Receptor (Ob-R), GLUT2 mRNA of the Hypothalamus and GLUT4 mRNA Expression of the Skeletal Muscle in OLETF Rats

PURPOSE The hypothalamus plays a central role in integrating hormonal and nutritional signals from the periphery and modulating food intake, energy balance. However, obesity and type 2 diabetes cause dysfunction of hypothalamus. This study was to investigate the effects of 8 weeks resistance exercise training on leptin receptor, GLUT2 mRNA of the hypothalamus and skeletal muscle GLUT4 mRNA expression in OLETF rats. METHODS Thirty male OLETF rats were randomly divided into three groups: LETO (CON, n=10), OLETF (OLETF+CON, n=10), OLETF+resistance exercise (OLETF+EXE, n=10). Exercise group conducted gradual ladder climbing exercise 3 days per week for 8 weeks. RESULTS The results of this study was following; In the hypothalamus, leptin receptor (Ob-R) and GLUT2 mRNA expression were significantly increased in OLETF+EXE group compared with OLETF group (p<.05). Activation of AMPK was significantly decreased in OLETF+EXE group compared to OLETF group (p<.05). GLUT4 mRNA expression of skeletal muscle was significantly increased in OLETF+EXE group compared with OLETF group (p<.05), and HOMA-IR index also was significantly lower in OLETF+EXE group than OLETF group (p<.05). CONCLUSIONS These results showed that 8 weeks resistance exercise training can improve leptin receptor mRNA expression and glucose sensing in hypothalamic. Also, these effects may contribute to energy homeostasis in peripheral such as skeletal muscle. 색인어: ì €í•­ì„± 운동, 시상하부, ì œ2형 당뇨병, ë ™í‹´ìˆ˜ìš©ì²´, GLUTs Keywords: Resistance exercise, Hypothalamus, Type 2 diabetes mellitus, Leptin receptor (Ob-R), GLUTs

Postnatal exposure to a high-carbohydrate diet interferes epigenetically with thyroid hormone receptor induction of the adult male rat skeletal muscle glucose transporter isoform 4 expression

Early life nutritional intervention causes adult-onset insulin resistance and obesity in rats. Thyroid hormone receptor (TR), in turn, transcriptionally enhances skeletal muscle Glut4 expression. We tested the hypothesis that reduced circulating thyroid-stimulating hormone and T4 concentrations encountered in postnatal (PN4–PN24) high-carbohydrate (HC) milk formula-fed versus the mother-fed controls (MF) would epigenetically interfere with TR induction of adult (100days) male rat skeletal muscle Glut4 expression, thereby providing a molecular mechanism mediating insulin resistance. We observed increased DNA methylation of the CpG island with enhanced recruitment of Dnmt3a, Dnmt3b and MeCP2 in the glut4 promoter region along with reduced acetylation of histone (H)2A.Z and H4 particularly at the H4.lysine (K)16 residue, which was predominantly mediated by histone deacetylase 4 (HDAC4). This was followed by enhanced recruitment of heterochromatin protein 1β to the glut4 promoter with increased Suv39H1 methylase concentrations. These changes reduced TR binding of the T3 response element of the glut4 gene (TREs; −473 to −450bp) detected qualitatively in vivo (electromobility shift assay) and quantified ex vivo (chromatin immunoprecipitation). In addition, the recruitment of steroid receptor coactivator and CREB-binding protein to the glut4 promoter–protein complex was reduced. Co-immunoprecipitation experiments confirmed the interaction between TR and CBP to be reduced and HDAC4 to be enhanced in HC versus MF groups. These molecular changes were associated with diminished skeletal muscle Glut4 mRNA and protein concentrations. We conclude that early postnatal exposure to HC diet epigenetically reduced TR induction of adult male skeletal muscle Glut4 expression, uncovering novel molecular mechanisms contributing to adult insulin resistance and obesity.

Effect of iron supplementation on glucose transporter 4 expression in adipose tissue and skeletal muscle of pregnant rats

Objective: The main goal of the present study was to investigate the effect of iron supplementation on glucose transporter 4 expressions in adipose tissue and skeletal muscle in female rats during pregnancy. Methods: Twenty-four pregnant Sprague-Dawley rats were randomly divided into 2 groups: a control group with a standard diet (containing iron 150 mg/kg) and an iron-supplementation group with a high-iron diet (containing iron 700 mg/kg) from day 0 to day 21 of pregnancy. Intraperitoneal glucose tolerance test was performed on gestational day 19. On gestational day 21, all of the pregnant rats from each group were sacrificed. The mean neonatal weights were measured and samples of maternal intraabdominal adipose tissue and skeletal muscle were taken to measure the expression of Glucose Transporter 4 (GLUT4) mRNA and protein. Results: Glucose tolerance decreased significantly in the iron supplementation group compared to the control group. The mean neonatal weights in the iron supplementation group were higher than that in the control group. Levels of GLUT4 mRNA in the adipose tissue were reduced by the administrations of high-iron diet. The skeletal muscle GLUT4 mRNA levels were not changed significantly by iron supplementation. Expression of GLUT4 protein both in the adipose tissue and skeletal muscle reduced significantly. Conclusion: These results suggest that iron supplementation during pregnancy would increase neonatal weights and could decrease maternal glucose tolerance by interfering GLUT4 expression in adipose tissue and skeletal muscle of rats.

Exercise increases skeletal muscle GLUT4 gene expression in patients with type 2 diabetes

The aim of the study was to determine the effect of a single bout of exercise on GLUT4 gene expression in muscle of patients with type 2 diabetes (T2D) and control subjects, matched for age and body mass index. Nine patients with T2D and nine control subjects performed 60 min of cycling exercise at ~55% peak power (W(max) ). Skeletal muscle biopsies were obtained at baseline, immediately post and 3-h post exercise. GLUT4 mRNA expression increased (p < 0.05) to a similar extent immediately post exercise in control (~60%) and T2D (~66%) subjects, and remained elevated (p < 0.05) 3-h post exercise with no differences between groups. Similarly, p-AMP-activated protein kinase, p38 mitogen-activated kinase and proliferator-activated receptor gamma co-activator-alpha mRNA expression were increased (p < 0.05) post exercise, and were not different between the groups. In conclusion, a single bout of exercise increased skeletal muscle GLUT4 mRNA expression in patients with T2D to a similar extent as in control subjects.

Histone Code Modifications Repress Glucose Transporter 4 Expression in the Intrauterine Growth-restricted Offspring

We examined transcriptional and epigenetic mechanism(s) behind diminished skeletal muscle GLUT4 mRNA in intrauterine growth-restricted (IUGR) female rat offspring. An increase in MEF2D (inhibitor) with a decline in MEF2A (activator) and MyoD (co-activator) binding to the glut4 promoter in IUGR versus control was observed. The functional role of MEF2/MyoD-binding sites and neighboring three CpG clusters in glut4 gene transcription was confirmed in C2C12 muscle cells. No differential methylation of these three and other CpG clusters in the glut4 promoter occurred. DNA methyltransferase 1 (DNMT1) in postnatal, DNMT3a, and DNMT3b in adult was differentially recruited with increased MeCP2 (methyl CpG-binding protein) concentrations to bind the IUGR glut4 gene. Covalent modifications of the histone (H) code consisted of H3.K14 de-acetylation by recruitment of histone deacetylase (HDAC) 1 and enhanced association of HDAC4 enzymes. This set the stage for Suv39H1 methylase-mediated di-methylation of H3.K9 and increased recruitment of heterochromatin protein 1alpha, which partially inactivates postnatal and adult IUGR glut4 gene transcription. Further increased interactions in the adult IUGR between DNMT3a/DNMT3b and HDAC1 and MEF2D and HDAC1/HDAC4 and decreased association between MyoD and MEF2A existed. We conclude that epigenetic mechanisms consisting of histone code modifications repress skeletal muscle glut4 transcription in the postnatal period and persist in the adult female IUGR offspring.

Metabolic homeostasis in mice with disruptedClockgene expression in peripheral tissues

The role of peripheral vs. central circadian rhythms and Clock in the maintenance of metabolic homeostasis and with aging was examined by using Clock(Delta19)+MEL mice. These have preserved suprachiasmatic nucleus and pineal gland rhythmicity but arrhythmic Clock gene expression in the liver and skeletal muscle. Clock(Delta19)+MEL mice showed fasting hypoglycemia in young-adult males, fasting hyperglycemia in older females, and substantially impaired glucose tolerance overall. Clock(Delta19)+MEL mice had substantially reduced plasma insulin and plasma insulin/glucose nocturnally in males and during a glucose tolerance test in females, suggesting impaired insulin secretion. Clock(Delta19)+MEL mice had reduced hepatic expression and loss of rhythmicity of gck, pfkfb3, and pepck mRNA, which is likely to impair glycolysis and gluconeogenesis. Clock(Delta19)+MEL mice also had reduced glut4 mRNA in skeletal muscle, and this may contribute to poor glucose tolerance. Whole body insulin tolerance was enhanced in Clock(Delta19)+MEL mice, however, suggesting enhanced insulin sensitivity. These responses occurred although the Clock(Delta19) mutation did not cause obesity and reduced plasma free fatty acids while increasing plasma adiponectin. These studies on clock-gene disruption in peripheral tissues and metabolic homeostasis provide compelling evidence of a relationship between circadian rhythms and the glucose/insulin and adipoinsular axes. It is, however, premature to declare that clock-gene disruption causes the full metabolic syndrome.

Nitric Oxide Influences AMPK Activity and the Induction of GLUT4 mRNA in Skeletal Muscle

BACKGROUND: The upregulation of GLUT4 expression is an important exercise-induced adaptation leading to improved glucose disposal in skeletal muscle. We have observed that nitric oxide synthase (NOS) inhibition (non-isoform-selective or nNOS-selective) attenuates the induction of GLUT4 mRNA mediated by AICAR (5-aminoimidazole-4-carboxamide riboside, 1mM) in L6 myotubes. Therefore, we sought to test whether AICAR-induced AMPK activity (i.e. phosphorylation of the AMPK substrate, Acetyl CoA Carboxylase, p-ACC) in L6 myotubes is dependent upon endogenous NOS activity, and if exogenous NO is sufficient to induce p-ACC. Secondly, we examined whether nNOS inhibition would prevent AICAR-induced upregulation of GLUT4 mRNA in rat skeletal muscle in vivo. METHODS: L6 myotubes were treated for 1h with AICAR (1mM), L-NAME (N(G)-L-nitro-arginine methyl ester,100μM and 500 μM), AICAR+L-NAME, or the NO donor, SNAP (1μM and 10μM). Separately, sixteen female Sprague-Dawley rats (10 mo old) were injected IP with: saline (control), AICAR (0.5mg/g body weight), TRIM (1-(2-trifluoromethyl-phenyl)-imidazole, 50 mg/g body weight), or AICAR+TRIM, and the plantaris muscles harvested after 12h. RESULTS: AICAR increased p-ACC by 2-fold (P<0.05), while co-treatment with L-NAME prevented this effect. Additionally, SNAP (10μM) increased p-ACC by 50% (P<0.05). However, the effect of L-NAME and SNAP on p-AMPK was variable and not statistically significant. In vivo, GLUT4 mRNA in the plantaris muscle was increased ∼80%, 12h after AICAR injection (P<0.01), while TRIM co-treatment inhibited this effect by ∼50%. CONCLUSION: These results suggest that NO influences AMPK activity. Further, nNOS activity is required for the full effect of AICAR on GLUT4 mRNA in vivo.Funded by AHA Grant #0530196N.

Nitric Oxide-dependent Regulation Of Glut4 Expression In L6 Myotubes

The upregulation of GLUT4 expression is one of the most important exercise-induced adaptations leading to improved glucose disposal in skeletal muscle. The GLUT4 gene is transcriptionally regulated and AMP-activated protein kinase (AMPK) is involved in this process. Previous studies in rats and humans have shown that AMPK is capable of phosphorylating and activating nitric oxide synthase (NOS). PURPOSE We hypothesized that NOS inhibition would prevent AMPK-induced upregulation in GLUT4 mRNA in L6 myotubes. METHODS Myoblasts were cultured in growth medium (10% FBS) until reaching 80% con.uence, then switched to differentiation medium (2%HoS) until myotubes were 80% confluent. At this point, myotubes were exposed to treatment medium (10%HoS + 5%FBS) for 16 h, with or without the AMPK activator, AICAR (5- aminoimidazole-4-carboxamide riboside, 1mM) or AICAR + the NOS inhibitor, LNAME (N(G)-L-nitro-arginine methyl ester,100μm). RESULTS AICAR induced ∼9-fold increase in GLUT4 mRNA levels in comparison to the control group (n=5–6/group; p = .0012), whereas co-treatment with L-NAME prevented this effect (p = .42). A dose-dependent positive effect of nitric oxide on GLUT4 expression was confirmed by incubation of myotubes with the nitric oxide donor, SNAP. Additionally, treatment with the cell permeable cGMP analog, 8-BrcGMP (1mM) for 16 h increased GLUT4 mRNA levels by 85% (n=6/group; p = .002). CONCLUSIONS These findings suggest that nitric oxide plays a role in the AMPK-induced upregulation of GLUT4 mRNA in skeletal muscle, and may do so by increasing intracellular levels of cGMP. Supported by UF Opportunity Fund.

Bidirectional regulation of uncoupling protein-3 and GLUT-4 mRNA in skeletal muscle by cold.

To elucidate the possible role of the mitochondrial uncoupling protein (UCP)-3 in skeletal muscle as a regulator of adaptive thermogenesis and energy balance, we examined the modulation by cold exposure (5 degrees C) of UCP-3 and glucose transporter isoform GLUT-4 mRNAs in male Sprague-Dawley rats. In skeletal muscle, UCP-3 and GLUT-4 mRNAs increased two- to threefold between 6 and 24 h of cold exposure and then decreased to 50% of the control value after 6 days in the cold. In contrast, skeletal muscle UCP-2 mRNA showed a small increase on day 3 and returned to normal after 6 days. The bidirectional regulation of UCP-3 and GLUT-4 mRNAs in skeletal muscle by cold suggests that UCP-3 may be a major mediator of acute adaptive thermogenesis but then is downregulated, along with GLUT-4, in the chronic state to preserve energy. In contrast, cold exposure caused only transient changes of UCP-2 and GLUT-4 mRNA in heart. These data are consistent with the necessity of the heart to continuously expend energy to maintain blood circulation, regardless of environmental conditions.

Influence of highly purified eicosapentaenoic acid ethyl ester on insulin resistance in the Otsuka Long-Evans Tokushima Fatty Rat, a model of spontaneous non-insulin-dependent diabetes mellitus

We investigated the effect of long-term administration of highly purified eicosapentaenoic acid ethyl ester (EPA-E), an n-3 polyunsaturated fatty acid derived from fish oil, in comparison to the effects of lard, olive oil, safflower oil, or distilled water as the control on the development of insulin resistance in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of spontaneous non—insulin-dependent diabetes mellitus (NIDDM) with obesity. After 17 or 18 weeks of treatment, the glucose infusion rate (GIR) in the euglycemic insulin-glucose clamp test only showed a significant increase in EPA-E-treated rats compared with control rats given distilled water alone as the vehicle. The GIR in EPA-E-treated animals was approximately three times greater than in the controls. This is the first report to display the influence of various fatty acids on the development of insulin resistance in OLETF rats. We demonstrated that EPA-E prevents the onset of insulin resistance, whereas olive oil and safflower oil have no effect and lard exacerbates insulin resistance. Fatty acid analysis of phospholipids in skeletal muscle showed a significant increase of the C18:2, C20:5, and C22:5 components in EPA-E-treated rats and, conversely, a significant decrease in C20:4. In addition, EPA-E-treated rats showed a significant increase in GLUT4 mRNA in skeletal muscle when compared with control rats. Our results indicate that the beneficial effect of EPA-E on insulin resistance in OLETF rats is likely to be dependent on modification of the phospholipid components of the skeletal muscle membrane. These findings suggest that dietary fatty acids may play a key role in the development of insulin resistance in patients with NIDDM.

THE EFFECTS OF IMMOBILIZATION AND SUBSEQUENT EXERCISE ON HUMAN MUSCLE GLUT4 mRNA LEVELS 1311

Muscle glucose transporter GLUT4 plays an important role in maintaining glucose homeostasis. Previous studies have shown that exercise increases muscle GLUT4 gene expression and protein level in animal models. The purpose of this study was to investigate the human skeletal muscle GLUT4 mRNA levels before and after immobilization and subsequent exercise. Four human subjects were immobilized with a full cast on the left leg for 3 weeks followed by 12 weeks of resistance exercise training. Total RNA was isolated from biopsy samples of the vastus lateralis muscle of the immobilized leg. Ribonuclease protection assay was used to measure the mRNA levels of GLUT4 transporter. Immobilization decreased GLUT4 mRNA levels to 60% of control(p<0.05). Subsequent resistance exercise training on the immobilized leg induced a 2.0-fold increase in GLUT4 mRNA levels vs immobilization(p<0.05). The results of this study suggest that human skeletal muscle glucose transporter GLUT4 can be regulated at the pre-translational level by immobilization and exercise.

Alterations in the level of insulin receptor and GLUT-4 mRNA in skeletal muscle from rats fed a kidney bean ( Phaseolus vulgaris) diet

1. 1. A decline in the level of circulating insulin was observed in rats fed a diet containing kidney bean 2. 2. Consumption of a diet containing kidney bean caused an increase in the level of mRNAs for the insulin receptor (327%) and GLUT-4 (185%) in the gastrocnemius muscle. In contrast there was only a small increase in the amount of actin mRNA (125%). Since the kidney bean-fed rats are euglycaemic the results suggest that insulin receptor and GLUT-4 mRNA levels are regulated in response to circulating insulin concentrations rather than glucose 3. 3. No increases in the level of insulin receptor and actin mRNA were evident in the soleus muscle of rats fed the diet containing kidney bean; however a decline was observed in the level of GLUT-4 mRNA 4. 4. It is proposed that a component of kidney beans, most likely the lectin phytohaemaglutinin, has systemic effects which lead to changes in expression of the insulin receptor and GLUT-4 genes and to the sensitivity of muscle to insulin.

Assessment of Glucose Transporter Gene Expression Using the Polymerase Chain Reaction

We used a novel adaptation of the polymerase chain reaction to examine relative levels of mRNA encoding two members of the facilitative glucose transporter gene family, the GLUT1 or erythrocyte/HepG2/brain isoform and the GLUT4 or insulin-regulatable isoform. The method was fast (vs. hybridization methods), required no specific probe, and used total RNA samples of less than 1 microgram. Taking advantage of regions of structural similarity and differences between the two isoforms, we designed a single set of oligonucleotide primers capable of amplifying both GLUT1 and GLUT4 cDNAs such that their respective products could be resolved on the basis of a 12 base pair size differential. Hence, reverse transcription and complementary DNA amplification could be carried out for both transcripts using identical primers in the same reaction tube. Using this methodology, we examined the relative amounts of GLUT4 and GLUT1 mRNAs in several rat tissues. As expected based on prior reports using Northern analysis, rat brain contained only GLUT1 mRNA and skeletal muscle contained a large predominance of GLUT4 mRNA. Both isoform mRNAs were found in adipose tissue whereas adipose cells, heart and diaphragm contained predominantly GLUT4 mRNA. Induction of diabetes with streptozocin decreased the GLUT4 to GLUT1 ratio in adipose tissue 4-fold and 24 h of insulin treatment of the diabetic rats increased this ratio 9- to 10-fold. Insulin treatment of normal rats increased this ratio by 70%. Hindlimb skeletal muscle GLUT4 mRNA was quantified in diabetic and insulin-treated diabetic rats as a function of brain GLUT1 mRNA added as an internal standard. Using this methodology, no significant difference in muscle GLUT4 mRNA was noted as a result of 24 h of insulin therapy. In summary, quantitative PCR may be used to compare mRNA levels encoding specific members of a gene family either within given cells or tissues or as affected by physiological perturbations. Subject to certain limitations discussed within, this methodology may be useful in future measurements of glucose transporter mRNA, especially when only small tissue or cell samples are available.