Nuclear factor 1 regulates adipose tissue-specific expression in the mouse GLUT4 gene

Previous studies demonstrated that an adipose tissue-specific element(s) (ASE) of the murine GLUT4 gene is located between −551 and −506 in the 5′-flanking sequence and that a high-fat responsive element(s) for down-regulation of the GLUT4 gene is located between bases −701 and −552. A binding site for nuclear factor 1 (NF1), that mediates insulin and cAMP-induced repression of GLUT4 in 3T3-L1 adipocytes is located between bases −700 and −688. To examine the role of NF1 in the regulation of GLUT4 gene expression in white adipose tissues (WAT) in vivo, we created two types of transgenic mice harboring mutated either 5′ or 3′ half-site of NF1-binding sites in GLUT4 minigene constructs. In both cases, the GLUT4 minigene was not expressed in WAT, while expression was maintained in brown adipose tissue, skeletal muscle, and heart. This was an unexpected finding, since a −551 GLUT4 minigene that did not have the NF1-binding site was expressed in WAT. We propose a model that explains the requirement for both the ASE and the NF1-binding site for expression of GLUT4 in WAT.     

Overexpression of GLUT4 in mice causes up-regulation of UCP3 mRNA in skeletal muscle

Mitochondrial uncoupling protein 3 (UCP3) is expressed in skeletal muscles. We have hypothesized that increased glucose flux in skeletal muscles may lead to increased UCP3 expression. Male transgenic mice harboring insulin-responsive glucose transporter (GLUT4) minigenes with differing lengths of 5'-flanking sequence (-3237, -2000, -1000 and -442 bp) express different levels of GLUT4 protein in various skeletal muscles. Expression of the GLUT4 transgenes caused an increase in UCP3 mRNA that paralleled the increase of GLUT4 protein in gastrocnemius muscle. The effects of increased intracellular GLUT4 level on the expression of UCP1, UCP2 and UCP3 were compared in several tissues of male 4 month-old mice harboring the -1000 GLUT4 minigene transgene. In the -1000 GLUT4 transgenic mice, expression of GLUT4 mRNA and protein in skeletal muscles, brown adipose tissue (BAT), and white adipose tissue (WAT) was increased by 1.4 to 4.0-fold. Compared with non-transgenic littermates, the -1000 GLUT4 mice exhibited about 4- and 1.8-fold increases of UCP3 mRNA in skeletal muscle and WAT, respectively, and a 38% decrease of UCP1 mRNA in BAT. The transgenic mice had a 16% increase in oxygen consumption and a 14% decrease in blood glucose and a 68% increase in blood lactate, but no change in FFA or beta-OHB levels. T3 and leptin concentrations were decreased in transgenic mice. Expression of UCP1 in BAT of the -442 GLUT4 mice, which did not overexpress GLUT4 in this tissue, was not altered. These findings indicate that overexpression of GLUT4 up-regulates UCP3 expression in skeletal muscle and down-regulates UCP1 expression in BAT, possibly by increasing the rate of glucose uptake into these tissues.     

Calpain system regulates muscle mass and glucose transporter GLUT4 turnover

The experiments in this study were undertaken to determine whether inhibition of calpain activity in skeletal muscle is associated with alterations in muscle metabolism. Transgenic mice that overexpress human calpastatin, an endogenous calpain inhibitor, in skeletal muscle were produced. Compared with wild type controls, muscle calpastatin mice demonstrated normal glucose tolerance. Levels of the glucose transporter GLUT4 were increased more than 3-fold in the transgenic mice by Western blotting while mRNA levels for GLUT4 and myocyte enhancer factors, MEF 2A and MEF 2D, protein levels were decreased. We found that GLUT4 can be degraded by calpain-2, suggesting that diminished degradation is responsible for the increase in muscle GLUT4 in the calpastatin transgenic mice. Despite the increase in GLUT4, glucose transport into isolated muscles from transgenic mice was not increased in response to insulin. The expression of protein kinase B was decreased by approximately 60% in calpastatin transgenic muscle. This decrease could play a role in accounting for the insulin resistance relative to GLUT4 content of calpastatin transgenic muscle. The muscle weights of transgenic animals were substantially increased compared with controls. These results are consistent with the conclusion that calpain-mediated pathways play an important role in the regulation of GLUT4 degradation in muscle and in the regulation of muscle mass. Inhibition of calpain activity in muscle by overexpression of calpastatin is associated with an increase in GLUT4 protein without a proportional increase in insulin-stimulated glucose transport. These findings provide evidence for a physiological role for calpains in the regulation of muscle glucose metabolism and muscle mass.     

CaMK activation during exercise is required for histone hyperacetylation and MEF2A binding at the MEF2 site on the Glut4 gene

The role of CaMK II in regulating GLUT4 expression in response to intermittent exercise was investigated. Wistar rats completed 5 x 17-min bouts of swimming after receiving 5 mg/kg KN93 (a CaMK II inhibitor), KN92 (an analog of KN93 that does not inhibit CaMK II), or an equivalent volume of vehicle. Triceps muscles that were harvested at 0, 6, or 18 h postexercise were assayed for 1) CaMK II phosphorylation by Western blot, 2) acetylation of histone H3 at the Glut4 MEF2 site by chromatin immunoprecipitation (ChIP) assay, 3) bound MEF2A at the Glut4 MEF2 cis-element by ChIP, and 4) GLUT4 expression by RT-PCR and Western blot. Compared with controls, exercise caused a twofold increase in CaMK II phosphorylation. Immunohistochemical stains indicated increased CaMK II phosphorylation in nuclear and perinuclear regions of the muscle fiber. Acetylation of histone H3 in the region surrounding the MEF2 binding site on the Glut4 gene and the amount of MEF2A that bind to the site increased approximately twofold postexercise. GLUT4 mRNA and protein increased approximately 2.2- and 1.8-fold, respectively, after exercise. The exercise-induced increases in CaMK II phosphorylation, histone H3 acetylation, MEF2A binding, and GLUT4 expression were attenuated or abolished when KN93 was administered to rats prior to exercise. KN92 did not affect the increases in pCaMK II and GLUT4. These data support the hypothesis that CaMK II activation by exercise increases GLUT4 expression via increased accessibility of MEF2A to its cis-element on the gene.     

Differential regulation of exonic regulatory elements for muscle-specific alternative splicing during myogenesis and cardiogenesis

Muscle-specific isoform of the mitochondrial ATP synthase gamma subunit (F(1)gamma) was generated by alternative splicing, and exon 9 of the gene was found to be lacking particularly in skeletal muscle and heart tissue. Recently, we reported that alternative splicing of exon 9 was induced by low serum or acidic media in mouse myoblasts, and that this splicing required de novo protein synthesis of a negative regulatory factor (Ichida, M., Endo, H., Ikeda, U., Matsuda, C., Ueno, E., Shimada, K., and Kagawa, Y. (1998) J. Biol. Chem. 273, 8492-8501; Hayakawa, M., Endo, H., Hamamoto, T., and Kagawa, Y. (1998) Biochem. Biophys. Res. Commun. 251, 603-608). In the present report, we identified a cis-acting element on the muscle-specific alternatively spliced exon of F(1)gamma gene by an in vivo splicing system using cultured cells and transgenic mice. We constructed a F(1)gamma wild-type minigene, containing the full-length gene from exon 8 to exon 10, and two mutants; one mutant involved a pyrimidine-rich substitution on exon 9, whereas the other was a purine-rich substitution, abbreviated as F(1)gamma Pu-del and F(1)gamma Pu-rich mutants, respectively. Based on an in vivo splicing assay using low serum- or acid-stimulated splicing induction system in mouse myoblasts, Pu-del mutation inhibited exon inclusion, indicating that a Pu-del mutation would disrupt an exonic splicing enhancer. On the other hand, the Pu-rich mutation blocked muscle-specific exon exclusion following both inductions. Next, we produced transgenic mice bearing both mutant minigenes and analyzed their splicing patterns in tissues. Based on an analysis of F(1)gamma Pu-del minigene transgenic mice, the purine nucleotide of this element was shown to be necessary for exon inclusion in non-muscle tissue. In contrast, analysis of F(1)gamma Pu-rich minigene mice revealed that the F(1)gamma Pu-rich mutant exon had been excluded from heart and skeletal muscle of these transgenic mice, despite the fact mutation of the exon inhibited muscle-specific exon exclusion in myotubes of early embryonic stage. These results suggested that the splicing regulatory mechanism underlying F(1)gamma pre-mRNA differed between myotubes and myofibers during myogenesis and cardiogenesis.     

Munc18c regulates insulin-stimulated glut4 translocation to the transverse tubules in skeletal muscle

To examine the intracellular trafficking and translocation of GLUT4 in skeletal muscle, we have generated transgenic mouse lines that specifically express a GLUT4-EGFP (enhanced green fluorescent protein) fusion protein under the control of the human skeletal muscle actin promoter. These transgenic mice displayed EGFP fluorescence restricted to skeletal muscle and increased glucose tolerance characteristic of enhanced insulin sensitivity. The GLUT4-EGFP protein localized to the same intracellular compartment as the endogenous GLUT4 protein and underwent insulin- and exercise-stimulated translocation to both the sarcolemma and transverse-tubule membranes. Consistent with previous studies in adipocytes, overexpression of the syntaxin 4-binding Munc18c isoform, but not the related Munc18b isoform, in vivo specifically inhibited insulin-stimulated GLUT4-EGFP translocation. Surprisingly, however, Munc18c inhibited GLUT4 translocation to the transverse-tubule membrane without affecting translocation to the sarcolemma membrane. The ability of Munc18c to block GLUT4-EGFP translocation to the transverse-tubule membrane but not the sarcolemma membrane was consistent with substantially reduced levels of syntaxin 4 in the transverse-tubule membrane. Together, these data demonstrate that Munc18c specifically functions in the compartmentalized translocation of GLUT4 to the transverse-tubules in skeletal muscle. In addition, these results underscore the utility of this transgenic model to directly visualize GLUT4 translocation in skeletal muscle.     

The MEF2A isoform is required for striated muscle-specific expression of the insulin-responsive GLUT4 glucose transporter

Previously, we have demonstrated that an MEF2 consensus sequence located between -473/-464 in the human GLUT4 gene was essential for both tissue-specific and hormonal/metabolic regulation of GLUT4 expression (Thai, M. V., Guruswamy, S., Cao, K. T., Pessin, J. E., and Olson, A. L. (1998) J. Biol. Chem. 273, 14285-14292). To identify the specific MEF2 isoform(s) responsible for GLUT4 expression, we studied the pattern of expression of the MEF2 isoforms in insulin-sensitive tissues. Both heart and skeletal muscle were found to express the MEF2A, MEF2C, and MEF2D isoforms but not MEF2B. However, only the MEF2A protein was selectively down-regulated in insulin-deficient diabetes. Co-immunoprecipitation with isoform-specific antibodies revealed that, in the basal state, essentially all of the MEF2A protein was presented as a MEF2A-MEF2D heterodimer without any detectable MEF2A-MEF2A homodimers or MEF2A-MEF2C and MEF2C-MEF2D heterodimers. Electrophoretic mobility shift assays revealed that nuclear extracts from diabetic animals had reduced binding to the MEF2 binding site compared with extracts from control or insulin-treated animals. Furthermore, immunodepletion of the MEF2A-MEF2D complex from control extracts abolished binding to the MEF2 element. However, addition of MEF2A to diabetic nuclear extracts fully restored binding activity to the MEF2 element. These data strongly suggest that the MEF2A-MEF2D heterodimer is selectively decreased in insulin-deficient diabetes and is responsible for hormonally regulated expression of the GLUT4 gene.     

AMP kinase is not required for the GLUT4 response to exercise and denervation in skeletal muscle

An acute bout of exercise increases muscle GLUT4 mRNA in mice, and denervation decreases GLUT4 mRNA. AMP-activated protein kinase (AMPK) activity in skeletal muscle is also increased by exercise, and GLUT4 mRNA is increased in mouse skeletal muscle after treatment with AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside(AICAR). These findings suggest that AMPK activation might be responsible for the increase in GLUT4 mRNA expression in response to exercise. To investigate the role of AMPK in GLUT4 regulation in response to exercise and denervation, transgenic mice with a mutated AMPK alpha-subunit (dominant negative; AMPK-DN) were studied. GLUT4 did not increase in AMPK-DN mice that were treated with AICAR, demonstrating that muscle AMPK is inactive. Exercise (two 3-h bouts of treadmill running separated by 1 h of rest) increased GLUT4 mRNA in both wild-type and AMPK-DN mice. Likewise, denervation decreased GLUT4 mRNA in both wild-type and AMPK-DN mice. GLUT4 mRNA was also increased by AICAR treatment in both the innervated and denervated muscles. These data demonstrate that AMPK is not required for the response of GLUT4 mRNA to exercise and denervation.     

Overexpression of CUG triplet repeat-binding protein, CUGBP1, in mice inhibits myogenesis

Accumulation of RNA CUG repeats in myotonic dystrophy type 1 (DM1) patients leads to the induction of a CUG-binding protein, CUGBP1, which increases translation of several proteins that are required for myogenesis. In this paper, we examine the role of overexpression of CUGBP1 in DM1 muscle pathology using transgenic mice that overexpress CUGBP1 in skeletal muscle. Our data demonstrate that the elevation of CUGBP1 in skeletal muscle causes overexpression of MEF2A and p21 to levels that are significantly higher than those in skeletal muscle of wild type animals. A similar induction of these proteins is observed in skeletal muscle of DM1 patients with increased levels of CUGBP1. Immunohistological analysis showed that the skeletal muscle from mice overexpressing CUGBP1 is characterized by a developmental delay, muscular dystrophy, and myofiber-type switch: increase of slow/oxidative fibers and the reduction of fast fibers. Examination of molecular mechanisms by which CUGBP1 up-regulates MEF2A shows that CUGBP1 increases translation of MEF2A via direct interaction with GCN repeats located within MEF2A mRNA. Our data suggest that CUGBP1-mediated overexpression of MEF2A and p21 inhibits myogenesis and contributes to the development of muscle deficiency in DM1 patients.     

The CHC22 Clathrin-GLUT4 Transport Pathway Contributes to Skeletal Muscle Regeneration

Mobilization of the GLUT4 glucose transporter from intracellular storage vesicles provides a mechanism for insulin-responsive glucose import into skeletal muscle. In humans, clathrin isoform CHC22 participates in formation of the GLUT4 storage compartment in skeletal muscle and fat. CHC22 function is limited to retrograde endosomal sorting and is restricted in its tissue expression and species distribution compared to the conserved CHC17 isoform that mediates endocytosis and several other membrane traffic pathways. Previously, we noted that CHC22 was expressed at elevated levels in regenerating rat muscle. Here we investigate whether the GLUT4 pathway in which CHC22 participates could play a role in muscle regeneration in humans and we test this possibility using CHC22-transgenic mice, which do not normally express CHC22. We observed that GLUT4 expression is elevated in parallel with that of CHC22 in regenerating skeletal muscle fibers from patients with inflammatory and other myopathies. Regenerating human myofibers displayed concurrent increases in expression of VAMP2, another regulator of GLUT4 transport. Regenerating fibers from wild-type mouse skeletal muscle injected with cardiotoxin also showed increased levels of GLUT4 and VAMP2. We previously demonstrated that transgenic mice expressing CHC22 in their muscle over-sequester GLUT4 and VAMP2 and have defective GLUT4 trafficking leading to diabetic symptoms. In this study, we find that muscle regeneration rates in CHC22 mice were delayed compared to wild-type mice, and myoblasts isolated from these mice did not proliferate in response to glucose. Additionally, CHC22-expressing mouse muscle displayed a fiber type switch from oxidative to glycolytic, similar to that observed in type 2 diabetic patients. These observations implicate the pathway for GLUT4 transport in regeneration of both human and mouse skeletal muscle, and demonstrate a role for this pathway in maintenance of muscle fiber type. Extrapolating these findings, CHC22 and GLUT4 can be considered markers of muscle regeneration in humans.     

GLUT4: a key player regulating glucose homeostasis? Insights from transgenic and knockout mice

Studies in which GLUT4 has been overexpressed in transgenic mice provide definitive evidence that glucose transport is rate limiting for muscle glucose disposal. Transgenic overexpression of GLUT4 selectively in skeletal muscle results in increased whole body glucose uptake and improves glucose homeostasis. These studies strengthen the hypothesis that the level of muscle GLUT4 affects the rate of whole body glucose disposal, and underscore the importance of GLUT4 in skeletal muscle for maintaining whole body glucose homeostasis. Studies in which GLUT4 has been ablated or 'knocked-out' provide proof that GLUT4 is the primary effector for mediating glucose transport in skeletal muscle and adipose tissue. Genetic ablation of GLUT4 results in impaired insulin tolerance and defects in glucose metabolism in skeletal muscle and adipose tissue. Because impaired muscle glucose transport leads to reduced whole body glucose uptake and hyperglycaemia, understanding the molecular regulation of glucose transport in skeletal muscle is important to develop effective strategies to prevent or reduce the incidence of Type II diabetes mellitus. In patients with Type II diabetes mellitus, reduced glucose transport in skeletal muscle is a major factor responsible for reduced whole body glucose uptake. Overexpression of GLUT4 in skeletal muscle improves glucose homeostasis in animal models of diabetes mellitus and protects against the development of diabetes mellitus. Thus, GLUT4 is an attractive target for pharmacological intervention strategies to control glucose homeostasis. This review will focus on the current understanding of the role of GLUT4 in regulating cellular glucose uptake and whole body glucose homeostasis.     

Expression and regulation of the human GLUT4/muscle-fat facilitative glucose transporter gene in transgenic mice.

To study the molecular basis of tissue-specific expression of the GLUT4/muscle-fat facilitative glucose transporter gene, we generated lines of transgenic mice carrying 2.4 kilobases of the 5'-flanking region of the human GLUT4 gene fused to a chloramphenicol acetyltransferase (CAT) reporter gene (hGLUT4[2.4]-CAT). This reporter gene construct was specifically expressed in tissues that normally express GLUT4 mRNA, which include both brown and white adipose tissues as well as cardiac, skeletal, and smooth muscle. In contrast, CAT reporter activity was not detected in brain or liver, two tissues that do not express the GLUT4 gene. In addition, the relative levels of CAT mRNA driven by the human GLUT4 promoter in various tissues of these transgenic animals mirrored those of the endogenous mouse GLUT4 mRNA. Since previous studies have observed alterations in GLUT4 mRNA levels induced by fasting and refeeding (Sivitz, W. I., DeSautel, S. L., Kayano, T., Bell, G. I., and Pessin, J. E. (1989) Nature 340, 72-74), the regulated expression the hGLUT4[2.4]-CAT transgene was also assessed in these animals. Fasting was observed to decrease CAT activity in white adipose tissue which was super-induced upon refeeding. These alterations in CAT expression occurred in parallel to the changes in endogenous mouse GLUT4 mRNA levels. Although CAT expression in skeletal muscle and brown adipose tissue was unaffected, the endogenous mouse GLUT4 mRNA was also refractory to the effects of fasting/refeeding in these tissues. These data demonstrate that 2.4 kilobases of the 5'-flanking region of the human GLUT4 gene contain all the necessary sequence elements to confer tissue-specific expression and at least some of the sequence elements controlling the hormonal/metabolic regulation of this gene.