GLUT4 Gene Transcription Research Articles

Regulation of glucose transporter GLUT-4 and hexokinase II gene transcription by insulin and epinephrine.

Transport of glucose across the plasma membrane by GLUT-4 and subsequent phosphorylation of glucose by hexokinase II (HKII) constitute the first two steps of glucose utilization in skeletal muscle. This study was undertaken to determine whether epinephrine and/or insulin regulates in vivo GLUT-4 and HKII gene transcription in rat skeletal muscle. In the first experiment, adrenodemedullated male rats were fasted 24 h and killed in the control condition or after being infused for 1.5 h with epinephrine (30 microg/ml at 1.68 ml/h). In the second experiment, male rats were fasted 24 h and killed after being infused for 2.5 h at 1.68 ml/h with saline or glucose (625 mg/ml) or insulin (39.9 microg/ml) plus glucose (625 mg/ml). Nuclei were isolated from pooled quadriceps, tibialis anterior, and gastrocnemius muscles. Transcriptional run-on analysis indicated that epinephrine infusion decreased GLUT-4 and increased HKII transcription compared with fasted controls. Both glucose and insulin plus glucose infusion induced increases in GLUT-4 and HKII transcription of twofold and three- to fourfold, respectively, compared with saline-infused rats. In conclusion, epinephrine and insulin may regulate GLUT-4 and HKII genes at the level of transcription in rat skeletal muscle.

Eccentric contractions decrease glucose transporter transcription rate, mRNA, and protein in skeletal muscle

We have recently shown that eccentric contractions (ECs; forced lengthening of active muscle) elicit a delayed decrease in glucose transporter (GLUT-4) protein content in rat skeletal muscle and a decrease in subsequent contraction-stimulated glucose transport. Here, we investigate whether this decrease in total GLUT-4 protein after prior ECs is due to changes in GLUT-4 gene transcription rate and GLUT-4 mRNA level. Furthermore, the effect of prior ECs on sarcolemmal GLUT-4 protein content in plasma membrane (PM) vesicles isolated from contraction-stimulated muscle was determined. Rat gastrocnemius muscle was electrically stimulated for ECs, and the contralateral muscle served, as unstimulated control (UC). Two days later, the total GLUT-4 protein content was decreased by 50% (P < 0.05) and 32% (P < 0.05) in the white and red gastrocnemius muscle, respectively. Furthermore, the GLUT-4 mRNA concentration was decreased by 41% (P < 0.05) in both the white and red gastrocnemius muscle. Moreover, the GLUT-4 transcription rate, determined by nuclear run-on analysis, was decreased by 75% (P < 0.05) in mixed EC gastrocnemius muscle compared with UC muscle. PM vesicles were isolated from EC and UC muscle after 15 min of isometric contractions. The PM GLUT-4 protein content was reduced by 51% (P < 0.05) in EC muscle compared with UC muscle. In conclusion, 2 days after ECs, the GLUT-4 transcription rate, GLUT-4 mRNA, and GLUT-4 protein content were decreased in rat skeletal muscle. Moreover, the PM GLUT-4 protein content in contraction-stimulated muscle was decreased. We suggest that eccentric muscle contractions decrease muscle GLUT-4 transcription rate, resulting in a lower GLUT-4 protein content, which in turn decreases the number of GLUT-4 transporters translocated to the sarcolemma, ultimately leading to decreased contraction-induced muscle glucose transport.

Myogenesis and MyoD Down-regulate Sp1: A MECHANISM FOR THE REPRESSION OF GLUT1 DURING MUSCLE CELL DIFFERENTIATION

Muscle cell differentiation caused a reduction of glucose transport, GLUT1 glucose transporter expression, and GLUT1 mRNA levels. A fragment of 2.1 kilobases of the rat GLUT1 gene linked to chloramphenicol acetyltransferase drove transcriptional activity in myoblasts, and differentiation caused a decrease in transcription. Transient transfection of 5' and 3' deletion constructs showed that the fragment -99/-33 of the GLUT1 gene drives transcriptional activity of the GLUT1 gene and participates in the reduced transcription after muscle differentiation. Electrophoretic mobility shift assays showed the binding of Sp1 protein to the fragment -102/-37 in the myoblast state but not in myotubes, and Sp1 was found to transactivate the GLUT1 promoter. Western blot analysis indicated that Sp1 was drastically down-regulated during myogenesis. Furthermore, the forced over-expression of MyoD in C3H10T1/2 cells mimicked the effects observed during myogenesis, Sp1 down-regulation and reduced transcriptional activity of the GLUT1 gene promoter. In all, these data suggest a regulatory model in which MyoD activation during myogenesis causes the down-regulation of Sp1, which contributes to the repression of GLUT1 gene transcription and, therefore, leads to the reduction in GLUT1 expression and glucose transport.

Lipid mediators of insulin resistance: ceramide signalling down-regulates GLUT4 gene transcription in 3T3-L1 adipocytes

We have previously demonstrated that chronic exposure of 3T3-L1 adipocytes to tumour necrosis factor-alpha (TNF) resulted in a marked decrease (approximately 90%) in cellular GLUT4 (insulin-responsive glucose transporter) mRNA content as a result of a decreased transcription rate of the GLUT4 gene (approximately 75%) and a reduced half-life of its mRNA (9 to 4.5 h). Investigation of the signalling mechanism responsible for this regulation demonstrated that in the 3T3-L1 adipocytes, sphingomyelin levels decreased to 50% of control levels within 40 min of exposure to TNF, consistent with activation of a sphingomyelinase. In the same manner as with TNF, treatment of the adipocytes with 1-3 microM C6-ceramide, a membrane-permeable analogue of ceramide, decreased GLUT4 mRNA content by approximately 60%. Subsequent investigations revealed that transcription of the GLUT4 gene was reduced by approximately 65% in response to C6-ceramide, demonstrating that the decrease in mRNA content is mediated by a reduction in the transcription of the genc. No effect on GLUT4 mRNA stability was observed after exposure of the adipocytes to C6-ceramide. These observations are interesting in light of our previous data demonstrating that TNF affects both GLUT4 transcription and mRNA stability in the 3T3-L1 adipocytes. In conclusion, the effect of ceramide on GLUT4 gene expression is at the level of transcription, suggesting that another pathway controls mRNA stability. These data establish that ceramide-initiated signal transduction pathways exist within the adipocyte, and provide a potential mechanism for control of GLUT4 gene expression.

Regulation of GLUT4 Gene Expression by Arachidonic Acid: EVIDENCE FOR MULTIPLE PATHWAYS, ONE OF WHICH REQUIRES OXIDATION TO PROSTAGLANDIN E2

We have previously described the ability of arachidonic acid (AA) to regulate GLUT4 gene expression (Tebbey, P.W., McGowan, K.M., Stephens, J.M., Buttke, T.M., and Pekala, P.H. (1994) J. Biol. Chem. 269, 639-644). Chronic exposure (48 h) of fully differentiated 3T3-L1 cells to AA resulted in an approximately 90% suppression of GLUT4 mRNA accumulation. This decrease was demonstrated to be due to a 50% decrease in GLUT4 gene transcription as well as a destabilization of the GLUT4 message (t1/2 decreased from 8.0 to 4.6 h). In the current study we have identified, at least in part, the mechanism by which AA exerts its effects on GLUT4 expression. Compatible with a cyclooxygenase mediated event, the AA-induced suppression of GLUT4 mRNA was abolished by pretreating the cells with the inhibitor, indomethacin. Consistent with this observation, exposure of the cells to 10 microM PGE2 mimicked the effect of AA, in contrast to products of the lipoxygenase pathway which were unable to suppress GLUT4 mRNA content. Quantification of the conversion of AA to PGE2 demonstrated a 50-fold increase in PGE2 released into the media within 7 h of AA addition. Cyclic AMP levels were also increased 50-fold with AA treatment consistent with PGE2 activation of adenylate cyclase. Various long chain fatty acids, including the nonmetabolizable analog of AA, eicosatetraenoic acid (ETYA), also decreased GLUT4 mRNA levels. The effect of ETYA, a potent inhibitor of both lipo- and cyclooxygenases and a potent activator of peroxisome proliferator activated receptors (PPARs), suggested the presence of a second pathway where non-metabolized fatty acid functioned to suppress GLUT4 mRNA levels. Further support for a PPAR-mediated mechanism was obtained by exposure of the cells to the classic PPAR activator, clofibrate, which resulted in a approximately 75% decrease in GLUT4 mRNA content. Nuclear extracts prepared from the adipocytes contained a protein complex that bound to the PPAR responsive element (PPRE) found in the promoter of the fatty acyl-CoA oxidase gene. When the adipocytes were treated with either AA or ETYA, binding to the PPRE was disrupted, consistent with an ability of these fatty acids to control gene expression by altering the occupation of a PPRE. However, a perfect PPRE has yet to be identified in the GLUT4 promoter, but this does not rule the possibility of a PPAR playing an indirect role in the AA-induced GLUT4 mRNA suppression.

Regulation of glucose transporters in cultured rat adipocytes: synergistic effect of insulin and dexamethasone on GLUT4 gene expression through promoter activation.

A triggering effect of insulin on GLUT4 expression in adipocytes is consistently observed in vivo, whereas GLUT1 is roughly unaffected. However, in cultured rat adipocytes, insulin increases GLUT1 but fails to increase GLUT4, suggesting that additional factors are involved in vivo. This prompted us to evaluate the potential role of glucocorticoids as coregulators with insulin of glucose transporter expression using 3T3-F442A adipose cells and primary cultured rat adipocytes. In both systems, insulin increased and dexamethasone decreased GLUT1 messenger RNA (mRNA) and protein, an effect inhibited by the glucocorticoid antagonist RU 38486. When the two hormones were added together, the effect of dexamethasone was dominant in 3T3-F442A cells, but was totally antagonized in rat adipocytes. Moreover, in rat adipocytes, the GLUT1 gene transcription rate (run-on) was identical in the absence or presence of the two hormones. With regard to GLUT4 expression, neither insulin nor dexamethasone alone had any significant effect after 2 days of treatment. In contrast, the combined hormones markedly increased GLUT4 mRNA (+550% in rat adipocytes; +130% in 3T3-F442A cells) and protein (+164% in rat adipocytes; +79% in 3T3-F442A cells) with a 24- to 48-h delay after mRNA induction. Studies of the molecular mechanism(s) showed that exposure of rat adipocytes to dexamethasone plus insulin did not affect GLUT4 mRNA stability, but increased the GLUT4 gene transcription rate 3-fold. Transient transfections of rat adipocytes with the 5'-flanking 2.2-kilobase sequence of the rat GLUT4 gene fused to luciferase demonstrated that promoter activity was unchanged by insulin, increased 50% by dexamethasone, and increased 3-fold in the presence of both. These data show that insulin elicits an increase in GLUT4 gene expression provided glucocorticoids are present. Our results indicate that the synergism between insulin and glucocorticoids on GLUT4 gene transcription is mediated through GLUT4 promoter activation.

Regulation of glucose transporters in cultured rat adipocytes: synergistic effect of insulin and dexamethasone on GLUT4 gene expression through promoter activation

A triggering effect of insulin on GLUT4 expression in adipocytes is consistently observed in vivo, whereas GLUT1 is roughly unaffected. However, in cultured rat adipocytes, insulin increases GLUT1 but fails to increase GLUT4, suggesting that additional factors are involved in vivo. This prompted us to evaluate the potential role of glucocorticoids as coregulators with insulin of glucose transporter expression using 3T3-F442A adipose cells and primary cultured rat adipocytes. In both systems, insulin increased and dexamethasone decreased GLUT1 messenger RNA (mRNA) and protein, an effect inhibited by the glucocorticoid antagonist RU 38486. When the two hormones were added together, the effect of dexamethasone was dominant in 3T3-F442A cells, but was totally antagonized in rat adipocytes. Moreover, in rat adipocytes, the GLUT1 gene transcription rate (run-on) was identical in the absence or presence of the two hormones. With regard to GLUT4 expression, neither insulin nor dexamethasone alone had any significant effect after 2 days of treatment. In contrast, the combined hormones markedly increased GLUT4 mRNA (+550% in rat adipocytes; +130% in 3T3-F442A cells) and protein (+164% in rat adipocytes; +79% in 3T3-F442A cells) with a 24- to 48-h delay after mRNA induction. Studies of the molecular mechanism(s) showed that exposure of rat adipocytes to dexamethasone plus insulin did not affect GLUT4 mRNA stability, but increased the GLUT4 gene transcription rate 3-fold. Transient transfections of rat adipocytes with the 5'-flanking 2.2-kilobase sequence of the rat GLUT4 gene fused to luciferase demonstrated that promoter activity was unchanged by insulin, increased 50% by dexamethasone, and increased 3-fold in the presence of both. These data show that insulin elicits an increase in GLUT4 gene expression provided glucocorticoids are present. Our results indicate that the synergism between insulin and glucocorticoids on GLUT4 gene transcription is mediated through GLUT4 promoter activation.

Stimulation of GLUT-1 glucose transporter expression in response to exposure to calcium ionophore A-23187.

We tested the hypothesis that an increase in cytosolic calcium concentration stimulates glucose transporter isoform (GLUT-1) gene expression. Exposure of a rat liver cell line (Clone 9) to 3 microM A-23187 for 12 h resulted in 3-, 5-, and 10-fold increases in cytochalasin B-inhibitable 3-O-methyl-D-glucose transport, GLUT-1 protein, and GLUT-1 mRNA content, respectively. The induction of GLUT-1 mRNA in response to A-23187 is not preceded by a significant decrease in cell ATP content. This induction is prevented by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in conjunction with ethylene glycol-bis(beta-aminoethyl ether)-N,N, N',N'-tetraacetic acid. To investigate the mechanism of GLUT-1 mRNA induction, we found that exposure to A-23187 stabilized GLUT-1 mRNA: with the employment of actinomycin D, GLUT-1 mRNA had a half-life of 1.5 and 5.5 h in control and A-23187-treated cells, respectively. In nuclear run-on assays, the rate of GLUT-1 gene transcription was stimulated 1.5- to 1.7-fold in nuclei isolated from cells exposed to A-23187 for either 30 min or 2 h. These results demonstrate that exposure to A-23187 stimulates GLUT-1 gene expression and that the increase in GLUT-1 mRNA content is mediated in part by enhanced GLUT-1 gene transcription as well as decreased GLUT-1 mRNA degradation. The increase in GLUT-1 mRNA content, in turn, is associated with increased cell GLUT-1 content and enhanced glucose transport.

DIFFERENTIAL REGULATION OF GLUCOSE-TRANSPORTER EXPRESSION IN HEMATOPOIETIC-CELLS BY ONCOGENIC TRANSFORMATION AND CYTOKINE STIMULATION

Malignant transformation by the v-src oncogene and mitogenic stimulation by interleukin-3 (IL-3) both increased glucose transport into a hematopoietic cell line. These increases were additive and correlated with elevations in the level of GLUT1 mRNA. Glucose transport and GLUT1 mRNA were dependent on the presence of a functional v-src gene product in the absence of IL-3. Nuclear run-on analyses and mRNA turnover experiments demonstrated that GLUT1 gene transcription was enhanced by v-src while IL-3 stabilized GLUT1 mRNA. Introduction of retroviruses overexpressing GLUT1 into factor-dependent cells did not abrogate factor-dependency. Thus, GLUT1 induction is necessary but not sufficient for mitogenesis.

Exercise induces a transient increase in transcription of the GLUT-4 gene in skeletal muscle.

Endurance exercise training elicits an increase in mitochondrial density as well as GLUT-4 glucose transporter protein content in skeletal muscle. Corresponding increases in mRNA for respiratory enzymes and GLUT-4 indicate that pretranslational control mechanisms are involved in this adaptive process. To directly test whether transcription of the GLUT-4 gene is activated in response to exercise training, nuclei were isolated from red hindlimb skeletal muscle of rats after 1 wk of exercise training (8% grade, 32 m/min, 40 min, twice/day). Rats were killed either 30 min, 3 h, or 24 h after the last training session. GLUT-4 transcription, determined by nuclear run-on analysis, was unaltered after 30 min, increased by 1.8-fold after 3 h, but was no longer different from controls 24 h after exercise. A similar transient increase in GLUT-4 transcription was evident, but less pronounced (1.4-fold), in untrained rats after a single bout of exercise, suggesting that the postexercise induction in GLUT-4 gene transcription is enhanced by exercise training. GLUT-4 protein content was increased 1.7-fold after 1 wk of training in the absence of any corresponding change in GLUT-4 mRNA, providing evidence that the initial increase in GLUT-4 expression involves translational and/or posttranslational control mechanisms. These findings demonstrate that muscle GLUT-4 expression in response to exercise training is subject to both transcriptional and posttranscriptional regulation. We propose that the increase in GLUT-4 mRNA evident with extended periods of training may result from a shift to pretranslational control and is the cumulative effect of repeated postexercise transient increases in GLUT-4 gene transcription.

Induction of GLUT1 mRNA in response to inhibition of oxidative phosphorylation.

In previous studies, we have shown that inhibition of oxidative phosphorylation in Clone 9 cells (a nontransformed rat liver cell line) by 5 mM azide results in a marked biphasic stimulation of glucose transport that is mediated by GLUT1 [M. Shetty, J. N. Loeb, and F. Ismail-Beige. Am.J. Physiol. 262 (Cell Physiol. 31): C527-C532, 1992]. The late phase of the response (at 8-24 h) is associated with a doubling of cell GLUT1 content and an 8- to 10-fold increment in GLUT1 mRNA abundance. To investigate the mechanisms mediating GLUT1 mRNA induction, we have examined the effect of incubation in the presence of azide on GLUT1 gene transcription. In nuclear run-on assays, the rate of GLUT1 gene transcription was increased 2.5 +/- 0.3-fold in nuclei from cells exposed to azide for 4 h. Additionally, GLUT1 mRNA turnover was decreased in cells treated with azide: upon inhibition of RNA synthesis by actinomycin D, GLUT1 mRNA content decreased with half-lives of 2.3 +/- 0.3 and 8.0 +/- 0.5 h in control cells and cells treated with azide for 4 h, respectively. GLUT1 mRNA half-life was most prolonged (> 12 h) when azide was added subsequent to the addition of actinomycin D, and the half-life continued to be prolonged (6.5 +/- 0.5 h) in cells exposed to azide for 16 h.(ABSTRACT TRUNCATED AT 250 WORDS)