SNAP-23 participates in SNARE complex assembly in rat adipose cells.
SNARE proteins are required for vesicle docking and fusion in eukaryotic cells in processes as diverse as homotypic membrane fusion and synaptic vesicle exocytosis [SNARE stands for SNAP receptor, where SNAP is soluble NSF attachment protein]. The SNARE proteins syntaxin 4 and vesicle-associated membrane protein (VAMP) 2/3 also participate in the insulin-stimulated translocation of GLUT4 from intracellular vesicles to the plasma membrane in adipose cells. We now report the molecular cloning and characterization of rat SNAP-23, a ubiquitously expressed homologue of the essential neuronal SNARE protein SNAP-25 (synaptosomal-associated protein of 25 kDa). Rat SNAP-23 is 86% and 98% identical respectively to human and mouse SNAP-23. Southern blot analysis reveals that the rat, mouse and human SNAP-23 genes encode species-specific isoforms of the same protein. Co-immunoprecipitation of syntaxin 4 and SNAP-23 shows association of these two proteins in rat adipose cell plasma membranes, and insulin stimulation does not alter the SNAP-23/syntaxin 4 complex. In addition, we demonstrate for the first time the participation of SNAP-23, along with syntaxin 4 and VAMP2/3, in the formation of 20S SNARE complexes prepared using rat adipose cell membranes and recombinant alpha-SNAP and NSF proteins. The stoichiometry of the SNARE complexes formed is essentially identical using membranes from either unstimulated or insulin-stimulated adipose cells. These data demonstrate that rat SNAP-23 associates with syntaxin 4 before insulin stimulation and is present in the SNARE complexes known to mediate the translocation of GLUT4 from intracellular vesicles to the plasma membrane of rat adipose cells. (+info)
Tumour necrosis factor-alpha regulates expression of the CCAAT-enhancer-binding proteins (C/EBPs) alpha and beta and determines the occupation of the C/EBP site in the promoter of the insulin-responsive glucose-transporter gene in 3T3-L1 adipocytes.
We have demonstrated previously that treatment of 3T3-L1 adipocytes with tumour necrosis factor-alpha (TNF) results in a rapid (4 h) and significant (75-80%) reduction in the rate of transcription of the GLUT4 gene. Control of GLUT4 gene transcription has been suggested at least in part to reside with the CCAAT-enhancer-binding protein (C/EBP) family (alpha, beta and delta isoforms) of transcription factors. Using electrophoretic mobility shift assays, we have examined the ability of TNF to alter the occupation of the C/EBP site in the GLUT4 promoter. The data suggest that in fully differentiated adipocytes the C/EBP site is a ligand for predominantly alpha/alpha homodimers; however, after exposure to TNF, a shift in occupancy of the site occurs and the ligands become alpha/beta heterodimers and beta/beta homodimers. Partner selection in dimer formation appears to be controlled by selective translocation of the beta-isoform from the cytosol to the nucleus after exposure of the cells to TNF. (+info)
Training in swimming reduces blood pressure and increases muscle glucose transport activity as well as GLUT4 contents in stroke-prone spontaneously hypertensive rats.
Exercise improves muscle insulin sensitivity and GLUT4 contents. We investigated the beneficial effects of swimming training on insulin sensitivity and genetic hypertension using stroke-prone hypertensive rats (SHRSP). We studied the relationship between genetic hypertension and insulin resistance in SHRSP and Wistar Kyoto rats (WKY) as a control. The systolic blood pressure of SHRSP was significantly reduced by 4-week swimming training (208.4 +/- 6.8 mmHg vs. 187.2 +/- 4.1 mmHg, p < 0.05). The swimming training also resulted in an approximately 20% increase in the insulin-stimulated glucose transport activity (p < 0.05) of soleus muscle strips and an approximately 3-fold increase in the plasma membrane GLUT4 protein expression (p < 0.01) in SHRSP. However, basal and insulin-stimulated glucose transport activity and GLUT4 contents were not significantly different between WKY and SHRSP. There was no difference in insulin resistance in skeletal muscle of SHRSP as compared with WKY. Our results indicated swimming training exercise improved not only hypertension but also muscle insulin sensitivity and GLUT4 protein expression in SHRSP. (+info)
Resistance training affects GLUT-4 content in skeletal muscle of humans after 19 days of head-down bed rest.
This study assessed the effects of inactivity on GLUT-4 content of human skeletal muscle and evaluated resistance training as a countermeasure to inactivity-related changes in GLUT-4 content in skeletal muscle. Nine young men participated in the study. For 19 days, four control subjects remained in a -6 degrees head-down tilt at all times throughout bed rest, except for showering every other day. Five training group subjects also remained at bed rest, except during resistance training once in the morning. The resistance training consisted of 30 isometric maximal voluntary contractions for 3 s each; leg-press exercise was used to recruit the extensor muscles of the ankle, knee, and hip. Pauses (3 s) were allowed between bouts of maximal contraction. Muscle biopsy samples were obtained from the lateral aspect of vastus lateralis (VL) muscle before and after the bed rest. GLUT-4 content in VL muscle of the control group was significantly decreased after bed rest (473 +/- 48 vs. 398 +/- 66 counts. min-1. microgram membrane protein-1, before and after bed rest, respectively), whereas GLUT-4 significantly increased in the training group with bed rest (510 +/- 158 vs. 663 +/- 189 counts. min-1. microgram membrane protein-1, before and after bed rest, respectively). The present study demonstrated that GLUT-4 in VL muscle decreased by approximately 16% after 19 days of bed rest, and isometric resistance training during bed rest induced a 30% increase above the value of GLUT-4 before bed rest. (+info)
Time-dependent and tissue-specific effects of circulating glucose on fetal ovine glucose transporters.
To determine the cellular adaptations to fetal hyperglycemia and hypoglycemia, we examined the time-dependent effects on basal (GLUT-1 and GLUT-3) and insulin-responsive (GLUT-4) glucose transporter proteins by quantitative Western blot analysis in fetal ovine insulin-insensitive (brain and liver) and insulin-sensitive (myocardium, skeletal muscle, and adipose) tissues. Maternal glucose infusions causing fetal hyperglycemia resulted in a transient 30% increase in brain GLUT-1 but not GLUT-3 levels and a decline in liver and adipose GLUT-1 and myocardial and skeletal muscle GLUT-1 and GLUT-4 levels compared with gestational age-matched controls. Maternal insulin infusions leading to fetal hypoglycemia caused a decline in brain GLUT-3, an increase in brain GLUT-1, and a subsequent decline in liver GLUT-1, with no significant change in insulin-sensitive myocardium, skeletal muscle, and adipose tissue GLUT-1 or GLUT-4 concentrations, compared with gestational age-matched sham controls. We conclude that fetal glucose transporters are subject to a time-dependent and tissue- and isoform-specific differential regulation in response to altered circulating glucose and/or insulin concentrations. These cellular adaptations in GLUT-1 (and GLUT-3) are geared toward protecting the conceptus from perturbations in substrate availability, and the adaptations in GLUT-4 are geared toward development of fetal insulin resistance. (+info)
Reconstitution of insulin-sensitive glucose transport in fibroblasts requires expression of both PPARgamma and C/EBPalpha.
Adipocyte differentiation is regulated by at least two major transcription factors, CCAAT/enhancer-binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma (PPARgamma). Expression of PPARgamma in fibroblasts converts them to fat-laden cells with an adipocyte-like morphology. Here, we investigate the ability of PPARgamma to confer insulin-sensitive glucose transport to a variety of murine fibroblast cell lines. When cultured in the presence of a PPARgamma ligand, Swiss-3T3 and BALB/c-3T3 cells ectopically expressing PPARgamma accumulate lipid droplets, express C/EBPalpha, aP2, insulin-responsive aminopeptidase, and glucose transporter isoform 4 (GLUT4), and exhibit highly insulin-responsive 2-deoxyglucose uptake. In contrast, PPARgamma-expressing NIH-3T3 cells, despite similar lipid accumulation, adipocyte morphology, and aP2 expression, do not express C/EBPalpha or GLUT4 and fail to acquire insulin sensitivity. In cells ectopically expressing PPARgamma, the development of insulin-responsive glucose uptake correlates with C/EBPalpha expression. Furthermore, ectopic expression of C/EBPalpha in NIH-3T3 cells converts them to the adipocyte phenotype and restores insulin-sensitive glucose uptake. We propose that the pathway(s) leading to fat accumulation and morphological changes are distinct from that leading to insulin-dependent glucose transport. Our results suggest that although PPARgamma is sufficient to trigger the adipogenic program, C/EBPalpha is required for establishment of insulin-sensitive glucose transport. (+info)
Endothelin stimulates glucose uptake and GLUT4 translocation via activation of endothelin ETA receptor in 3T3-L1 adipocytes.
Endothelin-1 (ET-1) is a 21-amino acid peptide that binds to G-protein-coupled receptors to evoke biological responses. This report studies the effect of ET-1 on regulating glucose transport in 3T3-L1 adipocytes. ET-1, but not angiotensin II, stimulated glucose uptake in a dose-dependent manner with an EC50 value of 0.29 nM and a 2.47-fold stimulation at 100 nM. ET-1 stimulated glucose uptake in differentiated 3T3-L1 cells but had no effect in undifferentiated cells, although ET-1 stimulated phosphatidylinositol hydrolysis to a similar degree in both. The 3T3-L1 cells expressed approximately 560,000 sites/cell of ETA receptor, which was not altered during differentiation. Western blot analysis and immunofluorescence staining show that ET-1 stimulated the translocation of insulin-responsive aminopeptidase and GLUT4 to the plasma membrane. The effect of ET-1 on glucose uptake was blocked by A-216546, an antagonist selective for the ETA receptor. ET-1 treatment did not induce phosphorylation of insulin receptor beta-subunit, insulin receptor substrate-1, or Akt but stimulated the tyrosyl phosphorylation of a 75-kDa protein. Genistein (100 microM), an inhibitor of tyrosine kinases, inhibited ET-1-stimulated glucose uptake. Our results show that ET-1 stimulates GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes via activation of ETA receptor. (+info)
Muscle fiber type-specific defects in insulin signal transduction to glucose transport in diabetic GK rats.
To determine whether defects in the insulin signal transduction pathway to glucose transport occur in a muscle fiber type-specific manner, post-receptor insulin-signaling events were assessed in oxidative (soleus) and glycolytic (extensor digitorum longus [EDL]) skeletal muscle from Wistar or diabetic GK rats. In soleus muscle from GK rats, maximal insulin-stimulated (120 nmol/l) glucose transport was significantly decreased, compared with that of Wistar rats. In EDL muscle from GK rats, maximal insulin-stimulated glucose transport was normal, while the submaximal response was reduced compared with that of Wistar rats. We next treated diabetic GK rats with phlorizin for 4 weeks to determine whether restoration of glycemia would lead to improved insulin signal transduction. Phlorizin treatment of GK rats resulted in full restoration of insulin-stimulated glucose transport in soleus and EDL muscle. In soleus muscle from GK rats, submaximal and maximal insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation and IRS-1-associated phosphatidylinositol (PI) 3-kinase activity were markedly reduced, compared with that of Wistar rats, but only submaximal insulin-stimulated PI 3-kinase was restored after phlorizin treatment. In EDL muscle, insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1-associated PI-3 kinase were not altered between GK and Wistar rats. Maximal insulin-stimulated Akt (protein kinase B) kinase activity is decreased in soleus muscle from GK rats and restored upon normalization of glycemia (Krook et al., Diabetes 46:2100-2114, 1997). Here, we show that in EDL muscle from GK rats, maximal insulin-stimulated Akt kinase activity is also impaired and restored to Wistar rat levels after phlorizin treatment. In conclusion, functional defects in IRS-1 and PI 3-kinase in skeletal muscle from diabetic GK rats are fiber-type-specific, with alterations observed in oxidative, but not glycolytic, muscle. Furthermore, regardless of muscle fiber type, downstream steps to PI 3-kinase (i.e., Akt and glucose transport) are sensitive to changes in the level of glycemia. (+info)