Dehydroascorbic acid transport by GLUT4 in Xenopus oocytes and isolated rat adipocytes.

Dehydroascorbic acid (DHA), the first stable oxidation product of vitamin C, was transported by GLUT1 and GLUT3 in Xenopus laevis oocytes with transport rates similar to that of 2-deoxyglucose (2-DG), but due to inherent difficulties with GLUT4 expression in oocytes it was uncertain whether GLUT4 transported DHA (Rumsey, S. C. , Kwon, O., Xu, G. W., Burant, C. F., Simpson, I., and Levine, M. (1997) J. Biol. Chem. 272, 18982-18989). We therefore studied DHA and 2-DG transport in rat adipocytes, which express GLUT4. Without insulin, rat adipocytes transported 2-DG 2-3-fold faster than DHA. Preincubation with insulin (0.67 micrometer) increased transport of each substrate similarly: 7-10-fold for 2-DG and 6-8-fold for DHA. Because intracellular reduction of DHA in adipocytes was complete before and after insulin stimulation, increased transport of DHA was not explained by increased internal reduction of DHA to ascorbate. To determine apparent transport kinetics of GLUT4 for DHA, GLUT4 expression in Xenopus oocytes was reexamined. Preincubation of oocytes for >4 h with insulin (1 micrometer) augmented GLUT4 transport of 2-DG and DHA by up to 5-fold. Transport of both substrates was inhibited by cytochalasin B and displayed saturable kinetics. GLUT4 had a higher apparent transport affinity (K(m) of 0.98 versus 5.2 mm) and lower maximal transport rate (V(max) of 66 versus 880 pmol/oocyte/10 min) for DHA compared with 2-DG. The lower transport rate for DHA could not be explained by binding differences at the outer membrane face, as shown by inhibition with ethylidene glucose, or by transporter trans-activation and therefore was probably due to substrate-specific differences in transporter/substrate translocation or release. These novel data indicate that the insulin-sensitive transporter GLUT4 transports DHA in both rat adipocytes and Xenopus oocytes. Alterations of this mechanism in diabetes could have clinical implications for ascorbate utilization.

Short-term exercise enhances insulin-stimulated GLUT-4 translocation and glucose transport in adipose cells.

This investigation examined the effects of short-term exercise training on insulin-stimulated GLUT-4 glucose transporter translocation and glucose transport activity in rat adipose cells. Male Wistar rats were randomly assigned to a sedentary (Sed) or swim training group (Sw, 4 days; final 3 days: 2 x 3 h/day). Adipose cell size decreased significantly but minimally (approximately 20%), whereas total GLUT-4 increased by 30% in Sw vs. Sed rats. Basal 3-O-methyl-D-[14C]glucose transport was reduced by 62%, whereas maximally insulin-stimulated (MIS) glucose transport was increased by 36% in Sw vs. Sed rats. MIS cell surface GLUT-4 photolabeling was 44% higher in the Sw vs. Sed animals, similar to the increases observed in MIS glucose transport activity and total GLUT-4. These results suggest that increases in total GLUT-4 and GLUT-4 translocation to the cell surface contribute to the increase in MIS glucose transport with short-term exercise training. In addition, the results suggest that the exercise training-induced adaptations in glucose transport occur more rapidly than previously thought and with minimal changes in adipose cell size.

Impaired glucose tolerance in mice with a targeted impairment of insulin action in muscle and adipose tissue.

Type 2 diabetes is a complex metabolic disorder characterized by peripheral insulin resistance and impaired beta cell function. Insulin resistance is inherited as a non-mendelian trait. In genetically predisposed individuals, resistance of skeletal muscle and adipose tissue to insulin action precedes the onset of clinical diabetes, and is thought to contribute to hyperglycaemia by leading to impaired beta cell function and increased hepatic glucose production. It is not clear whether beta cell and liver defects are also genetically determined. To test the hypothesis that insulin resistance in muscle and fat is sufficient to cause type 2 diabetes in the absence of intrinsic beta cell and liver abnormality, we generated transgenic mice that were insulin-resistant in skeletal muscle and adipose tissue. These mice developed all the prodromal features of type 2 diabetes but, despite the compounded effect of peripheral insulin resistance and a mild impairment of beta cell function, failed to become diabetic. These findings indicate the need for a critical re-examination of the primary site(s) of insulin resistance in diabetes.

Insulin stimulates both leptin secretion and production by rat white adipose tissue.

Leptin, the peptide encoded by the obese gene, is secreted by adipose cells and plays a role in regulating food intake, energy expenditure, and adiposity. Because earlier studies suggested that insulin increases the expression of leptin, we investigated the effect of insulin on leptin secretion by adipose tissue. Epididymal fat pads were incubated in vitro in the presence or absence of insulin over a 4-h time course. Insulin increased leptin secretion by about 80% at all time points studied. After 10 min of insulin treatment, the amount of tissue-associated leptin was lower in insulin-stimulated tissue, presumably due to the increased secretion. At later times, both tissue-associated leptin and total leptin production were higher in insulin-treated tissue. In untreated, isolated adipose cells, immunostaining of leptin was detected in the endoplasmic reticulum by confocal microscopy. After insulin treatment, there were two populations of cells. In many cells, leptin staining became fainter and was restricted to a narrow band near the plasma membrane. However, in other cells the leptin-staining pattern was unchanged. Leptin did not colocalize with GLUT4, the glucose transporter isoform found primarily in insulin-responsive cells, in either basal or insulin-stimulated adipose cells. In this study, insulin increased both secretion and production of leptin by adipose tissue fragments. Interestingly, insulin appeared to stimulate the transport of leptin from the endoplasmic reticulum rather than acting on a pool of regulated secretory vesicles. (Endocrinology 138: 4463-4472, 1997)

The inhibitory effect of staurosporine on insulin action is prevented by okadaic acid. Evidence for an important role of serine/threonine phosphorylation in eliciting insulin-like effects.

The serine/threonine phosphatase inhibitor, okadaic acid (OA), exerted several insulin-like effects in rat adipose cells and was, in part, synergistic with insulin. OA stimulated glucose transport activity, altered the electrophoretic mobility of IRS-1, increased the phosphorylation of the MAP-kinases ERK 1 and 2 on tyrosine sites, markedly increased MAP kinase activity and also acted synergistically with insulin in activating these enzymes. However, OA did not increase PI 3-kinase activity or the tyrosine phosphorylation of key upstream proteins in insulin's signaling cascade. Staurosporine virtually completely inhibited the insulin-stimulated glucose transport and MAP kinase activation in spite of a maintained high PI 3-kinase activity. In contrast, the effects of OA alone or in the presence of insulin were less, or not at all, affected. These data suggest that OA exerts an insulin-like effect through a serine/threonine-related pathway which is distinct from, but converges with, that of insulin downstream PI 3-kinase and upon which staurosporine exerts an inhibitory effect.

Effects of overexpressing wild-type and mutant PDGF receptors on translocation of GLUT4 in transfected rat adipose cells.

Activation of phosphatidylinositol 3-kinase (PI3K) by insulin is necessary for the effect of insulin to recruit GLUT4 to the cell surface in insulin target cells. In adipose cells, stimulation of endogenous PDGF receptors (PDGF-R) results in increased PI3K activity without causing recruitment of GLUT4. We overexpressed wild-type or mutant forms of the PDGF-R in rat adipose cells and examined their effects on PDGF- and insulin-stimulated recruitment of co-transfected epitope-tagged GLUT4. Control cells expressing only tagged GLUT4 had a 3-fold increase in cell surface GLUT4 upon insulin stimulation but no response to PDGF. Cells overexpressing wild-type PDGF-R maintained insulin responsiveness and, in addition, acquired the ability to recruit GLUT4 in response to PDGF. Surprisingly, overexpression of F740/ F751 (mutant PDGF-R unable to directly activate PI3K) led to similar results. Nevertheless, wortmannin (an inhibitor of PI3K) blocked effects of both PDGF and insulin to recruit GLUT4. Our data suggest that overexpression of PDGF-R mediates positive effects on GLUT4 translocation by a wortmannin sensitive pathway not dependent on direct interaction of the PDGF-R with PI3K.

Hormonal regulation of glucose transport in a brown adipose cell preparation isolated from rats that shows a large response to insulin.

Isolated brown adipose cells from rats are prepared whose viability is indicated by the expected stimulation of oxygen consumption by noradrenaline and counter-regulation of this oxygen consumption response by insulin. Insulin stimulates 3-O-methyl-D-glucose transport by approx. 15-fold in the absence of adenosine, and adenosine augments this response at least 2-fold. The insulin-stimulated translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane is readily detected by subcellular fractionation and Western blotting, and the appearance of GLUT4 on the cell surface in response to insulin is demonstrated by bis-mannose photolabelling. Isoprenaline also stimulates glucose transport activity but only by approx. 3-fold; this effect is not altered by adenosine. Isoprenaline increases insulin-stimulated glucose transport activity in the absence of adenosine but decreases it in the presence of adenosine. These results demonstrate that although the regulation of glucose transport by insulin in brown adipose cells is qualitatively similar to that in white adipose cells, counter-regulation by adenosine and isoprenaline is at least quantitatively and may be qualitatively different. Isolated brown adipose cells from rats thus represent an excellent model for further examination of the mechanism by which multiple hormone signalling pathways interact to control glucose transport and GLUT4 subcellular trafficking.

Roles of 1-phosphatidylinositol 3-kinase and ras in regulating translocation of GLUT4 in transfected rat adipose cells.

Insulin stimulates glucose transport in insulin target tissues by recruiting glucose transporters (primarily GLUT4) from an intracellular compartment to the cell surface. Previous studies have demonstrated that insulin receptor tyrosine kinase activity and subsequent phosphorylation of insulin receptor substrate 1 (IRS-1) contribute to mediating the effect of insulin on glucose transport. We have now investigated the roles of 1-phosphatidylinositol 3-kinase (PI 3-kinase) and ras, two signaling proteins located downstream from tyrosine phosphorylation. Rat adipose cells were cotransfected with expression vectors that allowed transient expression of epitope-tagged GLUT4 and the other genes of interest. Overexpression of a mutant p85 regulatory subunit of PI 3-kinase lacking the ability to bind and activate the p110 catalytic subunit exerted a dominant negative effect to inhibit insulin-stimulated translocation of epitope-tagged GLUT4 to the cell surface. In addition, treatment of control cells with wortmannin (an inhibitor of PI 3-kinase) abolished the ability of insulin to recruit epitope-tagged GLUT4 to the cell surface. Thus, our data suggest that PI 3-kinase plays an essential role in insulin-stimulated GLUT4 recruitment in insulin target tissues. In contrast, over-expression of a constitutively active mutant of ras (L61-ras) resulted in high levels of cell surface GLUT4 in the absence of insulin that were comparable to levels seen in control cells treated with a maximally stimulating dose of insulin. However, wortmannin treatment of cells overexpressing L61-ras resulted in only a small decrease in the amount of cell surface GLUT4 compared with that of the same cells in the absence of wortmannin. Therefore, while activated ras is sufficient to recruit GLUT4 to the cell surface, it does so by a different mechanism that is probably not involved in the mechanism by which insulin stimulates GLUT4 translocation in physiological target tissues.

Insulin receptor substrate 1 mediates the stimulatory effect of insulin on GLUT4 translocation in transfected rat adipose cells.

Insulin signaling is initiated at least in part by activation of the insulin receptor tyrosine kinase and subsequent phosphorylation of cellular substrates such as insulin receptor substrate 1 (IRS-1). Previous studies have focused on the role of IRS-1 in the mitogenic actions of insulin. We have now investigated the possible role of IRS-1 in mediating the effect of insulin to stimulate glucose transport in a physiologically relevant insulin target tissue. In this study, we transfected rat adipose cells in primary culture with an antisense ribozyme directed against rat IRS-1. Expression of the ribozyme in these cells caused a 4.4-fold increase in the concentration of insulin required to achieve half-maximal stimulation of the translocation of cotransfected epitope-tagged GLUT4 without changing the maximal insulin response. Overexpression of human IRS-1 increased the basal cell surface GLUT4 to nearly the maximal level in the absence of insulin. When the ribozyme (specific to rat IRS-1) was cotransfected along with human IRS-1, the insulin dose-response curve was shifted to the left when compared with cells transfected with the ribozyme alone. These data provide strong support for the hypothesis that IRS-1 plays a role in insulin-stimulated glucose transport in insulin-responsive cells.

Tyrosine kinase-deficient mutant human insulin receptors (Met1153-->Ile) overexpressed in transfected rat adipose cells fail to mediate translocation of epitope-tagged GLUT4.

Insulin regulates essential pathways for growth, differentiation, and metabolism in vivo. We report a physiologically relevant system for dissecting the molecular mechanisms of insulin signal transduction related to glucose transport. This is an extension of our recently reported method for transfection of DNA into rat adipose cells in primary culture. In the present work, cDNA coding for GLUT4 with an epitope tag (HA1) in the first exofacial loop is used as a reporter gene so that GLUT4 translocation can be studied exclusively in transfected cells. Insulin stimulates a 4.3-fold recruitment of transfected epitope-tagged GLUT4 to the cell surface. Cells cotransfected with the reporter gene and the human insulin receptor gene show an increase in cell surface GLUT4 in the basal state (no insulin) to levels comparable to those seen with maximal insulin stimulation of cells transfected with the reporter gene alone. In contrast, cells overexpressing a naturally occurring tyrosine kinase-deficient mutant insulin receptor (Met1153-->Ile) show no increase in the basal cell surface GLUT4 and no shift in the insulin dose-response curve relative to cells transfected with the reporter gene alone. These results demonstrate that insulin receptor tyrosine kinase activity is essential in insulin-stimulated glucose transport in adipose cells.

Comparison of body weight and adipose tissue in male C57Bl/6J mice fed diets with and without trans fatty acids.

The effect of a diet containing trans-fatty acids (tFA) on the fatty acid composition and fat accumulation in adipose tissue was investigated in mice. Male C57Bl/6J mice were fed Control or Trans Diets that were similar, except that 50% of the 18:1, which was all cis in the Control Diet, was replaced by tFA in the Trans Diet. At selected ages, body weight, epididymal fat pad weight, perirenal fat yield, adipose tissue cellularity and fatty acid composition were examined. Over the time period studied (2-24 mon), the proportion of 18:0 and 16:0 tended to decrease while cis-18:1 levels increased. Compared to the Control Diet, the Trans Diet resulted in adipose tissue lipids with higher percentages of 14:0 and 18:2n-6 and lower percentages of cis-18:1 and 20:4n-6. In polar lipids, tFA replaced saturated fatty acids, whereas tFA replaced cis-18:1 in the nonpolar lipids. Body weights at 16 and 24 mon of age and epididymal fat pad weights at 8-24 mon of age were lower in mice fed the Trans Diet as compared to those fed the Control Diet. At the ages studied, the Trans Diet also resulted in lower values for perirenal fat weights, triacylglycerol to polar lipid ratios, and adipose cell size. The data suggest that chronic consumption of tFA affects lipid metabolism and results in decreased fat accumulation in murine adipose tissue.

Glucose transporter recycling in rat adipose cells. Effects of potassium depletion.

Depletion of intracellular potassium (K+) induced a 4-fold increase in basal and 1 microM phorbol-12-myristate-13-acetate (PMA)-stimulated 3-O-methylglucose transport in rat adipose cells. K+ depletion had no effect on the maximum insulin (0.7 microM)-stimulated transport rate but enhanced the sensitivity to insulin 3-fold (EC50 = 0.05 versus 0.15 nM) by a mechanism that did not result from changes in the insulin receptor binding, autophosphorylation, or tyrosine kinase activity. Western blotting analysis revealed that K+ depletion induced a 2.2-fold increase in GLUT4 in plasma membranes from basal cells, enhanced the PMA-stimulated GLUT4 translocation by 4-fold, and increased the 5-fold insulin-stimulated GLUT4 translocation by 15%, indicating the presence of an inactive GLUT4 intermediate. The time course for insulin's stimulation of transport activity was accelerated by K+ depletion (t1/2 = 3 versus 1.5 min). Conversely, the reversal of transport activity, on removal of insulin, was delayed (t1/2 = 11 versus 22 min). The corresponding t1/2 values for the loss of GLUT4 were 22 min in control cells and 40 min in K(+)-depleted cells, again indicating the existence of an inactive intermediate. Photolabeling intact cells with the impermeant, exofacial photolabel 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4 - yloxy)-2-propylamine in the continuous presence of insulin revealed that K+ depletion had no effect on the GLUT4 externalization rate but halved the rate of internalization. K+ depletion elicited entirely analogous effects on the recycling of insulin-like growth factor II/mannose 6-phosphate receptor, strongly supporting the involvement of a coated pit mechanism in the recycling of GLUT4 transporters. An inactive conformation of GLUT4 has been detected in plasma membranes from insulin-stimulated cells, which is enhanced by K+ depletion, suggesting a limitation in the adipose cells' capacity to express active GLUT4 transporters.

Transfection of DNA into isolated rat adipose cells by electroporation: evaluation of promoter activity in transfected adipose cells which are highly responsive to insulin after one day in culture.

Isolated adipose cells are among the most insulin responsive cells with respect to glucose transport and metabolism. However, molecular biological techniques such as transfection of DNA have heretofore not been applied successfully in these cells in primary culture. We report a method for transfection of DNA into rat adipose cells by electroporation. Six shocks at 800 V and 25 microF in a 0.4 cm gap cuvette results in efficient transfection. We compared the ability of five promoters to drive expression of a luciferase reporter gene in transfected adipose cells. After one day in culture, promoter activity ranged from no expression to a very high level of expression. These transfected, cultured cells also displayed a 10-fold increase in 3-O-methylglucose transport with maximal insulin stimulation. The ability to transfect DNA into adipose cells which remain insulin responsive after one day in primary culture may be helpful for understanding adipose cell-specific gene regulation and elucidating the molecular mechanisms of insulin action.

Insulin resistant glucose transport activity in adipose cells from the SHR/N-corpulent rat.

Young male obese (cp/cp) and lean (cp/+ or +/+) littermates of the SHR/N-corpulent (cp) strain were fed purified diets containing 54% carbohydrate as either sucrose or cooked starch for 12 wk. A significant effect of phenotype (obese greater than lean) was observed on body weight, epididymal fat pad weight and fat cell size. A diet effect (sucrose greater than starch) was observed on body weight, fat pad weight, and fat cell size. No effect of phenotype or diet was observed on basal 3-O-methylglucose transport in isolated adipose cells. However, insulin-stimulated glucose uptake was decreased 70-80% in isolated adipose cells from obese SHR/N-cp rats. No effect of diet on insulin-stimulated glucose uptake was observed in obese SHR/N-cp rats. Scatchard analysis of insulin binding data demonstrated no differences in the dissociation constant (KD) for the insulin receptor:insulin complex. However, obese rats exhibited a decreased number of insulin receptors compared to lean SHR/N-cp rats. These data demonstrate that the obese SHR/N-cp rat exhibits insulin-resistant glucose transport. This altered insulin sensitivity may be one factor contributing to the development of noninsulin-dependent diabetes mellitus in these animals.

Effect of insulin upon the cellular character of rat adipose tissue.

The effect of insulin upon the lipid content, and the number and size of fat cells in the epididymal, retroperitoneal, and subcutaneous adipose tissue of a large number of rats were examined. Insulin administration began either in early life (birth, 1, or 3 wk of age) or during adulthood (age 10 wk). At different times during growth, groups of treated and control animals were killed and the size and number of fat cells in each of the three adipose depots were determined. Insulin-treated animals gained weight at an increased rate and had fatter epididymal, retroperitoneal, and subcutaneous adipose depots than untreated controls. In each site the expanded adipose tissue was accompanied by an increase in the lipid content per cell (cell size), but in no case was there an increase in the number of adipose cells. This was the case regardless of whether insulin treatment was initiated before weaning (birth, 1 wk of age), at weaning (3 wk), or post weaning (10 wk) and irrespective of the duration of the insulin treatment.