Role of protein kinase C in the regulation of glucose transport in the rat adipose cell. Translocation of glucose transporters without stimulation of glucose transport activity.

The possible role of protein kinase C in the regulation of glucose transport in the rat adipose cell has been examined. Both insulin and phorbol 12-myristate 13-acetate (PMA) stimulate 3-O-methylglucose transport in the intact cell ein association with the subcellular redistribution of glucose transporters from the low density microsomes to the plasma membranes, as assessed by cytochalasin B binding. In addition, the actions of insulin and PMA on glucose transport activity and glucose transporter redistribution are additive.

Subcellular distribution and phosphorylation state of insulin receptors from insulin- and isoproterenol-treated rat adipose cells.

Rat adipose cells treated with insulin followed by isoproterenol exhibit a change in glucose transporter intrinsic activity (lowered maximal activity) and a decrease in insulin sensitivity (rightward shift of the concentration-response curve) when assayed for 3-O-methylglucose transport. To investigate the latter phenomenon, the distribution and phosphorylation state of insulin receptors was examined. Isoproterenol augmented the effect of insulin to reduce cell surface receptors by 20-30%.

Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel. Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester.

A new impermeant photoaffinity label has been used for identifying cell surface glucose transporters in isolated rat adipose cells. This compound is 2-N-4(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4- yloxy)-2- propylamine. We have used this reagent in combination with immunoprecipitation by specific antibodies against the GLUT4 and GLUT1 glucose transporter isoforms to estimate the relative abundance of these two transporters on the surface of the intact adipose cell following stimulation by insulin and phorbol 12-myristate 13-acetate (PMA).

Differential regulation of two glucose transporters in adipose cells from diabetic and insulin-treated diabetic rats.

At least two genetically distinct glucose transporters (GTs) coexist in adipose cells, one cloned from human hepatoma cells and rat brain (HepG2/brain) and another from rat skeletal muscle, heart, and adipose cells (adipose cell/muscle). Here we demonstrate differential regulation of these two GTs in adipose cells of diabetic and insulin-treated diabetic rats and compare changes in the expression of each GT with marked alterations in insulin-stimulated glucose transport activity. Adipose cell/muscle GTs detected by immunoblotting with the monoclonal antiserum 1F8 (James, D.

Acute effects of cycloheximide on the translocation of glucose transporters in rat adipose cells.

Insulin's rapid action to increase glucose transport is believed to occur primarily through the translocation of glucose transporters from an intracellular pool to the plasma membrane. To better understand the mechanism involved, we studied the role of protein synthesis in glucose transporter translocation by using the protein synthesis inhibitor, cycloheximide. Isolated rat epididymal adipose cells were incubated in the presence or absence of cycloheximide (10 micrograms/ml) for a total of 120 min.

Regulation of glucose transporter-specific mRNA levels in rat adipose cells with fasting and refeeding. Implications for in vivo control of glucose transporter number.

Fasting in the rat is associated with a rapid and progressive decrease in insulin-stimulated glucose transport activity in adipose cells, which is not only restored to normal, but increased transiently to supranormal levels by refeeding. The mechanisms for these changes in glucose transport activity appear to involve alterations in both glucose transporter number and intrinsic activity (glucose turnover number). In this study, we use the human hepatoma Hep G2 glucose transporter complementary DNA clone to examine the molecular basis for these alterations.

Insulin-induced subcellular redistribution of insulin-like growth factor II receptors in the rat adipose cell. Counterregulatory effects of isoproterenol, adenosine, and cAMP analogues.

Insulin shifts the steady-state subcellular distribution of insulin-like growth factor II (IGF-II) receptors from a large intracellular pool to the plasma membrane in the rat adipose cell (Wardzala, L. J., Simpson, I.

Characterization of the stimulatory action of insulin on insulin-like growth factor II binding to rat adipose cells. Differences in the mechanism of insulin action on insulin-like growth factor II receptors and glucose transporters.

Insulin is known to increase the number of cell surface insulin-like growth factor II (IGF-II) receptors in isolated rat adipose cells through a subcellular redistribution mechanism similar to that for the glucose transporter. The effects of insulin on these two processes, therefore, have now been directly compared in the same cell preparations. 1) Insulin increases the steady state number of cell surface IGF-II receptors by 7-13-fold without affecting receptor affinity; however, insulin stimulates glucose transport activity by 25-40-fold.

Divergent mechanisms for the insulin resistant and hyperresponsive glucose transport in adipose cells from fasted and refed rats. Alterations in both glucose transporter number and intrinsic activity.

The effects of fasting and refeeding on the glucose transport response to insulin in isolated rat adipose cells have been examined using 3-O-methylglucose transport in intact cells and cytochalasin B binding and Western blotting in subcellular membrane fractions. After a 72-h fast, basal glucose transport activity decreases slightly and insulin-stimulated activity decreases greater than 85%. Following 48 h of fasting, insulin-stimulated glucose transport activity is diminished from 3.

Qualitative and quantitative comparison of glucose transport activity and glucose transporter concentration in plasma membranes from basal and insulin-stimulated rat adipose cells.

Conditions are described which allow the isolation of rat adipose-cell plasma membranes retaining a large part of the stimulatory effect of insulin in intact cells. In these membranes, the magnitude of glucose-transport stimulation in response to insulin was compared with the concentration of transporters as measured with the cytochalasin-B-binding assay or by immunoblotting with an antiserum against the human erythrocyte glucose transporter. Further, the substrate- and temperature-dependencies of the basal and insulin-stimulated states were compared.

Activity and phosphorylation state of glucose transporters in plasma membranes from insulin-, isoproterenol-, and phorbol ester-treated rat adipose cells.

The counterregulatory action of catecholamines on insulin-stimulated glucose transport and its relation to glucose transporter phosphorylation were studied in isolated rat adipose cells. Plasma membranes exhibiting reduced glucose transport activity were prepared as described previously (Joost, H. G.

Mechanism for markedly hyperresponsive insulin-stimulated glucose transport activity in adipose cells from insulin-treated streptozotocin diabetic rats. Evidence for increased glucose transporter intrinsic activity.

The effects of insulin therapy in streptozotocin diabetic rats on the glucose transport response to insulin in adipose cells have been examined. At sequential intervals during subcutaneous insulin infusion, isolated cells were prepared and incubated with or without insulin, and 3-O-methylglucose transport was measured. Insulin treatment not only reversed the insulin-resistant glucose transport associated with diabetes, but resulted in a progressive hyperresponsiveness, peaking with a 3-fold overshoot at 7-8 days (12.

Mechanism for enhanced glucose transport response to insulin in adipose cells from chronically hyperinsulinemic rats. Increased translocation of glucose transporters from an enlarged intracellular pool.

The mechanism for the increased glucose transport response to insulin in adipose cells from chronically hyperinsulinemic rats was examined. Rats were infused with insulin s.c.

Regulation of insulin-stimulated glucose transport in the isolated rat adipocyte. cAMP-independent effects of lipolytic and antilipolytic agents.

This paper examines the modulation of insulin-stimulated glucose transport activity in rat adipose cells by ligands for receptors (R) that mediate stimulation (Rs; lipolytic) or inhibition (Ri; antilipolytic) of adenylate cyclase. The changes in glucose transport activity and cAMP, as assessed by 3-O-methylglucose uptake and (-/+) cAMP-dependent protein kinase (A-kinase) activity ratios, respectively, were monitored under conditions that maintain steady-state A-kinase activity ratios (Honnor, R. C.

Body composition, adipocyte size, free fatty acid concentration, and glucose tolerance in children of diabetic pregnancies.

Previous studies show that children of women who are diabetic during pregnancy are more obese and have a higher prevalence of non-insulin-dependent diabetes mellitus (NIDDM) than children of women who first developed NIDDM greater than 1 yr after the pregnancy (prediabetic mothers) and children of women who have never developed diabetes (nondiabetic mothers). To determine whether lean and obese children of glucose-intolerant pregnancies can be distinguished from similar children of glucose-tolerant pregnancies, we measured body composition, abdominal and gluteal adipocyte size, fasting free fatty acid (FFA), and fasting and stimulated glucose and insulin concentrations during an oral glucose tolerance test in prepubertal children of glucose-intolerant and prediabetic mothers. Each group ranged in adipocity from 6 to 40% body fat.

Insulin-stimulated glucose transport in rat adipose cells. Modulation of transporter intrinsic activity by isoproterenol and adenosine.

The mechanism of modulation of insulin-stimulated glucose transport activity in isolated rat adipose cells by lipolytic and antilipolytic agents has been examined. We have measured glucose transport activity in intact cells with 3-O-methylglucose and in plasma membranes with D-glucose, and the concentration of glucose transporters in plasma membranes using a cytochalasin B binding assay. In intact cells, isoproterenol reduced insulin-stimulated transport activity by 60%.

Glucose deprivation and hexose transporter polypeptides of murine fibroblasts.

The effect of Glc deprivation (starvation) on hexose transporter (GT) polypeptide(s) (pp) was studied in 3T3-C2 murine fibroblasts. Cells deprived of Glc exhibit 5-fold increases in hexose transport and Glc-displaceable cytochalasin B binding. Immunoblots of membranes reveal a Mr 55,000 GT pp in fed (4 g of Glc/liter) cells and Mr 55,000 and Mr 42,000 GT pp in starved cells.

Biochemical and functional heterogeneity of rat adipocyte glucose transporters.

We have studied the biochemical mechanism of insulin action on glucose transport in the rat adipocyte. Plasma membranes and low-density microsomes were prepared by differential ultracentrifugation of basal and insulin-stimulated cells. The photochemical cross-linking agent hydroxysuccinimidyl-4-azidobenzoate was used to covalently bind [3H]cytochalasin B to the glucose transporter which migrated as a 45-50-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Regulation of glucose utilization in adipose cells and muscle after long-term experimental hyperinsulinemia in rats.

The effects of chronic insulin administration on the metabolism of isolated adipose cells and muscle were studied. Adipose cells from 2 and 6 wk insulin-treated and control rats, fed either chow or chow plus sucrose, were prepared, and insulin binding, 3-O-methylglucose transport, glucose metabolism, and lipolysis were measured at various insulin concentrations. After 2 wk of treatment, adipose cell size and basal glucose transport and metabolism were unaltered, but insulin-stimulated transport and glucose metabolism were increased two- to threefold when cells were incubated in either 0.

Integral membrane protein translocations in the mechanism of insulin action.

The subcellular distributions of insulin and insulin-like growth factor type II (IGF-II) receptors, and glucose transporters, have been examined in basal and insulin-stimulated rat adipose cells. Plasma membranes (PM), high-density microsomes (HDM) and low-density microsomes (LDM) were prepared by differential ultracentrifugation. Insulin receptors were quantified by 125I-insulin binding or lactoperoxidase 125I-iodination and immunoprecipitation, IGF-II receptors by 125I-IGF-II binding, and glucose transporters by specific D-glucose-inhibitable [3H]cytochalasin B binding.

Subcellular translocation of glucose transporters: role in insulin action and its perturbation in altered metabolic states.

In this article we have described the hypothesis that insulin stimulates glucose transport through glucose transporter translocation from an intracellular pool to the plasma membrane. In addition, we have shown that changes in the numbers and subcellular distributions of glucose transporters correlate with alterations in insulin-stimulated glucose transport activity in several experimental models of insulin resistance and hyperresponsiveness. However, in experiments with counterregulatory hormones and with hyperresponsive states induced by nutritional repletion following deprivation, changes in insulin responsiveness cannot be fully explained by such alterations in the numbers and/or subcellular distribution of glucose transporters.