Effect of insulin on glucose metabolism in adipocytes from virgin and late-pregnant rats.

Under basal conditions (zero insulin), paraovarian adipocytes from 19-day-pregnant rats exhibited the same rates of [U-14C]glucose conversion into CO2 and total lipids as did those from age-matched virgin rats. The dose-response curves for insulin stimulation of glucose metabolism were similar in both groups: maximal response (+100% over basal values) and high sensitivity (half-maximal effect at 0.05 nM-insulin). The present results suggest that the insulin resistance in vivo that occurs during late pregnancy may involve circulating factors lost in vitro.     

Anomeric specificity of D-glucose metabolism in rat adipocytes

The anomeric specificity of D-glucose metabolism was investigated in rat adipocytes exposed for 60 min at 8 degrees C to pure alpha- or beta-D-glucose or to equilibrated D-glucose. The rate of D-[5-3H]glucose utilization was higher with alpha- than beta-D-glucose. However, as judged from the oxidation of D-[1-14C]glucose and D-[6-14C]glucose anomers, the fraction of D-glucose catabolism occurring via the pentose cycle was higher with beta- than alpha-D-glucose. In the presence of equilibrated D-glucose, the utilization of alpha-D-[5-3H]glucose and the oxidation of both alpha-D-[1-14C]glucose and alpha-D-[6-14C]glucose were higher, relative to the anomer concentration, than the corresponding values for beta-D-glucose. It is concluded that the anomeric specificity of D-glucose metabolism is operative in adipocytes, even when they are exposed to equilibrated D-glucose.     

Effect of lactation on insulin sensitivity of glucose metabolism in rat adipocytes

During lactation glucose metabolism in paraovarian adipocytes is characterized by a 40 and 80% decrease of glucose incorporation into CO2 and fatty acids in the presence of insulin. In contrast with the stimulation by insulin of glucose incorporation into lactate, glycerol remains unchanged. As a result, insulin sensitivity of total glucose metabolism (oxidation and lipid synthesis) is not altered in adipocytes from lactating rats.     

Effects of insulin and norepinephrine on glucose transport and metabolism in rat brown adipocytes. Potentiation by insulin of norepinephrine-induced glucose oxidation

Glucose is an important fuel for rat brown adipose tissue in vivo and its utilization is highly sensitive to insulin. In this study, the different glucose metabolic pathways and their regulation by insulin and norepinephrine were examined in isolated rat brown adipocytes, using [6-14C]glucose as a tracer. Glucose utilization was stimulated for insulin concentrations in the range of 40-1000 microU/ml. Furthermore, the addition of adenosine deaminase (200 mU/ml) or adenosine (10 microM) did not alter insulin sensitivity of glucose metabolism. The major effect of insulin (1 mU/ml) was a respective 7-fold and 5-fold stimulation of lipogenesis and lactate synthesis, whereas glucose oxidation remained very low. The 5-fold stimulation of total glucose metabolism by 1 mU/ml of insulin was accompanied by an 8-fold increase in glucose transport. In the presence of norepinephrine (8 microM), total glucose metabolism was increased 2-fold. This was linked to a 7-fold increase of glucose oxidation, whereas lipogenesis was greatly inhibited (by 72%). In addition, norepinephrine alone did not modify glucose transport. The addition of insulin to adipocytes incubated with norepinephrine, induced a potentiation of glucose oxidation, while lipogenesis remained very low. In conclusion, in the presence of insulin and norepinephrine glucose is a oxidative substrate for brown adipose tissue. However the quantitative importance of glucose as oxidative fuel remains to be determined.     

Sulphonylureas-induced increase in insulin binding and glucose metabolism by isolated rat adipocytes

The effects of oral hypoglycaemic drugs, SPC-703 (n-/p-toluenesulphonyl/-5-methyl-2-pirazoline-1-carbonami de) and tolbutamide on insulin binding and glucose metabolism by isolated adipocytes were studied. After 10 days of administration of both sulphonylurea derivatives, no differences were observed in insulin concentration between both experimental and the control groups of animals, despite a significant fall in blood glucose level. SPC-703 and tolbutamide in concentrations of 1 mM added in vitro to the suspension of adipocytes had no effect on insulin binding or on basal and insulin simulated glucose metabolism. Daily administration of 300 mg/kg body weight of SPC-703 or tolbutamide for 10 days resulted in 48% and 34% increase of specific binding of insulin by adipocytes, respectively. From the Scatchard plot analysis we noted that the increase of binding resulted from increased affinity of insulin receptors for hormone. Simultaneous increase in basal and insulin stimulated glucose metabolism by adipocytes, as measured by 14CO2 production and 14C incorporation into cellular lipids, was observed. The results indicate that hypoglycaemic action of sulphonylureas may be explained by increased affinity of insulin receptors and the stimulating action of these compounds on peripheral glucose metabolism.     

The influence of insulin on glucose permeability and metabolism of human granulocytes.

Viable human polymorphonuclear leukocytes isolated from peripheral blood were incubated for 1 h at 37 degrees C with variable concentrations of insulin in a saline medium buffered at pH 7.4. The hormone increased glucose consumption by about 40% without influencing the permeability of the membranes to glucose, whose uptake followed a passive diffusion process. The measurement of intermediates localized activation of glycolysis by insulin, down to 0.36 nM, at the phosphofructokinase step. However, the spectrophotometric measurement showed no activation of phosphofructokinase after preincubation with insulin of either intact granulocytes or crude or ultracentrifuged homogenates. The level of cyclic AMP, which is known to activate phosphofructokinase, was not modified by insulin; cyclic GMP did not activate the enzyme in the granulocyte extracts: neither of the two nucleotides can therefore be considered as a direct messenger of the action of insulin on phosphofructokinase. An important fraction of the extra glucose consumed under the influence of insulin was recovered as neither glycogen nor lactate, nor was it oxidized in the Krebs cycle. It might be assumed to have been converted into glycerolipids. However, insulin produced no detectable accumulation of triglycerides and activated neither the pentose phosphate pathway nor oxidative decarboxylation of pyruvate. The fate of the extra glucose consumed under the influence of insulin therefore remains questionable.     

Effects of insulin on glucose metabolism in isolated human fat cells

Isolated fat cells were used for the study of in vitro effects of insulin on glucose metabolism in human and rat adipose tissue. In human subcutaneous fat cells, effects of insulin could be detected at concentrations of glucose in the medium from 1 to 10 micro moles/ml. Cellular responsiveness was inversely proportional to the glucose level. At a constant concentration of 6 micro moles of glucose per ml, the effects of insulin at various concentrations up to 500 micro U/ml were investigated. At the highest concentration, which gave the maximal response, there was a 100% increase in the conversion of glucose-U-(14)C to glyceride-glycerol and a 40% increase in glucose oxidation. The dose-response curve was steepest between 2 and 20 micro U/ml. Rat epididymal fat cells were much more responsive to insulin. Glucose lipogenesis and pentose cycle activity could also be demonstrated in rat cells, whereas these activities could not be shown in fat cells from human omental and subcutaneous tissue. The findings for human cells are attributed to changes in cellular activity during preparation.     

Transport function and subcellular distribution of purified human erythrocyte glucose transporter reconstituted into rat adipocytes.

In order to delineate the insulin-independent (constitutive) and insulin-dependent regulations of the plasma membrane glucose transporter concentrations in rat adipocytes, we introduced purified human erythrocyte GLUT-1 (HEGT) into rat adipocytes by poly(ethylene glycol)-induced vesicle-cell fusion and its transport function and subcellular distribution in the host cell were measured. HEGT in adipocytes catalysed 3-O-methylglucose equilibrium exchange with a turnover number that is indistinguishable from that of the basal adipocyte transporters. However, insulin did not stimulate significantly the HEGT function in adipocytes where it stimulated the native transporter function by 7-8-fold. The steady state distribution and the transmembrane orientation assays revealed that more than 85% of the HEGT that were inserted in the physiological, cytoplasmic side-in orientation at the adipocytes plasma membrane were moved into low-density microsomes (LDM), while 90% of the HEGT that were inserted in the wrong, cytoplasmic side-out orientation were retained in the plasma membrane. Furthermore, more than 70% of the LDM-associated HEGT were found in a small subset of LDM that also contained 80% of the LDM-associated GLUT-4, the insulin-regulatable, native adipocyte glucose transporter. However, insulin did not cause redistribution of HEGT from LDM to the plasma membrane under the condition where it recruited GLUT-4 from LDM to increase the plasma membrane GLUT-4 content 4-5-fold. These results demonstrate that the erythrocyte GLUT-1 introduced in adipocytes transports glucose with an intrinsic activity similar to that of the adipocyte GLUT-1 and/or GLUT-4, and enters the constitutive GLUT-4 translocation pathway of the host cell provided it is in physiological transmembrane orientation, but fails to enter the insulin-dependent GLUT-4 recruitment pathway. We suggested that the adipocyte plasma membrane glucose transporter concentration is constitutively kept low by a mechanism where a cell-specific constituent interacts with a cytoplasmic domain common to GLUT-1 and GLUT-4, while the insulin-dependent recruitment requires a cytoplasmic domain specific to GLUT-4.     

Catecholamine-induced insulin resistance of glucose transport in isolated rat adipocytes.

The effects of pre-incubation with isoprenaline and noradrenaline on insulin binding and insulin stimulation of D-glucose transport in isolated rat adipocytes are reported. (1) Pre-incubation of the cells with isoprenaline (0.1-10 microM) in Krebs-Ringer-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid] buffer (30 min, 37 degrees C) at D-glucose concentrations of 16 mM, in which normal ATP levels were maintained, caused a rightward-shift in sensitivity of D-glucose transport to insulin stimulation by 50% and a decrease in maximal responsiveness by 30% (2) [A14-125I]insulin binding was reduced significantly by 35% at insulin concentrations less than 100 mu-units/ml and Scatchard analysis showed that this consisted mainly of a decrease in high-affinity binding. (3) Pre-incubation with catecholamines under the same conditions but at low glucose concentrations (0-5 mM) caused a fall in intracellular ATP levels of 65 and 45% respectively. (4) The fall in ATP additionally lowered insulin binding by 50% at all insulin concentrations and a parallel shift of the binding curves in the Scatchard plot showed that this was due to a decrease in the number of receptors. (5) At low and high ATP concentrations the insulin stimulation of D-glucose transport was inhibited to a similar extent. (6) Pre-incubation with catecholamines thus inhibited insulin stimulation of D-glucose transport in rat adipocytes mainly by a decrease in high-affinity binding of insulin, which was not mediated by low ATP levels. This mechanism may play a role in the pathogenesis of catecholamine-induced insulin resistance in vivo.     

The effect of glucose concentrations in the medium on expression of insulin receptors in human lymphocytes B and T: an in vitro study

Context: Insulin is one of the most-known factors that influence the intensity of cell-bound glucose transport. However, in order to react to this hormone, a cell needs specific receptors present in its membrane. The aim of this work was to investigate the insulin receptor expression in B and T cells under incubation with pathological glucose concentrations, respond hyperglycemia and hypoglycemia.

Materials and Methods: Isolated B and T cells were cultivated in different concentrations of glucose (high, low and normal). The expression of insulin receptors was investigated using methods of immunocytochemistry and flow cytometry.

Results: Incubation for 24?h of lymphocytes in pathological glucose concentrations seems to only have a slight influence on the expression of insulin receptors. No insulin receptor expression has been found in lymphocytes T incubated in both pathological concentrations of glucose. Different concentrations of glucose in the incubation medium were found to only marginally influence expression of insulin receptors in lymphocytes B.

Conclusions: Pathological concentrations of glucose in medium cause a decrease in the percent of cells which show expression of insulin receptors in comparison with normal glucose concentration. Thus, it appears highly probable that the insulin receptors did not arise under pathological glucose concentration in these cells de novo, but in little percent lymphocytes have existed there earlier, before the incubation.     

The effect of glucose concentrations in the medium on expression of insulin receptors in human lymphocytes B and T: an in vitro study

Context: Insulin is one of the most-known factors that influence the intensity of cell-bound glucose transport. However, in order to react to this hormone, a cell needs specific receptors present in its membrane. The aim of this work was to investigate the insulin receptor expression in B and T cells under incubation with pathological glucose concentrations, respond hyperglycemia and hypoglycemia. Materials and Methods: Isolated B and T cells were cultivated in different concentrations of glucose (high, low and normal). The expression of insulin receptors was investigated using methods of immunocytochemistry and flow cytometry. Results: Incubation for 24?h of lymphocytes in pathological glucose concentrations seems to only have a slight influence on the expression of insulin receptors. No insulin receptor expression has been found in lymphocytes T incubated in both pathological concentrations of glucose. Different concentrations of glucose in the incubation medium were found to only marginally influence expression of insulin receptors in lymphocytes B. Conclusions: Pathological concentrations of glucose in medium cause a decrease in the percent of cells which show expression of insulin receptors in comparison with normal glucose concentration. Thus, it appears highly probable that the insulin receptors did not arise under pathological glucose concentration in these cells de novo, but in little percent lymphocytes have existed there earlier, before the incubation.     

Insulin and insulin mutants stimulate glucose uptake in rat adipocytes

A simple method to determine thein vitro biological activity of insulin by measuring glucose uptake in the rat adipocytes is presented here. In the presence of insulin, the glucose uptake is 5–6 times more than the basal control. And the uptake of D-[3-³H]-glucose is linear as the logarithm of insulin concentration from 0.2 ώg/L to 1.0 ώg/L. Glucose and 3-O-methyl-glucose inhibit D-[3-³H]-glucose uptake into adipocytes. By this method, thein vitro biological activity of [B₂-Lys]-insulin and [B₃-Lys]-insulin was measured to be 61.6% and 154% respectively, relative to that of insulin.     

Time course of termination of the glucose transport action of insulin in adipocytes.

Rapid sequence measures of changes in the rate of 14CO2 production from [14C]glucose bathing the cells was abruptly reduced from 20 to 4 microunits/ml. Interpretation of the data in terms of glucose transport was based on calibration experiments that described the time course of change in 14CO2 production when [14C]glucose entry into adipocytes was slowed by reducing the specific activity of [14C]glucose in the incubation medium. All experiments were performed at 37 degrees in Krebs-Ringer bicarbonate buffer at pH 7.4. Termination of the glucose transport action of insulin (which includes insulin-receptor disassociation and all other steps leading to decelerated glucose entry) began within 2 min and was complete within 30 min. The transition from one steady state rate of glucose transport to the other could be approximated by an exponential process occurring with a half-time of 14 min. For comparison, the time course of initiation of the glucose transport action of insulin was measured under the same conditions. The transition curve was virtually identical.     

Effect of GLP-1 on lipid metabolism in human adipocytes.

We have studied the effect of several doses of GLP-1, compared to that of insulin and glucagons, on lipogenesis, lipolysis and cAMP cellular content, in human adipocytes isolated from normal subjects. In human adipocytes, GLP-1 exerts a dual action, depending upon the dose, on lipid metabolism, being lipogenic at low concentrations of the peptide (ED50, 10(-12) M), and lipolytic only at doses 10-100 times higher (ED50, 10(-10) M); both effects are time- and GLP-1 concentration-dependent. The GLP-1 lipogenic effect is equal in magnitude to that of equimolar amounts of insulin; both hormones apparently act synergically, and their respective action is abolished by glucagon. The lipolytic effect of GLP-1 is comparable to that of glucagon, apparently additive to it, and the stimulated value induced by either one is neutralized by the presence of insulin. In the absence of IBMX, GLP-1, at 10(-13) and 10(-12) M, only lipogenic doses, does not modify the cellular content of cAMP, while from 10(-11) M to 10(-9) M, also lipolytic concentrations, it has an increasing effect; in the presence of IBMX, GLP-1 at already 10(-12) M increased the cellular cAMP content. In human adipocytes, GLP-1 shows glucagon- and also insulin-like effects on lipid metabolism, suggesting the possibility of GLP-1 activating two distinct receptors, one of them similar or equal to the pancreatic one, accounting cAMP as a second messenger only for the lipolytic action of the peptide.