Phospholipase C mimics insulin action on pyruvate dehydrogenase and insulin mediator generation but not glucose transport or utilization

This study investigated the extent to which a purified phosphatidylinositol-specific and a commercial non-specific phospholipase C mimicked acute insulin action in rat adipocytes. The enzymes mimicked insulin stimulation of pyruvate dehydrogenase (PDH) and breakdown of a glycophospholipid proposed as a precursor for an intracellular mediator of insulin action, but were much less effective in stimulating glucose transport and utilization. These observations corroborate recent suggestions that insulin may activate a phospholipase C to generate a mediator that can account for insulin activation of PDH from a mediator precursor with a phosphatidylinositol anchor. This mediator precursor is probably an outer membrane component since effects were obtained with intact cells. It is unlikely that this mechanism accounts fully for insulin action since phosphatidylinositol-specific and commercial phospholipase C stimulation of glucose transport was significantly less than that elicited by insulin.     

Diacylglycerols modulate insulin action in rat adipocytes

Effect of 1,2-diacylglycerols on the insulin receptor function and insulin action in rat adipocytes was studied. 1,2-dioctanoylglycerol (100 micrograms/ml) did not alter insulin binding but it did stimulate phosphorylation of the beta-subunit of the insulin receptor as well as its tyrosine kinase activity. However, dioctanoylglycerol inhibited insulin-stimulated receptor autophosphorylation. This concentration of dioctanoylglycerol inhibited insulin-stimulated CO2 metabolism, lipogenesis and 3-O-methyl-glucose transport in a dose-dependent manner but did not alter any of these bioeffects in absence of insulin. While there was no direct link between diacylglycerol effect on tyrosine kinase activity of the insulin receptor and insulin action in rat adipocytes, the parallel inhibition of insulin-stimulated receptor autophosphorylation and insulin bioeffects by dioctanoylglycerol suggests its direct or indirect role in insulin signalling in rat fat cells.     

Effect of purified phospholipases on glucose transport, insulin binding, and insulin action in isolated rat adipocytes

The influence of alterations in phospholipid structure by phospholipase treatment on insulin action and glucose transport in rat adipocytes was studied. It appeared that phospholipase A2 from bee venom caused a breakdown of approximately 50% of phosphotidylcholine without lysis of the cells. Because of this treatment, insulin binding was increased, resulting in an increased sensitivity of glucose transport towards lower insulin concentrations. Moreover, an increased affinity of the transport system for 2-deoxyglucose was observed. Phospholipase C from Clostridium welchii caused complete lysis of adipocytes. Phospholipase A2 from Crotalus adamenteus was without effect.     

Pervanadate [peroxide(s) of vanadate] mimics insulin action in rat adipocytes via activation of the insulin receptor tyrosine kinase

Both vanadate and hydrogen peroxide (H2O2) are known to have insulin-mimetic effects. We previously reported that the mixture of vanadate plus H2O2 results in the generation of a peroxide(s) of vanadate, which strongly enhances IGF-II binding to rat adipocytes (Kadota et al., 1987b). We now report that pervanadate mimics insulin in isolated rat adipocytes to (1) stimulate lipogenesis, (2) inhibit epinephrine-stimulated lipolysis, and (3) stimulate protein synthesis. The efficacy of pervanadate is comparable to that of insulin. However, it is 10(2)-10(3) times more potent than vanadate alone. Exposure of intact rat adipocytes to pervanadate was found to activate the WGA-purified insulin receptor tyrosine kinase assayed with the exogenous substrate poly(Glu80/Tyr20) in a dose-dependent manner to a maximum of 1464% of control at 10(-3) M compared with a maximum insulin effect of 1046% at 10(-6) M. In contrast, in vitro assayed autophosphorylation of the WGA-purified extract was increased 3-fold after exposure of intact cells to insulin but not significantly increased after pervanadate. Furthermore, high concentrations of pervanadate (10(-5) M) inhibited subsequent in vitro added insulin-stimulated autophosphorylation. In vitro addition of pervanadate to WGA-purified receptors could not stimulate autophosphorylation or exogenous tyrosine kinase activity and did not inhibit insulin-stimulated autophosphorylation. Labeling of intact adipocytes with [32P]orthophosphate followed by exposure to 10(-4) M pervanadate increased insulin receptor beta-subunit phosphorylation (7.9 +/- 3.0)-fold, while 10(-7) M insulin and 10(-4) vanadate increased labeling (5.3 +/- 1.8)- and (1.1 +/- 0.2)-fold, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Insulin and insulin mutants stimulate glucose uptake in rat adipocytes

A simple method to determine the in 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 μg/L to 1.0 μg/L. Glucose and 3-O-methyl-glucose inhibit D-[3-3H]-glucose uptake into adipocytes. By this method, the in 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.     

Adenosine effects upon insulin action on lipolysis and glucose transport in human adipocytes

The dose response effect of a new adenosine analogue, GR 79236 (N-[1S trans-2-hydroxycyclopentyl] adenosine) upon insulin sensitivity was examined in human adipocytes. The influence of adenosine upon insulin sensitivity for suppression of lipolysis and stimulation of glucose transport was examined. Removal of adenosine by use of adenosine deaminase stimulated lipolysis to the same extent as did 10^–9 M noradrenaline. GR79236 brought about dose dependent inhibition of lipolysis with half-maximal effect at 11.3±7.8×10^–9 M. When lipolysis was stimulated by noradrenaline alone the subsequent inhibition of lipolysis brought about by GR79236 was significantly greater than that of insulin. To examine adenosine effects on the insulin signalling pathway separately from those on lipolysis, the insulin sensitivity of glucose transport was examined. Removal of adenosine brought about a small but significant increase in the concentration of insulin required for half-maximal stimulation of glucose transport. Adenosine agonists offer promise as new agents for the modulation of metabolism in diabetes and other states of insulin resistance.     

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.     

The effect of triiodothyronine on insulin binding and action in rat adipocytes

The effects of a short term (2 hour) incubation of 5 microM triiodothyronine (T3) on 125I-insulin binding and insulin stimulated (14C)-2-deoxy-D-glucose uptake in rat adipocytes was investigated. In the presence of 5 microM T3, (14C)-2-deoxy-D-glucose uptake was significantly decreased by 11 to 24% at insulin concentrations of 5 to 1000 microU/ml. The concentration of insulin eliciting a half maximal response for insulin stimulated (14C)-2-deoxy-D-glucose uptake was 11.5 microU/ml in the control, and 14.3 microU/ml in the T3 treated cells (p less than 0.01). T3 treated adipocytes bound 9 to 22% less 125I-labeled insulin yet the concentration of native insulin necessary to displace 50% of the bound 125I-labeled insulin was the same in the control and T3 treated cells (75 and 70 ng/ml, respectively). These studies indicate that the decreased sensitivity of T3 treated cells to insulin is in accordance with a decreased number of receptors with the same binding characteristics as those of the control cells. The decreased maximal uptake of (14C)-2-deoxy-D-glucose at saturating insulin levels is likely to be independent of receptor number and result from a second, undetermined alteration in the hexose transport system of adipocytes treated with T3.     

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.     

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.     

The potentiations by insulin-stimulating peptide from bovine serum albumin of the effects of insulin mimickers and insulin in stimulating glucose utilization by rat adipocytes

An insulin-stimulating peptide derived from bovine serum albumin by digestion with trypsin was shown to inhibit insulin degradation. Addition of this peptide (1.2 microM) to the medium of isolated rat adipocytes markedly inhibited the degradation of insulin in the medium, but had a little effect on degradation of cell-associated insulin. Moreover, this peptide did not prevent dissociation of cell-associated insulin, suggesting that it is a bacitracin-type, not a chloroquine-type inhibitor of insulin degradation. The peptide also potentiated the stimulation by insulin mimickers of glucose oxidation by rat adipocytes, strongly indicating that it has some other effects besides inhibition of insulin degradation. Therefore, the effect of the peptide on activation of pyruvate dehydrogenase (PDH), one of the postbinding actions of insulin, was studied. Addition of the peptide (4 microM) to adipocytes was found to activate PDH in the absence or presence of insulin. This stimulatory effect of the peptide on PDH was dose-dependent and was observed in both whole cells and subcellular fractions of rat adipocytes. The peptide also stimulated PDH in a subcellular system of either plasma membranes and mitochondria or mitochondria only. Sodium fluoride, an inhibitor of phosphatase, blocked the action of the peptide almost completely, suggesting that the stimulatory effect of the peptide on PDH activity is at least partly due to its activation of PDH phosphatase. The mechanisms of action of the peptide are discussed. The peptide should be useful in studies on modulation of the action of insulin.     

In vitro effects of glucocorticoid on glucose transport in rat adipocytes: evidence of a post-receptor coupling defect in insulin action

The in vitro effect of glucocorticoid on insulin binding and glucose transport was studied with rat adipocytes. Isolated rat adipocytes were incubated with or without 0.70 microgram/ml (1.9 mumol) of hydrocortisone in TCM 199 medium at 37 degrees C, 5% CO2/95% air (v/v), pH 7.4, for 2, 4, and 8 h, and then fat cell insulin binding and insulin-stimulated 3-O-methylglucose transport were measured. Hydrocortisone did not affect insulin binding in terms of affinity or receptor number. Glucose transport in the absence of insulin was significantly decreased at the incubation time of 2 h and continued to decrease up to 8 h of incubation with hydrocortisone. Decreased insulin sensitivity of glucose transport (i.e., a right-ward shift of the dose response curve) was also demonstrated after 2 h incubation with hydrocortisone, and the ED50 of insulin was maximally increased at 4 h of incubation (0.53 ng/ml for treated vs. 0.22 ng/ml for control cells). Maximal insulin responsiveness was also significantly decreased in treated cells after 8 h incubation with hydrocortisone. When percent maximum glucose transport was expressed relative to receptor-bound insulin, the ED50 values of treated and control cells were 10.5 and 7.2 pg of bound insulin, per 2 X 10(5) cells, respectively. Thus, it was evident that glucocorticoid induced a post-receptor coupling defect in the signal transmission of insulin-receptor complex.     

Effect of depletion of phosphate and bicarbonate ions on insulin action in rat adipocytes

The effect of insulin on rat adipocytes was studied in isotonic buffers (pH 7.4) containing NaCl, CaCl2, MgSO4, KCl, and bovine serum albumin but no phosphate or bicarbonate anions. In phosphate- and bicarbonate-free buffers the dose-response curve to insulin is shifted to the right, the effects of the hormone on hexose uptake, glucose metabolism, and inhibition of lipolysis being observed at much higher (nearly 2 orders of magnitude) concentrations of insulin. The insulin binding capacity of the cells is only slightly changed. The dose-response curve for isoproterenol which stimulates lipolysis in the same cell type is almost the same in both Krebs-Ringer bicarbonate buffer and phosphate- and bicarbonate-free buffers. The dose-response curves for agents that mimic the action of insulin such as wheat germ agglutinin or vanadate ions are also shifted to the right. The dose-response curve to insulin can be returned to "normal" by readdition of either bicarbonate or phosphate. Almost complete recovery is obtained at either 10 mM bicarbonate or 24 mM phosphate, respectively. External Ca2+ ions which are not required for the proper action of insulin in fat cells maintained in Krebs-Ringer bicarbonate buffer, become essential for insulin action in bicarbonate-free buffer. The study indicates that depletion of bicarbonate and, to a lesser extent, phosphate anions, interferes with an essential insulin-dependent post-binding event. Also, in bicarbonate-free medium, external Ca2+ ions are essential for insulin-mediated processes. The implications of this study to the mode of action of insulin, and to physiological and clinical states of insulin desensitization are discussed.     

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.     

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.     

Effect of insulin on the glucose utilization in isolated cardiac myocytes from adult rat

The acute effects of insulin on glucose utilization in isolated rat quiescent cardiac myocytes were studied. Insulin (80 nM) increased the rate of glucose clearance by 2-3 times in the presence of glucose ranging from 0.3 microM to 5.5 mM. Glucose transport, which was measured in terms of both D-glucose uptake in the presence of 0.3 microM D-glucose and initial rate of uptake of 3-O-methylglucose, was stimulated 3-fold in the presence of insulin. At higher glucose concentrations (greater than 100 microM), a decrease in glucose clearance rate due to a shift of the rate-limiting step from glucose transport to a post-transport step in the pathway of glucose metabolism was observed. At the physiological concentration of glucose (5.5 mM), about 73% of glucose was metabolized into lactate, about 10% was oxidized into CO2 and the rest (17%) remained inside the cells. The pentose phosphate pathway did not contribute to the glucose metabolism in these cells. Insulin (80 nM) significantly increased the uptake of glucose (112%), and the conversions of glucose into lactate (16%), glycogen (64%), and triglyceride (18%), but not into CO2 (3%). Insulin transiently increased the percentage of I-form of glycogen synthase by 16% above basal, but did not affect the percentage of a-form of glycogen phosphorylase. The content of glucose 6-phosphate in the cells was increased by 46% above the basal value in the presence of insulin. These results indicate that insulin has different acute stimulatory effects on various steps in the metabolic pathway of glucose in isolated quiescent cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)