Cyclic AMP modulates insulin binding and induces post-receptor insulin resistance of glucose transport in isolated rat adipocytes

The effect of cAMP on insulin binding and insulin stimulation of glucose transport was investigated in isolated rat adipocytes. Preincubation for 30 min in medium containing 16 mmol/l glucose and either db-cAMP or bromo-cAMP in concentrations of 10(-4)-10(-3) M inhibited high affinity binding of insulin by 15 to 30% and glucose transport by 30 to 50%. Preincubation with IBMX (10(-4)-10(-3) M) reduced insulin binding by 25% and glucose transport by 70%. Closer analysis of these data indicated that preincubation with these compounds caused not only a decrease in insulin binding but also a post-receptor resistance. High intracellular cyclic AMP-levels seem therefore to induce insulin resistance at both receptor and post-receptor levels.     

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.     

In vivo and in vitro effect of p-chlorophenoxyisobutyrate on insulin binding and glucose transport in isolated rat adipocytes

We studied the in vivo and in vitro effect of p-chlorophenoxyisobutyrate (CPIB) on insulin binding and glucose transport in isolated rat adipocytes. In the in vitro study, adipocytes were incubated with 1mM of CPIB for 2 h at 37 degrees C, pH 7.4, and then insulin binding (37 degrees C, 60 min) and 3-0-methylglucose transport (37 degrees C, 2s) were measured. Incubation with CPIB did not affect either insulin binding or glucose transport in the cells. The addition of insulin (10 ng/ml) with CPIB to the incubation media also did not affect the following insulin binding and glucose transport. In the in vivo study, rats were fed a high sucrose-diet containing 0.25% CPIB for 7 days. Serum cholesterol, plasma free fatty acid, and insulin levels were significantly decreased in the CPIB-treated rats. The treated rats demonstrated an almost 2 fold increased maximal binding capacity for insulin (189,000 sites/cell for treated vs 123,000 sites/cell for control cells). Basal glucose transport (glucose transport in the absence of insulin) significantly decreased in the CPIB-treated rats, although insulin-stimulated glucose transport was comparable in treated and control cells. Thus, CPIB might have no direct effect on glucose transport and insulin binding, as determined by the in vitro studies. Furthermore, a relatively short-term in vivo treatment with CPIB, such as 7 days, did not stimulate glucose transport.     

Glucose oxidation,glucose transport and insulin binding in isolated rat epididymal adipocytes in the presence of anesthetics

Insulin binding and glucose oxidation were measured in isolated rat adipocytes in the presence of several anesthetics; ethanol, n-octanol, pentobarbital, chlorpromazine and tetracaine. Ethanol and chlorpromazine, at anesthetic and pentobarbitol at sub-anesthetic concentrations are inhibitory to both basal and insulin stimulated rates of glucose oxidation. At all concentrations of ethanol, pentobarbital or chlorpromazine tested binding of insulin is not affected. Since anesthetics may alter membrane fluidity, it is suggested that an anesthetic-induced increase in membrane fluidity beyond that which occurs at 37°C is detrimental to glucose oxidation. Of the 5 anesthetics examined, only chlorpromazine (10 μM or less) and tetracaine (500 μM) stimulate glucose oxidation. These two agents are known to bind to a cell's cytoskeletal system; the binding of chlorpromazine to microtubules is entropy driven. The temperature and concentration dependence of chlorpromazine stimulation of glucose oxidation (transport) are consistent with this form of binding. It is proposed that chlorpromazine binds to the cytoskeletal system of the adipocyte and that this system is normally restrictive to the motion of membrane proteins. Disruption of the cytoskeletal system by chlorpromazine or tetracaine would increase the frequency of insulin-receptor and glucose-carrier contact. Activation of glucose transport could ensue.     

Regulation of glucose transport in isolated adipocytes by levamisole.

When isolated rat adipocytes were incubated with increasing concentrations of levamisole (0.5-5 mM), basal glucose oxidation decreased by almost 50% and insulin-stimulated glucose oxidation decreased by 90%. The decrease in glucose oxidation correlated with an inhibition of glucose transport, since levamisole at 5.0 mM decreased basal 3-O-methylglucose transport by 60% and insulin-stimulated transport by 80%. Diamide-stimulated glucose transport was also inhibited approximately 80% by 5.0 mM levamisole. Levamisole at concentrations up to 5.0 mM had no effect on phosphofructokinase activity. The present results suggest that levamisole inhibits glucose utilization by inhibiting glucose transport in a concentration-dependent manner.     

Metabolic requirements for deactivation of insulin-stimulated glucose transport in isolated rat adipocytes

We have studied the rate of deactivation of the insulin-stimulated glucose transport system following the removal of insulin. Under all conditions, dissociation of insulin from its receptor proceeded much more rapidly than deactivation of the glucose transport system, indicating that deactivation was not simply a passive process reflecting a decline in receptor occupancy. The results demonstrate that deactivation of the glucose transport system is dependent upon ongoing cellular metabolism, and that this process occurs in a normal manner when a variety of substrates (glucose, fructose, or pyruvate) are available to the cells. When no substrate was present, then transport remained at or near the fully stimulated level. In an attempt to localize which metabolic sequence is involved in mediating glucose transport deactivation, studies were performed in the presence of a variety of substrates, inhibitors, and combinations of the two. NaF and citrate had marked effects to inhibit the normal rate of deactivation in the presence of glucose, whereas DNP had no effect on the rate of deactivation in the presence of added glucose. Pyruvate is a substrate which enters the glycolytic pathway distal to the site of action of NaF or citrate in the glycolytic pathway, and in the presence of pyruvate, the inhibiting effects of NaF and citrate on the rate of deactivation were abolished. These results demonstrate that deactivation of the insulin-stimulated glucose transport system is an active process dependent upon some aspect of cellular glucose metabolism. It is likely that the important metabolic step is distal to the point at which pyruvate enters the glycolytic pathway and possibly proximal to the step at which DNP inhibits mitochondrial oxidative phosphorylation.     

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.     

Endotoxin-induced alterations in glucose transport in isolated adipocytes

2-Deoxyglucose and $\text{3-O-}\text{methyglucose}$ were used to assess endotoxin-induced changes in glucose transport in rat adipocytes. 6 h after Escherichia coli endotoxin injection insulin-stimulated 2-deoxyglucose uptake was significantly depressed ($\text{V}\text{decreased}\text{, K}{}_{\mathrm{<mtext>m</mtext>}}\text{unaltered}$), phosphorylation of 2-deoxyglucose was seemingly unimpaired; basal 3-methylglucose entry was significantly increased, insulin-stimulated uptake was unaltered. Insulin significantly reduced $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ in control and endotoxin-treated cells. Cytochalasin B-insensitive uptake of both 2-deoxyglucose and 3-methylglucose, a small fraction of total transport, increased significantly in endotoxic cells. Endotoxin reduced spermine- and insulin-stimulated 2-deoxyglucose uptake to a similar extent. Results are consistent with the hypotheses that (1) a site of endotoxin-induced insulin resistance is at the cell membrane level and may reflect a decrease in number or activity of effective carrier units, rather than alterations in affinity, (2) endotoxin does not compromise the hexokinase system, (3) the cell membrane-localized effect of endotoxin on hexose transport is not necessarily mediated by the insulin receptor and (4) the entry of 2-deoxyglucose and 3-methylglucose may involve two separate transport systems.     

Glucose and insulin co-regulate the glucose transport system in primary cultured adipocytes. A new mechanism of insulin resistance

We have previously shown in primary cultured rat adipocytes that insulin acts at receptor and multiple postreceptor sites to decrease insulin's subsequent ability to stimulate glucose transport. To examine whether D-glucose can regulate glucose transport activity and whether it has a role in insulin-induced insulin resistance, we cultured cells for 24 h in the absence and presence of various glucose and insulin concentrations. After washing cells and allowing the glucose transport system to deactivate, we measured basal and maximally insulin-stimulated 2-deoxyglucose uptake rates (37 degrees C) and cell surface insulin binding (16 degrees C). Alone, incubation with D-glucose had no effect on basal or maximal glucose transport activity, and incubation with insulin, in the absence of glucose, decreased maximal (but not basal) glucose transport rates only 18% at the highest preincubation concentration (50 ng/ml). However, in combination, D-glucose (1-20 mM) markedly enhanced the long-term ability of insulin (1-50 ng/ml) to decrease glucose transport rates in a dose-responsive manner. For example, at 50 ng/ml preincubation insulin concentration, the maximal glucose transport rate fell from 18 to 63%, and the basal uptake rate fell by 89%, as the preincubation D-glucose level was increased from 0 to 20 mM. Moreover, D-glucose more effectively promoted decreases in basal glucose uptake (Ki = 2.2 +/- 0.4 mM) compared with maximal transport rates (Ki = 4.1 +/- 0.4 mM) at all preincubation insulin concentrations (1-50 ng/ml). Similar results were obtained when initial rates of 3-O-methylglucose uptake were used to measure glucose transport. D-glucose, in contrast, did not influence insulin-induced receptor loss. In other studies, D-mannose and D-glucosamine could substitute for D-glucose to promote the insulin-induced changes in glucose transport, but other substrates such as L-glucose, L-arabinase, D-fructose, pyruvate, and maltose were without effect. Also, non-metabolized substrates which competitively inhibit D-glucose uptake (3-O-methylglucose, cytochalasin B) blocked the D-glucose plus insulin effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Phorbolesters enhance basal D-glucose transport but inhibit insulin stimulation of D-glucose transport and insulin binding in isolated rat adipocytes

The C-kinase activating phorbolester TPA (12-O-Tetradecanoyl-beta-phorbol-13-acetate) and PdBu (4-beta-Phorbol-12,13,dibutyrate) stimulated D-glucose transport twofold in isolated rat adipocytes but inhibited high affinity insulin binding and the responsiveness of D-glucose transport to insulin stimulation by about 30%. Phorbolesters have therefore insulin-like effects but antagonize insulin on receptor and postreceptor level.     

Insulin stimulation of glucose transport in isolated rat adipocytes. Functional evidence for insulin activation of intrinsic transporter activity within the plasma membrane.

We examined the effects of the membrane-impermeant amino-group-modifying agent fluorescein isothiocyanate (FITC) on the basal and insulin-stimulated hexose-transport activity of isolated rat adipocytes. Pre-treatment of cells with FITC causes irreversible inhibition of transport measured in subsequently washed cells. Transport activity was inhibited by approx. 50% with 2 mM-FITC in 8 min. The cells respond to insulin, after FITC treatment and removal, and the fold increase in transport above the basal value caused by maximal concentrations of insulin was independent of the concentration of FITC used for pre-treatment over the range 0-2 mM, where basal activity was progressively inhibited. The ability of FITC to modify selectively hexose transporters accessible only to the external milieu was evaluated by two methods. (1) Free intracellular FITC, and the distribution of FITC bound to cellular components, were assessed after dialysis of the homogenate and subcellular fractionation on sucrose gradients by direct spectroscopic measurement of fluorescein. Most (98%) of the FITC was associated with the non-diffusible fractions. Equilibrium sucrose-density-gradient centrifugation of the homogenate demonstrated that the subcellular distribution of the bound FITC correlated with the density distribution of a plasma-membrane marker, but not markers for Golgi, endoplasmic reticulum, mitochondria or protein. Exposing the cellular homogenate, rather than the intact cell preparation, to 2 mM-FITC resulted in a 4-5-fold increase in total bound FITC, and the density-distribution profile more closely resembled the distribution of total protein. (2) Incubation of hexokinase preparations with FITC rapidly and irreversibly inactivates this protein. However, both intracellular hexokinase total activity and its apparent Michaelis constant for glucose were unaffected in FITC-treated intact cells. Further control experiments demonstrated that FITC pre-treatment of cells had no effect on the intracellular ATP concentration or the dose-response curve of insulin stimulation of hexose transport. Since the fold increase of hexose transport induced by insulin is constant over the range of inhibition of surface-labelled hexose transporters, we suggest that insulin-induced insertion of additional transporters into the plasma membrane may not be the major locus of acceleration of hexose transport by the hormone.     

Effects of islet-activating protein on insulin- and isoprenaline-stimulated glucose transport in isolated rat adipocytes

The effects of islet-activating protein (IAP), a Bordetella pertussis toxin, on insulin- and isoprenaline-stimulated glucose transport were studied in isolated rat adipocytes. Basal as well as insulin-stimulated glucose transport were not affected when cells were pretreated with IAP. In contrast, IAP pretreatment abolished the stimulatory effect of isoprenaline. When IAP-pretreated cells were exposed to a combination of insulin and isoprenaline, the catecholamine significantly reduced the stimulatory effect of insulin. Since IAP is supposed to specifically block the inhibitory component Ni of adenylate cyclase, the results suggest that: (a) the effect of insulin is unrelated to the regulation of adenylate cyclase; (b) isoprenaline may exert both stimulatory and inhibitory effects depending on activation of Ni. The inhibitory regulation of adenylate cyclase may thus be a pivotal link in the regulation of glucose transport.     

Effects of decavanadate and insulin enhancing vanadium compounds on glucose uptake in isolated rat adipocytes

The effects of different vanadium compounds namely pyridine-2,6-dicarboxylatedioxovanadium(V) (V5-dipic), bis(maltolato) oxovanadium(IV) (BMOV) and amavadine, and oligovanadates namely metavanadate and decavanadate were analysed on basal and insulin stimulated glucose uptake in rat adipocytes. Decavanadate (50 μM), manifest a higher increases (6-fold) on glucose uptake compared with basal, followed by BMOV (1 mM) and metavanadate (1 mM) solutions (3-fold) whereas V5 dipic and amavadine had no effect. Decavanadate (100 μM) also shows the highest insulin like activity when compared with the others compounds studied. In the presence of insulin (10 nM), only decavanadate increases (50%) the glucose uptake when compared with insulin stimulated glucose uptake whereas BMOV and metavanadate, had no effect and V5 dipic and amavadine prevent the stimulation to about half of the basal value. Decavanadate is also able to reduce or eradicate the suppressor effect caused by dexamethasone on glucose uptake at the level of the adipocytes. Altogether, vanadium compounds and oligovanadates with several structures and coordination spheres reveal different effects on glucose uptake in rat primary adipocytes.     

Mechanisms of insulin degradation by isolated rat adipocytes

Summary We have examined some of the chemical and biological characteristics of the insulin-derived cell-associated radioactivity following incubation of isolated adipocytes with ¹²⁵I-insulin (10^–10 M) for one hour at 37 °C S ephadex G-50 chromatography of the cell-associated radioactivity demonstrated three peaks: peak I eluted with the void volume and consisted of large molecular weight material; peak II comigrated with 1251-insulin; and peak III consisted of small molecular weight degradation products (probably iodotyrosine). When the insulin peak (peak II) was divided into fourths, it was found that the binding and biologic activity of this material was not homogenous; thus, binding and biologic activity (relative to native insulin) fell markedly from the earliest to the latest eluting fractions of this peak. Furthermore, when the entire peak 11 material was applied to DEAE-Sephacel and eluted with a 0.01–0.2 M NaCl gradient, three distinct peaks were observed. These peaks were all 90% TCA precipitable, whereas the ability of the latter two eluting peaks to precipitate with anti-insulin antiserum was markedly reduced. When similar experiments were performed with chloroquine-treated cells, a large increase in cell-associated radioactivity was observed, and Sephadex G-50 chromatography demonstrated that this increase was entirely confined to peaks I and II. When the insulin peak (peak II) was divided into fourths, it was found that chloroquine markedly inhibited the decreased binding and biologic activity, from the earliest to the latest eluting fraction of this peak. Furthermore, when the peak II material (Sephadex G-50) from chloroquine-treated cells was chromatographed on DEAE-Sephacel, this material eluted in a single peak which was 95% TCA precipitable and 106% precipitable by anti-insulin antiserum. In conclusion, these studies demonstrate that: 1) intermediate insulin-derived products with reduced binding and biologic activity are generated in the process of cellular insulin degradation, and 2) the formation of these intermediate products is mediated by a chloroquine-sensitive pathway.     

Stimulation of glucose transport by guanine nucleotides in permeabilized rat adipocytes.

Effects of guanine nucleotides on glucose transport were studied in permeabilized rat epididymal fat cells. GTP gamma S and Gpp(NH)p, but not App(NH)p, stimulated 3-O-methylglucose transport. Effect of GTP gamma S was dose-dependent, being detectable at 0.1 mM, and 1.0 mM GTP gamma S stimulated glucose transport to the same extent as insulin. GTP gamma S (0.3 mM) enhanced insulin-stimulated glucose transport while 1 mM GTP gamma S did not affect insulin-mediated transport. GDP beta S had no effect on glucose transport by itself but rather enhanced insulin action. NaF, which is known to activate trimeric G proteins, increased glucose transport to the same extent as insulin. Likewise, mastoparan augmented glucose transport. These results indicate that a certain type of trimeric G protein(s) is involved in the regulation of glucose transport.     

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.