Development of insulin-sensitivity at weaning in the rat. Role of the nutritional transition.

This study was undertaken to determine the factors involved in the development of insulin-sensitivity at weaning. Glucose kinetics were studied in suckling rats and in rats weaned on to a high-carbohydrate (HC) or a high-fat (HF) diet, in the basal state and during euglycaemic-hyperinsulinaemic-clamp studies. These studies were coupled with the 2-deoxyglucose technique, allowing a measure of glucose utilization by individual tissues. In the basal state, the glycaemia was higher in HF-weaned rats (124 +/- 4 mg/dl) than in suckling (109 +/- 1 mg/dl) and HC-weaned rats (101 +/- 3 mg/dl). Glucose turnover rates were similar in the three groups of animals (14 mg/min per kg). Nevertheless, basal metabolic glucose clearance rate was 20% lower in HF-weaned rats than in the other groups. During the euglycaemic-hyperinsulinaemic experiments, hepatic glucose production was suppressed by 90% in HC-weaned rats, whereas it remained at 40% of basal value in suckling and HF-weaned rats, indicating an insulin resistance of liver of these animals. Glucose clearance rate during the clamp was 18.3 +/- 0.9 ml/min per kg in suckling rats, whereas it was 35.3 +/- 1.2 ml/min per kg in HC-weaned rats and 27.8 +/- 1.1 ml/min per kg in HF-weaned rats, indicating an insulin resistance of glucose utilization in suckling, and to a lower extent, in HF-weaned rats. The deoxyglucose technique showed that peripheral insulin resistance was localized in muscles and white adipose tissue of suckling and HF-weaned rats. These results indicate that the switch from milk to a HC diet is an important determinant of the development of insulin-sensitivity at weaning in the rat.     

Effect of insulin on glucose transport and metabolism in isolated fat-cells of gonadal adipose tissue from mature age-matched male and female rats.

Insulin action on glucose transport and metabolism was studied in paraovarian adipocytes from 3-month-old female rats and compared with insulin action in epididymal adipocytes from closely age-matched males. At maximal insulin concentrations the stimulations of 2-deoxyglucose uptake (4-fold the basal value) and of [U-14C]glucose incorporation into CO2 and total lipids (3- and 2-fold the basal values respectively) were similar in adipocytes from rats of both sexes. At submaximal insulin concentrations (less than 0.2 nM) the ability of paraovarian adipocytes to transport and to metabolize glucose was higher than that of epididymal adipocytes; accordingly an increase in insulin binding was observed in paraovarian adipocytes as compared with epididymal adipocytes. These results show that paraovarian adipocytes from mature female rats were highly responsive to insulin, and exhibited a higher sensitivity to the hormone than did epididymal adipocytes from male rats of the same age.     

Late effects of postnatal administration of monosodium glutamate on insulin action in adult rats

Early postnatal administration of monosodium glutamate (MSG) to rats induces obesity, hyperinsulinemia and hyperglycemia in adulthood, thus suggesting the presence of insulin resistance. We therefore investigated the effects of insulin on glucose transport and lipogenesis in adipocytes as well as insulin binding to specific receptors in the liver, skeletal muscle and fat tissues. An increase of plasma insulin, glucose and leptin levels was found in 3-month-old rats treated with MSG during the postnatal period. The attenuation of insulin stimulatory effect on glucose transport was observed in MSG-treated rats. Despite the lower basal and insulin-stimulated glucose uptake, the incorporation of glucose into lipids was significantly higher in MSG-treated rats, suggesting a shift in glucose metabolism towards lipid synthesis in fat tissue. Insulin binding to plasma membranes from the liver, skeletal muscle and adipocytes was decreased in MSG-treated rats. This is in agreement with the lower insulin effect on glucose transport in these animals. Furthermore, a decreased amount of GLUT4 protein was found in adipocytes from MSG-treated obese rats. The results demonstrated an attenuation of insulin effect on glucose transport due to a lower insulin binding and lower content of GLUT4 protein in MSG-treated rats. However, the effect of insulin on lipogenesis was not changed. Our results indicated that early postnatal administration of MSG exerts an important effect on glucose metabolism and insulin action in adipocytes of adult animals.     

Mechanism of insulin resistance in the post receptor events in sheep: 3-O-methylglucose transport in ovine adipocytes

A severe resistance to the stimulatory action of insulin on glucose metabolism has been shown in ruminant adipose tissue or isolated adipocytes as compared to that of rats. To elucidate the mechanism of insulin resistance in ruminants, we measured the stimulatory effect of insulin on 3-O-methylgulose transport and on intracellular glucose metabolism in isolated adipocytes from sheep and rats. At a glucose concentration (0.1 mM) where transport is thought to be rate-limiting for metabolism, lipogenesis from [U-14C]glucose by ovine adipocytes was markedly less than by rat adipocytes in both the basal state and at all insulin concentrations. The responsiveness to insulin assessed by percent increase above basal was reduced to about 15% of that in rat adipocytes, but the insulin sensitivity was similar, because the insulin concentration giving half-maximal stimulation, ED50, did not differ significantly between ovine and rat adipocytes. The maximal insulin-stimulated 3-O-methylglucose transport in ovine adipocytes per cell was less than 20% of that in rat adipocytes, with a significant lowering in basal rates of transport. However, when data was expressed per 3-O-methylglucose equilibrium space no significant differences were found between ovine and rat in the basal transport rates, but a lowered ability of insulin to stimulate glucose transport was still seen in ovine adipocytes. The dose-response curve for glucose transport was slightly shifted to the right in ovine adipocytes compared to rat adipocytes, indicating a small decrease in insulin sensitivity. The decrease in glucose transport was due to 60% reduction in the maximum velocity in the insulin--stimulated state, with no change in the Km.     

Mechanism of methaemoglobin reduction by human erythrocytes.

The effect of alterations to the insulin receptor on the insulin sensitivity of isolated adipocytes was studied. Receptor changes were induced by treatment of adipocytes with either phospholipase C or trypsin. After enzyme treatment, binding of insulin to insulin receptors and insulin-mediated glucose metabolism were examined. Exposure of adipocytes to phospholipase C (2 units/ml) significantly increased insulin binding to the cells, but destroyed the ability of the cells to oxidize glucose. After treatment with trypsin (500 micrograms/ml) for 5 min, insulin binding to the adipocytes was significantly increased. This was shown to be due to an increase in insulin-receptor affinity. Metabolic studies showed that trypsin treatment led to an increase in basal glucose transport but markedly decreased the response to insulin at all concentrations tested. Adipocytes treated with trypsin showed no significant difference in basal glucose oxidation rates when compared with controls, but were less sensitive to insulin at low insulin concentrations, and showed a decreased maximum response at high insulin concentrations. In conclusion, these findings indicate a dissociation between induced changes in binding of insulin to insulin receptors and subsequent hormone action. The importance of post-receptor events in the biological action of insulin is highlighted.     

Proposed mechanism of insulin-resistant glucose transport in the isolated guinea pig adipocyte. Small intracellular pool of glucose transporters

A marked resistance to the stimulatory action of insulin on glucose metabolism has previously been shown in guinea pig, compared to rat, adipose tissue and isolated adipocytes. The mechanism of insulin resistance in isolated guinea pig adipocytes has, therefore, been examined by measuring 125I-insulin binding, the stimulatory effect of insulin on 3-0-methylglucose transport and on lipogenesis from [3-3H]glucose, the inhibitory effect of insulin on glucagon-stimulated glycerol release, and the translocation of glucose transporters in response to insulin. The translocation of glucose transporters was assessed by measuring the distribution of specific D-glucose-inhibitable [3H]cytochalasin B binding sites among the plasma, and high and low density microsomal membrane fractions prepared by differential centrifugation from basal and insulin-stimulated cells. At a glucose concentration (0.5 mM) where transport is thought to be rate-limiting for metabolism, insulin stimulates lipogenesis from 30 to 80 fmol/cell/90 min in guinea pig cells and from 25 to 380 fmol/cell/90 min in rat cells with half-maximal effects at approximately 100 pM in both cell types. Insulin similarly stimulates 3-O-methylglucose transport from 0.40 to 0.70 fmol/cell/min and from 0.24 to 3.60 fmol/cell/min in guinea pig and rat fat cells, respectively. Nevertheless, guinea pig cells bind more insulin per cell than rat cells, and insulin fully inhibits glucagon-stimulated glycerol release. In addition, the differences between guinea pig and rat cells in the stimulatory effect of insulin on lipogenesis and 3-O-methylglucose transport cannot be explained by the greater cell size of the former compared to the latter (0.18 and 0.09 micrograms of lipid/cell, respectively). However, the number of glucose transporters in the low density microsomal membrane fraction prepared from basal guinea pig cells is markedly reduced compared to that from rat fat cells (12 and 70 pmol/mg of membrane protein, respectively) and the translocation of intracellular glucose transporters to the plasma membrane fraction in response to insulin is correspondingly reduced. These results suggest that guinea pig adipocytes are markedly resistant to the stimulatory action of insulin on glucose transport and that this resistance is the consequence of a relative depletion in the number of intracellular glucose transporters.     

Relation of insulin receptors to insulin resistance.

The status of insulin-receptor interactions in a variety of insulin-resistant states is reviewed. Utilizing large adipocytes from adult rats and small fat cells from young rats, we have conducted a series of in vitro experiments in an attempt to determine the cellular alteration(s) responsible for the insulin resistance associated with obesity. Stimulation of glucose oxidation by insulin is reduced in large cells. Studies using a mimicker of insulin action, spermine, as well as measurements of 125I-insulin binding to large and small cells indicate that receptor number and affinity are not responsible for hormone resistance. Furthermore, when rapid and direct measurements of sugar uptake were made, insulin stimulation was virtually identical in both cell types. These findings indicate that large adipocytes have an efficient insulin-responsive D-glucose transport system and suggest that the apparent hormone resistance may be due to alterations in intracellular glucose metabolism. It has been proposed that altered insulin-receptor interaction underlies the insulin resistance of human obesity. We have investigated this particular aspect of insulin action by 125I-insulin binding studies. Similar numbers of insulin receptors per cell and affinity for insulin were observed in adipocytes obtained from normal weight subjects and morbidly obese patients. Thus, the initial step in insulin action is unaltered in human obesity.     

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.     

Effect of exercise on insulin binding and glucose transport in adipocytes of normal humans

Acute exercise increases insulin binding to its receptors on blood cells. Whether the enhanced insulin binding explains the exercise-induced increase in glucose uptake is unclear, since insulin binding and glucose uptake have not been measured simultaneously in a target tissue of insulin. In this study, we determined insulin binding and the rate of glucose transport in adipocytes obtained by needle biopsy from 10 healthy men before and after 3 h of cycle-ergometric exercise. During the exercise, plasma glucose (P less than 0.01) and insulin (P less than 0.001) fell and serum free fatty acid level rose 4.3-fold (P less than 0.001). 125I-insulin binding to adipocytes remained unchanged during exercise. The rate of basal glucose transport clearance fell from 28.1 +/- 5.7 fl.cell-1.s-1 to 22.9 +/- 5.6 fl.cell-1.s-1 (P less than 0.005), and the insulin-stimulated increase in glucose transport rate rose from 196 +/- 26 to 279 +/- 33% (P less than 0.025) during the exercise. Thus, in the adipocytes during exercise, the basal glucose transport rate and the responsiveness of glucose transport to insulin changed in the absence of alterations in insulin binding. These data indicate that the exercise-induced changes in insulin binding show tissue specificity and do not always parallel alterations in glucose transport.     

Stimulation of glucose transport by semicarbazide-sensitive amine oxidase activity in adipocytes from diabetic rats

Semicarbazide-sensitive amine oxidase (SSAO) is highly expressed in adipose cells, and substrates of SSAO such as benzylamine in combination with low concentrations of vanadate strongly stimulate glucose transport and GLUT4 recruitment in mouse 3T3-L1 adipocytes and in isolated rat adipocytes. Here we examined whether this combination of molecules also stimulates glucose transport in adipocytes from streptozotocin-induced diabetic rats and from Goto-Kakizaki diabetic rats. As previously reported, adipocytes obtained from streptozotocin-induced diabetic rats, showed a reduced stimulation of glucose transport in response to insulin. Under these conditions, the combination of benzylamine and vanadate caused a marked stimulation of glucose transport that was similar to the stimulation detected in control adipocytes. Adipocytes isolated from Goto-Kakizaki diabetic rats also showed a defective response to insulin; however, acute incubation in the presence of benzylamine and vanadate stimulated glucose transport in these cells to the same extent than in adipocytes from non-diabetic rats. These data indicate that adipocytes obtained from two different models of animal diabetes do not show resistance to the activation of glucose transport by SSAO activity, which is in contrast to the well reported resistance to insulin action. It seems to suggest that SSAO activity in combination with vanadate triggers a glucose transport-activating intracellular pathway that remains intact in the diabetic state. Further, our data support the view that the combination of benzylamine and vanadate could be an effective therapy in diabetes.     

Changes in the properties of cytosolic acetyl-CoA carboxylase studied in cold-clamped liver samples from fed, starved and starved-refed rats.

The mechanism responsible for the insulin resistance described in vivo in brown adipose tissue (BAT) of lactating rats was investigated. The effect of insulin on glucose metabolism was studied on isolated brown adipocytes of non-lactating and lactating rats. Insulin stimulation of total glucose metabolism is 50% less in brown adipocytes from lactating than from non-lactating rats. This reflects a decreased effect of insulin on glucose oxidation and lipogenesis. However, the effect of noradrenaline (8 microM) on glucose metabolism was preserved in brown adipocytes from lactating rats as compared with non-lactating rats. The number of insulin receptors is similar in BAT of lactating and non-lactating rats. The insulin-receptor tyrosine kinase activity is not altered during lactation, for receptor autophosphorylation as well as tyrosine kinase activity towards the synthetic peptide poly(Glu4-Tyr1). The defect in the action of insulin is thus localized at a post-receptor level. The insulin stimulation of pyruvate dehydrogenase activity during euglycaemic/hyperinsulinaemic clamps is 2-fold lower in BAT from lactating than from non-lactating rats. However, the percentage of active form of pyruvate dehydrogenase is similar in non-lactating and lactating rats (8.6% versus 8.9% in the basal state, and 37.0% versus 32.3% during the clamp). A decrease in the amount of pyruvate dehydrogenase is likely to be involved in the insulin resistance described in BAT during lactation.     

Polymyxin B is an inhibitor of insulin-induced hypoglycemia in the whole animal model. Studies on the mode of inhibitory action

The cyclic decapeptide, polymyxin B (PMXB), was found to inhibit hypoglycemia in mice receiving exogenous insulin (Amir, S., and Shechter, Y. (1985) Eur. J. Pharmacol. 110, 283-285). In this study, we have extended this observation to rats. Insulin-dependent hypoglycemia in rats is efficiently blocked at a 12:1 molar ratio of PMXB to insulin. This effect is highly specific, as it could not be mimicked by a variety of antibiotics or positively charged substances. Chemical modifications of PMXB have revealed that the ring structure, rather than the tail structure, is important for anti-insulin-like activity. Colistin A, which differs from PMXB by one conservative amino acid substitution in the ring structure, is devoid of this activity. Polymyxin B does not interact with insulin, nor does it alter the rate of insulin absorption and/or degradation, or the ability of insulin to bind to target tissues. This peptide inhibits hypoglycemia by blocking insulin-dependent activation of the hexose transport mechanism, as deduced by in vitro studies. The effect of insulin in stimulating hexose uptake (and subsequent glucose metabolism) in both isolated muscle tissue and adipocytes is blocked with little or no effect on the basal activities of these processes. Colistin A has no significant inhibiting effect. Other insulin-dependent activities, such as inhibition of lipolysis in adipocytes or synthesis of DNA in muscle cells, are not inhibited. It is concluded that PMXB inhibits, in a highly specific manner, the action of insulin in stimulating hexose transport and subsequent glucose metabolism, both in vitro and in the whole animal model.     

Depot-specific effects of early growth retardation on adipocyte insulin action.

Male offspring of rats protein restricted during pregnancy and lactation are growth restricted and have changes in insulin action on epididymal adipocytes. Adipocytes from different anatomical depots are thought to have distinct metabolic functions. The aim of the present study was to determine whether the alterations in metabolism of adipocytes from early growth restricted rats is depot-specific or more generalised. Epididymal, intra-abdominal and subcutaneous adipocytes were studied from three-month-old male offspring of control and protein restricted dams. Basal glucose uptakes were higher in low protein adipocytes (p<0.01) compared to controls. However, insulin stimulation was less in the low protein group in all depots compared to controls (p<0.05). Isoproterenol-stimulated lipolysis was greater in low protein adipocytes (p<0.0001), but the magnitude was greater in epididymal (p<0.0001) and intra-abdominal (p<0.0001) adipocytes than in subcutaneous adipocytes. Epididymal and intra-abdominal adipocytes from low protein offspring were also resistant to the anti-lipolytic action of insulin. These results suggest that certain changes associated with early growth retardation are depot-specific, being enhanced in the more metabolically active intra-abdominal and epididymal tissues.     

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.     

Glucose metabolism in isolated fat cells: enhanced response of larger adipocytes from older rats to epinephrine and adrenocorticotropin.

The effects of insulin and of two lipolytic hormones (epinephrine and ACTH1) on the rate and pattern of glucose metabolism were compared during incubation of isolated fat cells, obtained from epididymal fat pads of rats of varying age and degrees of adiposity. Glucose metabolism and the intracellular free fatty acid levels were expressed on a per cell basis and in relation to adipocyte size. The data for total glucose metabolism show that, in contrast to the declining insulin effect observed with adipocyte enlargement, the stimulation of glucose uptake and metabolism by these lipolytic hormones was significantly greater in the larger fat cells from the older fatter rats than in the smaller ones from the younger leaner rats. Lipolytic hormones suppressed, whereas insulin enhanced, fatty acid synthesis; moreover the lipolytic hormones stiumlated glucose ce effect of epinephrine on the intracellular free fatty acid levels was greater in the small fat cells than in the large ones; this effect of epinephrine was markedly curtained by the presence of glucose in the incubation medium, making it unlikely that acceleration of glucose metabolism by the lipolytic stimulus was mediated by an elevation of the intracellular free fatty acid level. The present results show a markedly enhanced capacity of the large adipocytes to accelerate glucose metabolism in response to these liplytic hormones. Thus, in contrast to prevailing notions of declining hormonal responsiveness with expanding fat cell size in older and more obese animals, this study documents an instance of increased hormonal response in enlarged adipocytes and points to the need for a more comprehensive reevaluation of the various hormonal effects in adipocytes of different size.     

Cadmium-induced stimulation of lipogenesis from glucose in rat adipocytes

Exposure of adipocytes of rats to CdCl2 caused acceleration of [3-3H]glucose incorporation into lipid maximally at 500 microM in Krebs-Ringer bicarbonate buffer, pH 7.4, containing 0.2% albumin. T.l.c. of the lipids extracted from adipocytes showed that Cd2+ increased labelling of di- and tri-[14C]acylglycerols predominantly. With increasing concentrations of glucose the apparent Km value was not affected by Cd2+, but the V value was increased, similarly to the effect of insulin. In the presence of insulin, Cd2+ (5 microM) exerted a consistent additive effect with a stimulatory effect of insulin on lipogenesis at all concentrations of insulin tested (5-50 mu units/ml). The stimulation was observed at a high concentration of glucose, suggesting that Cd2+ accelerated intracellular metabolism of glucose, mimicking insulin. However, although Zn2+ and Mn2+ stimulated the transport at a rate similar to that observed with insulin (200 mu units/ml), Cd2+ had no stimulating effect on the membrane transport of 3-O-methylglucose. The biological potency of Cd2+ and the insulin-like effects of Zn2+, both of which metals belong to the same group in the Periodic Table, are similar towards glucose metabolism, but quite different towards glucose transport.     

Effect of physical training on glucose transporters in fat cell fractions

Physical training increases maximally insulin-stimulated glucose assimilation and 3-O-methylglucose transport in epididymal fat cells. In the present report, glucose-inhibitable cytochalasin B binding in subcellular fractions of epididymal adipocytes was measured to assess changes in number of glucose transporters induced by training. Groups of rats trained by swimming were compared to control groups of the same age, matched with respect to body weight by restricted feeding. It was found that in trained rats the number of glucose transporters in the low density microsome fractions from non-insulin-stimulated fat cells was larger than in untrained rats. In both groups of rats, insulin stimulation of adipocytes decreased the number of glucose transporters in low-density microsomes by about 60% and increased the number of glucose transporters in the plasma membrane fractions. The number of glucose transporters in the plasma membrane fractions from maximally insulin-stimulated fat cells was larger in trained rats than in control rats. [U-¹⁴C]Glucose incorporation into lipids varied in proportion to plasma membrane cytochalasin B binding per cell under all conditions tested. The results explain the enhancing effect of training on insulin responsiveness transport of hexose in fat cells.     

Insulin resistance in soleus muscle from obese Zucker rats. Involvement of several defective sites

1. The effect of insulin upon glucose transport and metabolism in soleus muscles of genetically obese (fa/fa) and heterozygote lean Zucker rats was investigated at 5–6 weeks and 10–11 weeks of age. Weight-standardized strips of soleus muscles were used rather than the intact muscle in order to circumvent problems of diffusion of substrates. 2. In younger obese rats (5–6 weeks), plasma concentrations of immunoreactive insulin were twice those of controls, whereas their circulating triacylglycerol concentrations were normal. Insulin effects upon 2-deoxyglucose uptake and glucose metabolism by soleus muscles of these rats were characterized by both a decreased sensitivity and a decrease in the maximal response of this tissue to the hormone. 3. In older obese rats (10–11 weeks), circulating concentrations of insulin and triacylglycerols were both abnormally elevated. A decrease of 25–35% in insulin-binding capacity to muscles of obese rats was observed. The soleus muscles from the older obese animals also displayed decreased sensitivity and maximal response to insulin. However, at a low insulin concentration (0.1m-i.u./ml), 2-deoxyglucose uptake by muscles of older obese rats was stimulated, but such a concentration was ineffective in stimulating glucose incorporation into glycogen, and glucose metabolism by glycolysis. 4. Endogenous lipid utilization by muscle was calculated from the measurements of O₂ consumption, and glucose oxidation to CO₂. The rate of utilization of fatty acids was normal in muscles of younger obese animals, but increased in those of the older obese rats. Increased basal concentrations of citrate, glucose 6-phosphate and glycogen were found in muscles of older obese rats and may reflect intracellular inhibition of glucose metabolism as a result of increased lipid utilization. 5. Thus several abnormalities are responsible for insulin resistance of muscles from obese Zucker rats among which we have observed decreased insulin binding, decreased glucose transport and increased utilization of endogenous fatty acid which could inhibit glucose utilization.     

Calcium as modulator of the hormonal-receptors-biological-response coupling system. Effects of Ca2+ ions on the insulin activated 2-deoxyglucose transport in rat fat cells.

Activation of 2-deoxyglucose transport in isolated rat fat cells by insulin is dependent upon the presence of Ca2+ in the external medium. When calcium concentration is kept below 100 micron, insulin acts like a partial agonist, giving only half of the maximal activation obtained normally with a millimolar concentration of this ion. Oxytocin, whose insulin-like action on adipocytes activates glucose oxidation by these cells, was found to be unable to affect the rate of 2-deoxyglucose transport. This, together with previous observations, suggests that calcium ions play a role in the mechanism of insulin action possibly by binding selectively to membrane sites involved in the transmission of the hormonal message to the glucose carrier. Oxytocin seems to trigger only intracellular glucose metabolism and it appears that there is an absolute requirement for calcium ions in the activation of a still unknown membrane signal.     

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.