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 O(2) consumption, and glucose oxidation to CO(2). 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.

The effect of insulin on glucose transport and glucose transporters was studied in perfused rat heart. Glucose transport was measured by the efflux of labelled 3-O-methylglucose from hearts preloaded with this hexose. Insulin stimulated 3-O-methylglucose transport by: (a) doubling the maximal velocity (Vmax); (b) decreasing the Kd from 6.9 to 2.7 mM; (c) increasing the Hill coefficient toward 3-O-methylglucose from 1.9 to 3.1; (d) increasing the efficiency of the transport process (k constant). Glucose transporters in enriched plasma and microsomal membranes from heart were quantified by the [3H]cytochalasin-B-binding assay. When added to normal hearts, insulin produced the following changes in the glucose transporters: (a) it increased the translocation of transporters from an intracellular pool to the plasma membranes; (b) it increased (from 1.6 to 2.7) the Hill coefficient of the transporters translocated into the plasma membranes toward cytochalasin B, suggesting the existence of a positive co-operativity among the transporters appearing in these membranes; (c) it increased the affinity of the transporters (and hence, possibly, of glucose) for cytochalasin B. The data provide evidence that the stimulatory effect of insulin on glucose transport may be due not to the sole translocation of intracellular glucose transporters to the plasma membrane, but to changes in the functional properties thereof.

Aspects of the regulation of the glucose transport by perfused hearts of normal rats have been studied by measuring glucose transport (via the efflux of labelled 3-O-methyl-D-glucose) and glucose transporters (via the labelled cytochalasin B binding assay). Similarly to what is observed with insulin, increasing workload (by raising perfusion pressure from 50 to 100 mm Hg) stimulated glucose transport 7 to 8-fold. Glucose (via its analog 3-O-methylglucose, used at 15 mmol/l) stimulated its own transport 4-fold. The three stimuli favored the translocation of glucose transporters from an intracellular pool (microsomes) to the plasma membrane. Insulin increased the apparent affinity (decreased dissociation constant values) of plasma membrane transporters for cytochalasin, as well as the Hill coefficient, indicating the occurrence of a positive cooperativity amongst plasma membrane transporters. Workload increased only the Hill coefficient, glucose only the apparent affinity for cytochalasin of plasma membrane transporters. This study shows that insulin, workload and glucose itself stimulate glucose transport by favouring the translocation process of glucose transporter as well as by changing, albeit by a different mechanism, the functional properties of the transporters once translocated to the plasma membrane.

Summary. Overall D-glucose metabolism and 3-0-methylglucose transport were measured in the perfused heart preparation of lean and genetically obese (fa/fa) rats. Absolute values of basal and insulin-stimulated glucose metabolism were decreased in hearts of 15-week-old obese rats when compared to lean age-matched controls. Basal and maximally stimulated (i. e., by the combined addition of insulin and increasing perfusion pressure) 3-0-methylglucose transport was normal in hearts from young obese rats (5-week-old). However, when only one stimulus was used (insulin or increasing perfusion pressure alone), 3-0-methylglucose transport was stimulated to values that were lower than those of lean rats. Basal 3-0-methylglucose transport was four times lower in hearts from older obese rats (15-week-old) than in lean ones of the same age. At this age, stimulation of 3-0-methylglucose transport by insulin alone, by increasing perfusion pressure alone or by the combination of both stimuli, reached values in obese rats that were only half those of lean animals. It is concluded that: (a) in the early phase of the syndrome, the basal glucose transport system in hearts of obese rats is normal, but its response to stimulation becomes abnormal and; (b) at a later phase of obesity, the glucose transport system becomes abnormal even under basal conditions and its responsiveness to various stimuli is markedly impaired.Key words: Perfused heart; genetically obese rats; glucose transport; insulin; perfusion pressure.Obesity in man and laboratory animals is usually associated with hyperinsulinaemia and insulin resistance [1]. It was initially thought that the major cause of insulin resistance in obese hyperinsulinaemic animals was the decreased ability of plasma membranes of liver [2][3][4][5], adipose tissue [6] and muscle [4,7] to bind insulin, an abnormality that could be accounted for by a decrease in specific insulin receptor number [8]. Subsequent studies have suggested that additional defects unrelated to the insulin receptors (i.e. post-receptor defects) also contributed to insulin resistance [% 9, 10]. In insulinresistant muscles, several such post-receptor defects have been described [4,7,[10][11][12][13][14]. In isolated soleus muscles of the genetically obese Zucker (fa/fa) rat the following post-receptor abnormalities have been shown: (a) increased utilization of endogenous fatty acids inhibitory to glycolysis [7]; (b) decreased uptake of the D-glucose analogue, 2-deoxy-D-glucose (2-DG) [4,7,12]. In the perfused hindquarter of obese hyperglycaemic (db/db) mice the existence of a post-receptor defect at the level of glucose transport (again measured with 2-DG) has been proposed to be the major cause of insulin resistance [15]. Thus the evidence that insulin resistance could be partly attributed to defectual glucose handling has been indirect, based either on overall glucose metabolism or on 2-DG glucose uptake.Studies carried out with 2-DG have two major drawbacks: (a) 2-DG is not only taken up but phosphorylated [16]; (b) t...

In perfused lean rat hearts, the activator of protein kinase C phorbol myristate acetate (PMA), when present alone, stimulates glucose transport but inhibits the insulin stimulation of this transport. PMA also inactivates glycogen synthase in hepatocytes. In contrast, none of these effects are observed in hearts and hepatocytes of obese animals, indicating an impaired protein kinase C activation in these tissues, which are insulin resistant. Direct measurements of protein kinase C activity in lean rat hearts revealed that PMA provokes a translocation of the enzyme from a soluble to a particulate fraction. In obese rat hearts, the basal distribution of protein kinase C is altered (more activity is found in the soluble and less in the particulate fraction), and the translocation induced by PMA is impaired. Pretreatment of lean rats with PMA in vivo, aimed at downregulating protein kinase C, induces the same defects (i.e., insulin resistance and unresponsiveness to PMA) as those observed in hearts of untreated obese animals. The results indicate that part of the insulin resistance might be the consequence of altered modulation of insulin action by protein kinase C.