Mechanisms of insulin resistance in human obesity: evidence for receptor and postreceptor defects.

UNLABELLED: To assess the mechanisms of the insulin resistance in human obesity, we have determined, using a modification of the euglycemic glucose clamp technique, the shape of the in vivo insulin-glucose disposal dose-response curves in 7 control and 13 obese human subjects. Each subject had at least three euglycemic studies performed at insulin infusion rates of 15, 40, 120, 240, or 1,200 mU/M2/min. The glucose disposal rate was decreased in all obese subjects compared with controls (101 +/- 16 vs. 186 +/- 16 mg/M2/min) during the 40 mU/M2/min insulin infusion. The mean dose-response curve for the obese subjects was displaced to the right, i.e., the half-maximally effective insulin concentration was 270 +/- 27 microU/ml for the obese compared with 130 +/- 10 microU/ml for controls. In nine of the obese subjects, the dose-response curves were shifted to the right, and maximal glucose disposal rates (at a maximally effective insulin concentration) were markedly decreased, indicating both a receptor and a postreceptor defect. On the other hand, four obese patients had right-shifted dose-response curves but reached normal maximal glucose disposal rates, consistent with decreased insulin receptors as the only abnormality. When the individual data were analyzed, it was found that the lease hyperinsulinemic, least insulin-resistant patients displayed only the receptor defect, whereas those with the greatest hyperinsulinemia exhibited the largest post-receptor defect, suggesting a continuous spectrum of defects as one advances from mild to severe insulin resistance. When insulin's ability to suppress hepatic glucose output was assessed, hyperinsulinemia produced total suppresssion in all subjects. The dose-response curve for the obese subjects was shifted to the right, indicating a defect in insulin receptors. Insulin binding to isolated adipocytes obtained from the obese subjects was decreased, and a highly significant inverse linear relationship was demonstrated between insulin binding and the serum insulin concentration required for halfmaximal stimulation of glucose disposal. IN CONCLUSION: (a) decreased cellular insulin receptors contribute to the insulin resistance associated with human obesity in all subjects; (b) in the least hyperinsulinemic, insulin-resistant patients, decreased insulin receptors are the sole defect, whereas in the more hyperinsulinemic, insulin-resistant patients, the insulin resistance is the result of a combination of receptor and postreceptor abnormalities; (c) all obese patients were insensitive to insulin's suppressive effects on hepatic glucose output; this was entirely the result of decreased insulin receptors; no postreceptor defect in this insulin effect was demonstrated.

Effect of a high carbohydrate diet on insulin binding to adipocytes and on insulin action in vivo in man.

We studied the effects of short-term (5 days) and long-term (2 wk) high carbohydrate (75%) feedings on insulin binding to isolated adipocytes and insulin sensitivity in vivo in normal subjects. Ingestion of the high carbohydrate diet led to daylong hyperinsulinemia in both short- and long-term groups. Insulin binding to isolated adipocytes was decreased in both groups; in the short-term groups this decrease in insulin binding was caused by a decrease in the receptor affinity, whereas in the long-term group it was caused by a decrease in receptor number. On the other hand, despite this decrease in insulin binding, total in vivo insulin sensitivity was markedly improved in both groups. In conclusion, (1) the short-term adaptive response of the insulin receptor is a decrease in binding affinity whereas the long-term response is a decrease in receptor number, (2) sustained and chronic hyperinsulinemia can lead to a decrease in the number of cellular insulin receptors, (3) high carbohydrate diets lead to a general increase in insulin's ability to promote glucose removal from plasma, and (4) the paradox of enhanced insulin sensitivity in the face of decreased insulin binding can be explained if high carbohydrate diets also lead to an increase in the activity of steps in glucose metabolism distal to the insulin receptor.

The effects of dietary carbohydrate content on insulin binding and glucose metabolism by isolated rat adipocytes.

The effects of changes in the amount of dietary carbohydrate (CHO) on cellular insulin and glucose metabolism have been assessed in rat adipocytes. Feeding animals a 67% CHO (fat-free) diet resulted in decreased insulin binding but enhanced activity of both the glucose transport system and intracellular pathways of glucose metabolism. Feeding rats a 67% fat (CHO-free) diet resulted in decreased insulin receptors as well as decreased activity of the glucose transport system and intracellular glucose metabolism. Therefore, the in vivo insulin resistance caused by a high fat, low CHO diet seems to be adequately explained, since all aspects of insulin's cellular action were depressed. On the other hand, at first approximation, the increased in vivo insulin response caused by a high CHO diet appears contradictory to the observed decrease in insulin binding. However, a probable explanation for this apparent paradox is provided by the enhanced activity of the cellular insulin effector systems distal to the insulin receptor. Therefore, the increased in vivo insulin responsiveness after high CHO feedings is most likely due to post receptor increases in various aspects of glucose metabolism.

Effect of a high carbohydrate diet on adipocyte glucose metabolism in spontaneously obese rats and insulin-deficient diabetic rats.

We have studied the effects of increased dietary carbohydrate content on glucose transport and intracellular glucose metabolism in adipocytes from spontaneously obese rats and from streptozotocin-induced insulin-deficient diabetic rats. Four groups of animals were studied: 1) obese rats on the control diet, 2) obese rats on the high carbohydrate diet, 3) diabetic rats on the control diet, and 4) diabetic rats on the high carbohydrate diet. Compared to the control diet, the high carbohydrate diet led to an increase in insulin secretion in the obese rats, whereas the diabetic animals were unable to respond to the diet with enhanced insulin output. In the diabetic animals, the rates of adipocyte glucose transport, glucose oxidation, and lipogenesis were low relative to those in nondiabetic controls and were not influenced by the high carbohydrate diet. In the obese animals on the control diet, absolute rates of glucose transport were similar to those of lean controls, but glucose oxidation and lipogenesis were depressed. On the high carbohydrate diet, all aspects of cellular glucose transport and metabolism were markedly increased. Thus, when plasma insulin levels were allowed to increase (obese animals), rates of cellular glucose metabolism increased; when plasma insulin was kept constant (diabetic rats) cellular glucose metabolism was unchanged. In conclusion, 1) a high carbohydrate diet leads to augmented rates of glucose transport, oxidation, and lipogenesis, provided the animal can respond to the increased dietary carbohydrate with increased insulin secretion, and 2) the diet-induced relative hyperinsulinemia most probably mediates the changes in cellular metabolism.

Insulin resistance and diabetes due to a genetic defect in insulin receptors.

A 14-yr-old woman presented with fasting hyperglycemia (269 mg/dl), fasting hyperinsulinemia (45 microU/ml), acanthosis nigricans, and insulin resistance. The patient's circulating insulin was normal by physical and biological criteria, and insulin receptor antibodies were not detected. Both the patient's in vivo dose-response curve for insulin-stimulated glucose transport in isolated adipocytes were shifted to the right and showed marked decreases in the maximal insulin response. Basal hepatic glucose output was significantly increased, and the in vivo dose-response curve for insulin-mediated suppression of basal hepatic glucose output was shifted to the right. Insulin binding to the patient's erythrocytes, monocytes, and adipocytes was markedly decreased. To confirm that the severe reduction in cellular insulin receptors was a primary rather than an acquired defect, similar studies were conducted using cultured fibroblasts. No detectable binding of insulin to these cells was observed. Further studies showed that the patient's mother and two sisters were hyperinsulinemic and insulin resistant, and had comparable, although less severe, changes in insulin binding. The patient was also demonstrated to have an insulin secretory defect both to both oral and iv glucose challenges. We thus conclude that this family demonstrates a genetic deficiency of insulin receptors, resulting in insulin resistance and, in this patient, severe diabetes mellitus.

Semisynthesis and biological activity of porcine [LeuB24]insulin and [LeuB25]insulin.

Two analogs of porcine insulin with substitutions of leucine for phenylalanine in the COOH-terminal region of the insulin B chain have been prepared by a combination of solid-phase synthesis and semisynthesis. Solid-phase synthesis of the substituted octapeptides B23-B30 bearing the trifluoracetyl group on lysine-B29, enzymatic coupling of the octapeptides to bis(tertiary-butyloxycarbonyl)desoctapeptide insulin by trypsin, and deprotection of the corresponding adducts in formic acid and piperidine resulted in two insulin derivatives, one with leucine at position B24 and the other with leucine at position B25. These analogs had only about 10% and 1%, respectively, of the activity of porcine insulin in competing for the binding of [125I]iodoinsulin to both rat adipocytes and human IM-9 lymphocytes. The relative potencies of the analogs in stimulating glucose oxidation by rat adipocytes decreased in the order porcine insulin > [LeuB24]insulin > [LeuB25]insulin. However, at high concentrations both analogs had full agonists activity. Experiments in which the semisynthetic insulins were mixed with the native hormone showed that [LeuB24]insulin, but not [LeuB25]insulin, was an active antagonist of insulin action. These results suggest that the antagonistic activity of a human insulin variant having leucine at position B24 or B25 can be assigned to the molecule with the sequence Gly-Leu-Phe-Tyr (residues B23-B26) in its active site.

Relationship between negative cooperativity and insulin action.

We have compared the respective abilities of porcine insulin and [LeuB25]insulin to enhance the rate of dissociation of receptor-bound [125I]insulin from IM-9 lymphocytes and isolated rat adipocytes and to increase the rate of adipocyte glucose transport and oxidation. Although porcine insulin (100 ng/mL) greatly enhanced the dissociation rate of previously bound [125I]insulin, [LeuB25]insulin (at a concentration yielding equivalent receptor occupancy) had no effect. Nevertheless, the analogue fully stimulated adipocyte glucose transport and oxidation at concentrations consistent with its reduced (1.7% of normal) intrinsic binding affinity. Activation of glucose transport by the analogue was rapid, and the corresponding rate of activation was indistinguishable from that produced by native insulin. The increased dissociation rate observed with increasing receptor occupancy by native porcine insulin has been interpreted as evidence for negative cooperative site-site interactions between occupied receptors. According to this formulation, [LeuB25]insulin is a "noncooperative" insulin analogue. Since this [LeuB25]insulin retains full biologic activity, the enhancement of the insulin dissociation rate at high levels of receptor occupancy does not reflect a phenomenon inherent in insulin's action to augment glucose metabolism.