A B S T R A C T 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 insulinglucose 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 ef: fective instulin concentration was 270+27 ,uU/ml for the obese coompared with 130±10 ,U/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 least hyperinsulinemic, least insulin-resistant patients displayed only the receptor defect, whereas those with the greatest hyperinsulinemia exhibited the largest postreceptor 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 suppression in all subjects. The doseresponse curve for the obese subjects was shifted to the right, indicating a defect in insulin receptors. InReceived for publication 27 August 1979 and in revised form 30 January 1980. 1272 sulin binding to isolated adipocytes obtained from the obese subjects was decreased, and a highly significanit inverse linear relationship was demonstrated between insulin binding andl the serum instulin concentrationl re(luired f'or halfrmaximnal stimulation of glucose disposal. In conclusioni: (a) decreased cellular insulin receptors contrilbute 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.

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 trifluoroacetyl group on lysine-B29, enzymatic coupling of the octapeptides to bis(tertiaiy-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%, re- 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.Studies of naturally occurring and chemically modified insulins have shown that the COOH-terminal region of the B chain lies within the site conferring activity to the hormone (1-5). Although residues B23-B26 (Gly-Phe-Phe-Tyr) have remained invariant during animal evolution, we recently identified an abnormal human insulin in which this portion of the molecule had been modified: the variant, isolated in equimolar amounts with normal insulin from the pancreas of a diabetic but hyperinsulinemic patient, appeared to contain a substitution of leucine for phenylalanine at position 24 or 25 of the insulin B chain (6). The mixture of normal and abnormal hormones had lower than expected activity both in competing for the binding of [1251]iodoinsulin to membrane receptors on rat adipocytes and in stimulating 2-deoxyglucose uptake and glucose oxidation by the same cells (6). Importantly, the biological activity of the hormone preparation (15% of normal) was much lower than its apparent binding activity (45% of normal). These results, and others on the biological effectiveness of the insulin mixture present in plasma (7) and of the insulin variant purified by cellular adsorption (8), were consistent with the patient's clinical features (7) and suggested that the abnormal hormone was an active antagonist of insulin action. Further studies on placing the amino acid substitution in sequence and on determining the mechanism by which the variant might interfere with a cellular response to insulin were prevented, however, by the small amount of material available.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3181The pioneering work of Inouye et al. (9), which describes the semisynthesis of insulin derivatives by trypsin-catalyzed peptide bond formation at Arg-B22, now permits the preparation of analogs containing amino acid substitutions in the COOHterminal region of the insulin B chain. In this report we describe the solid-phase synthesis of the [LeuB , trifluoroacetyl (TFA)-LysB29] and [...

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.

We have studied the short term regulation of insulin receptors by serially measuring insulin binding to erythrocytes during 5 h of infusions of glucose and insulin. Two infusion protocols were employed: (1) Hyperinsulinemic study. Subjects were infused with insulin (80 mU/min) to induce sustained hyperinsulinemia, while euglycemia was approximated by infusion of glucose (8 mg/kg/min). (2) Hyperglycemic study. Subjects were infused with glucose (7 mg/kg/min) and a small amount of insulin (10 mU/min), while endogenous insulin secretion was inhibited with epinephrine and propranolol. Insulin binding to erythrocyte insulin receptors was measured serially during both of these infusion protocols for 5 h. The results demonstrated no change in insulin binding during the first 3 h of either infusion. However, by 5 h of either infusion, a striking 36% decrease in insulin binding, from 6.3 ± 0.5% to 4.0 ± 0.6%, was observed. Scatchard analysis and average affinity analysis of the binding data demonstrated that this decrease in insulin binding was entirely caused by a decrease in receptor affinity. Insulin binding to circulating monocytes was also measured, and comparable effects were observed. When cells were removed from the in vivo environment at 3 h and were incubated in vitro for a subsequent 2 h, decreased insulin binding developed during the incubation. Thus, this short term regulation of insulin receptor affinity occurred in vivo or in vitro, and cells that were programmed during the first 3 h of the infusion study could develop a decrease in insulin receptor affinity in vitro in the absence of plasma factors.

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.