Studies on the mechanism of insulin resistance in the liver from humans with noninsulin-dependent diabetes. Insulin action and binding in isolated hepatocytes, insulin receptor structure, and kinase activity.

Abstract

We have developed a method to isolate insulin-responsive human hepatocytes from an intraoperative liver biopsy to study insulin action and resistance in man.Hepatocytes from obese patients with noninsulin-dependent diabetes were resistant to maximal insulin concentration, and those from obese controls to submax- imal insulin concentration in comparison to nonobese controls.Insulin binding per cell number was similar in all groups.How- ever, insulin binding per surface area was decreased in the two obese groups because their hepatocytes were larger.In addition, the pool of detergent-extractable receptor was further decreased in diabetics.Insulin receptors in all groups were unaltered as determined by affinity-labeling methods.However, insulin-stim- ulated insulin receptor kinase activity was decreased in diabetics.Thus, in obesity, decreased surface binding could explain re- sistance to submaximal insulin concentrations.In diabetes, di- minished insulin-stimulated protein kinase activity and decreased intracellular pool of receptors could provide an explanation for postinsulin-binding defect(s) of insulin action in human liver. Methods Materials. a-[ I 14C]Aminoisobutyric acid (51.6 mCi/mmol), carrier-freeNa 125I, [methoxyl-3H]inulin (186.4 mCi/g), 3-0-['4C] methyl-D-glucose (58.0 mCi/mmol), L-[U-_4C]leucine (300 mCi/mmol), and [I-_4C]acetic acid, sodium salt (56.0 mCi/mmol) were obtained from New England Nuclear, Boston, MA.Crude collagenase (type IV, lots 124F-6807, 25F-6806, 25F-68 17, 44F-68 12) and deoxyribonuclease were obtained from Sigma Chemical Co., St. Louis, MO; dispase was purchased from Boeh- ringer Mannheim Biochemicals, Indianapolis, IN; and soybean trypsin inhibitor from Worthington Biomedical Corp., Freehold, NJ.Fraction V bovine serum albumin (BSA) was obtained from Armour Pharma- ceutical, Co., Kankakee, IL, and it was further purified by charcoal treat- ment (23).Crystalline porcine insulin was kindly provided by Dr. Ronald Chance of Eli Lilly & Co., Indianapolis, IN.All other chemicals were reagent grade.Human subjects.11 morbidly obese patients with NIDDM and an equal number of morbidly obese patients without diabetes were studied.The clinical and biochemical data ofthese patients are presented in Table I.These patients were admitted to the hospital to undergo gastric bypass for the treatment of morbid obesity.We also studied a nonobese non- diabetic group composed of four subjects who had uncomplicated gallstone disease and were admitted for elective cholecystectomy, and five brain-dead organ donors.The biochemical data on the liver ofthese two clinically different subgroups of nonobese subjects were identical.Thus, they have been combined and referred to as the nonobese comparison group.The value ofthe organ donor's liver should be underscored because we have the entire liver from the donors.This tissue has been fundamentalInsulin Resistance in Liverfrom Noninsulin-dependent Diabetes 249