Stimulation by Insulin of Protein Synthesis in Isolated Fat Cells. ∗

Abstract

This communication presents evidence that insulin, in physiological concentration, stimulates synthesis of protein from amino acids by individual fat cells of rat epididymal adipose tissue.

Methods. The isolated fat cell preparation is obtained by a modification of Rodbell's technique (1). Male Sprague-Dawley rats, weighing 110-210 g, were utilized. Eight animals were used in each experiment. The rats, in a fed state, were sacrificed by decapitation. Distal segments of epididymal adipose tissue were quickly excised, weighed, and added to 8-10 ml of Krebs-Ringer-bicarbonate solution containing 1 mg/ml glucose, 4% bovine albumin, and 20 mg of collagenase.† The albumin was previously purified by filtering through #42 Whatman paper followed by dialysis, utilizing Krebs-Ringer solution, for 18-24 hours. Before incubation, the pH of the medium was adjusted precisely to 7.4. Following incubation for one hour at 37°C in a Dubnoff metabolic shaker, the adipose tissue preparation was filtered through surgical gauze. The filtrate was suspended in 8-10 ml of Krebs-Ringer-bicarbonate‡ solution and centrifuged for 1-2 minutes at 600X. The infranate, containing blood vessels and connective tissue, was then aspirated. Resuspension of the preparation, centrifugation, and aspiration were repeated twice. The fat cell preparation was finally suspended in 6-8 ml of Krebs-Ringer-bicarbonate solution.‡ An amino acid mixture§ and Krebs-Ringer-bicarbonate,‡ with or without insulin, were added in 0.05 ml volumes each to plastic vials (“Analocup,” Aloe Co.). The final insulin¶ concentration was 800 micro-units/ml. With constant agitation, 0.5 ml of the fat cell preparation and approximately 0.1 microcurie of radioactive Amino Acid-C¹⁴ (U.L.) Mixture‖ were added to each plastic vial. A 0.1 ml aliquot was taken after 5 minutes had elapsed, and the mixture was then incubated at 37°C in a Dubnoff metabolic shaker for 2 hours, when a second 0.1 ml aliquot was taken.