Characterization of skeletal muscle insulin receptor.

The insulin receptor from rat skeletal muscle was characterized. Treatment of muscle membranes with the photoactive insulin analog, 125I[N-epsilonB29-monoazidobenzoyl]-insulin revealed a single protein band of 135,000 Da, the alpha subunit. Iodination of total membrane protein followed by Triton X-100 solubilization and immunoprecipitation demonstrated the presence of a protein band of 90,000 Da, the beta subunit, together with a protein band of 190,000 Da, which may be the receptor precursor. In partially purified receptor preparations, the beta subunit exhibited dose-dependent, insulin-stimulated phosphorylation with incorporation of phosphate solely into tyrosine residues, which was also observed in the 190,000-Da receptor precursor. Purified plasma membranes contained a large amount of insulin-degrading activity which had to be inactivated prior to performing insulin-binding studies. If degradation of insulin was not prevented, apparent enhanced binding in the presence of unlabeled insulin was observed.

A novel mechanism for the insulin-like effect of vanadate on glycogen synthase in rat adipocytes.

Vanadate activated rat adipocyte glycogen synthase similarly to insulin in a dose- and time-dependent manner. No additional effect was observed when insulin and vanadate were added together. Vanadate also partially counteracted the effect of epinephrine to activate rat adipocyte glycogen phosphorylase similarly to insulin. Inhibition of Na+K+ATPase or stimulation of hydrogen peroxide generation were shown not to be the mechanisms of the insulin-like action of vanadate on glycogen synthase. Vanadate stimulated the phosphorylation of the 95,000-dalton subunit of the insulin receptor on tyrosine residues both in intact adipocytes and in a solubilized insulin receptor fraction. Vanadate also stimulated the phosphorylation of the 95,000-dalton subunit of a highly purified insulin receptor from human placenta. Neither the insulin receptor fraction from rat adipocyte nor the highly purified insulin receptor from human placenta contained any detectable phosphotyrosine phosphatase activity. Potassium fluoride had no stimulatory effect on the phosphorylation of the insulin receptor. Vanadate caused a 10-fold decrease in the Km for ATP, for tyrosine kinase, and enhanced the phosphorylation of histone H2B. These results demonstrate that vanadate enhances the phosphorylation of the insulin receptor by stimulating the kinase reaction in a similar but not identical manner to insulin.

Selective inhibition of the insulin-stimulated phosphorylation of the 95,000 dalton subunit of the insulin receptor by TAME or BAEE.

Added N alpha-p-tosyl-l-arginine methyl ester or N alpha-benzoyl-l-arginine ethyl ester inhibited the stimulation by insulin of phosphorylation of the 95,000 dalton subunit of the insulin receptor both in a partially purified insulin receptor fraction from rat adipocytes and in a highly purified insulin receptor preparation from human placenta. N-alpha-p-tosyl-l-lysine chloromethyl ketone, N alpha-p-tosyl-l-lysine methyl ester, or N-acetyl-l-phenylalanine ethyl ester were much less potent, while N-benzoyl-1-alanine methyl ester was without effect. Inhibition of the phosphorylation by the arginine analogues did not require preincubation of the insulin receptor with inhibitors in the presence of insulin prior to phosphorylation. Inhibition by N alpha-p-tosyl-l-arginine methyl ester was decreased by preincubation of the receptor fraction with cold ATP and MnCl2. These results suggest that N alpha-p-tosyl-l-arginine methyl ester inhibits an initial ATP and Mn2+ dependent reaction in insulin-stimulated phosphorylation process.

Characterization of the chromobindins. Soluble proteins that bind to the chromaffin granule membrane in the presence of Ca2+.

A group of proteins that bind to the chromaffin granule membrane in the presence of Ca2+ has been isolated by affinity chromatography of bovine adrenal medullary cytosol on granule membranes coupled to Sepharose 4B. Twenty-two of these proteins were resolved into classes depending upon the Ca2+ concentration at which they were eluted from the affinity column (40 or 0.1 microM), upon their affinities for native granule membranes or for liposomes prepared from extracted granule lipids, and upon the requirement of seven of the proteins for ATP in the cytosol fraction and column buffers to promote binding. The molecular weights and isoelectric points of these proteins were determined by two-dimensional electrophoresis. Two of the granule-binding proteins were identified: synexin and calmodulin. Calmodulin was found to bind to seven specific granule membrane proteins after diffusion of 125I-labeled calmodulin into an acrylamide gel of membrane proteins separated by electrophoresis in the presence of sodium dodecyl sulfate. A phospholipid-activated protein kinase activity, possibly due to protein kinase C, was present in the granule-binding fraction. Two major granule-binding proteins were found to present a pattern in two-dimensional electrophoresis that was very similar to but shifted slightly toward the basic end of the gel from the pattern generated by light chains associated with clathrin in adrenal medullary coated vesicles. In the chromaffin cell, these proteins, by associating with the granule membrane in the presence of an increased cytosolic Ca2+ concentration, might play a variety of roles in the process of exocytosis.

Energy balance and regulation of body weight after intestinal bypass surgery in rats.

This study evaluated the possibility that intestinal bypass surgery alters energy balance and regulation of body weight. In two sets of experiments, male Sprague-Dawley rats underwent bypass or sham bypass surgery. In experiment 1, half of each group was overfed and half was underfed. Bypass reduced net calorie intake (intake - fecal loss) at 2 wk but not at 6 wk. Body weights were maintained at a much lower level in the bypass rats, yet apparent energy expenditure was greater. In experiment 2, bypass rats were compared with sham bypass and sham bypass who were food restricted for paired-weight-loss to bypass (PWL sham). Net calorie intake and body weights of bypass and sham bypass followed a similar pattern as in experiment 1. At similar levels of body weight postoperatively, bypass rats required more calories per kilogram than did the PWL shams, suggesting that bypass resets downward the level at which body weight is regulated. Calorie requirements correlated with food intake better than with absolute body weight. Further studies to determine the mechanism of altered energy expenditure may provide methods for weight loss without extensive surgery.

Role of the small bowel in regulating food intake in rats.

This study tested the hypothesis that exposure of the lower intestine to nutrients decreases subsequent food intake. Eight male Sprague-Dawley rats underwent jejunoileal bypass surgery with 8 cm of proximal jejunum anastomosed to 5 cm of terminal ileum in an end-to-side fashion. Eight control rats had sham bypass surgery with similar intestinal incisions that were reanastomosed in normal continuity. Another group of eight rats underwent ileal transposition (IT). A 10-cm segment of terminal ileum was transected, leaving the mesentery and blood supply intact, and transposed to the jejunum 8 cm from the ligament of Treitz. Thus total intestinal length was intact. Eight control rats had similar intestinal transections reanastomosed in normal continuity. A similar pattern of decreased food intake in the first 1-2 wk occurred in both bypass and IT rats. The values were significantly different from the respective sham surgery control groups. Body weights decreased, but the fall was greater in bypass rats. These data suggest that partially digested food in the lower intestine decreases food intake.

Altered dietary preference for fat and sucrose after intestinal bypass surgery in rats.

We studied changes in dietary preference for sucrose and fat before and after intestinal bypass surgery or sham bypass surgery in Sprague-Dawley rats. During 6-d test periods before and after surgery, rats were offered both ground regular rat chow (chow) and ground chow adulterated with 20 percent fat as vegetable oil (Expt 1) or ground chow adulterated with 25 percent sucrose (Expt 2). A control group was fed only ground chow throughout (Expt 3). Before surgery in both experiments with adulterated chow, the rats preferred the fat or sucrose adulterated chow over regular chow by at least a 7:1 margin. After surgery, the bypass rats continued to prefer the adulterated diets, but by only 2.5:1 and 4:1 margins, respectively. The sham bypass rats continued their preoperative pattern of intake. Total calorie intake decreased by 25 percent to 36 percent in the bypass rats fed fat and sucrose chow. In both experiments the decrease came from decreased intake of the adulterated foods only. Bypass rats in the control group decreased food intake by only 9 percent. Sham bypass rats maintained their preoperative calorie intake and patterns of intake after surgery. Body weights of bypass rats decreased significantly compared to preoperative weights and compared to weights of the sham bypass rats. These data suggests that preference for both fat and sweet foods decreases in the initial period of rapid weight loss after bypass and this decreased preference may play a role in weight loss after bypass.