Modulation by glucose of insulin secretion and glucose phosphorylating activity in cultured pancreatic islets from obese (fa/fa) Zucker rats.

OBJECTIVE: In normal B-cells, glucokinase activity is regulated by glucose. We hypothesized that chronic exposure to low or high glucose levels would regulate glucokinase function and insulin secretion differently in islets of fa/fa compared with lean rats. SUBJECTS, DESIGN, and MEASUREMENTS: Islets isolated from lean and fa/fa rats (8-12 wk old) were cultured for 1-7 days in low (3.3 mM), moderate (12.5 mM) or supraphysiological (25.0 mM) glucose-supplemented medium. Sensitivity to glucose of hexokinase, glucokinase (by enzyme assay and kinetic analysis), and the insulin response (by radioimmunoassay) were assessed in each group of islets. RESULTS: Islets of fa/fa rats cultured in 12.5 mM glucose for 1-7 days demonstrated a left-shift in both the EC50 of the insulin response and the Km of glucokinase to glucose. The glucokinase Vmax of fa/fa rat islets was lower under all conditions tested, thereby limiting the potential increase in insulin secretion. When cultured in 3.3 mM glucose for 1-7 days, fa/fa rat islets retained responsiveness to glucose longer and the estimated EC50 for glucose actually declined. However, the glucokinase Km for glucose increased three-fold in both phenotypes cultured in low glucose. Lean and fa/fa rat islets cultured in 25.0 mM glucose demonstrated a paradoxical hypersecretion of insulin to basal glucose concentrations and desensitization to stimulation by high concentrations of glucose. Islets from fa/fa rats were more easily desensitized, with significant effects in 25.0 mM glucose by 3 days compared with 7 days for the lean rat islets. Culture in high glucose erased the phenotype differences in glucokinase Km that were observed in 12.5 mM glucose cultured islets. CONCLUSIONS: Differences in fa/fa rat islet glucokinase were observed only at moderate, near physiological glucose conditions. Glucokinase activity was similarly affected by low or high glucose in the two phenotypes, although differences in insulin secretion pattern were still detected, leading to the conclusion that factors other than glucokinase contribute to altered insulin secretion in the fa/fa rat. Further study of the glucose desensitization phenomenon in fa/fa rat islets might help unravel the factors that increase susceptibility to development of diabetes mellitus in some phenotypes.

Improved sensitivity of the mouse interstitial cell testosterone assay with the addition of forskolin.

Modification of the mouse interstitial cell testosterone assay by the addition of 1.5 microM forskolin to the incubation medium has improved the sensitivity of this luteinizing hormone bioassay from approximately 100 to 3 pg/tube of NIH rLH RP-2. Luteinizing hormone can be clearly detected in 1 microL of serum from rats castrated 1 week previously and 5 microL of serum from intact rats. Parallelism was noted between dilution curves of serum from intact and castrated rats, and the luteinizing hormone standard curve. Luteinizing hormone detected in serum samples from 30 intact rats by the improved bioassay and by radioimmunoassay was significantly correlated (r = 0.85). Secretion patterns of circulating luteinizing hormone in individual rats were similar when detected by either bioassay or radioimmunoassay. Thus, with the addition of forskolin, the mouse interstitial cell testosterone assay has been improved so that luteinizing hormone can be detected in small volumes of serum or plasma from male rats.

An investigation of the requirement for increased LH secretion during the compensatory response in androgen secretion after unilateral castration of the adult rat.

The endocrine mechanisms underlying the response to unilateral castration were examined by determining systemic androgen and luteinizing hormone (LH) concentrations, as well as testicular vein androgen at 3, 6, 12, and 24 hours after sham surgery and castration. Systemic androgen was significantly depressed 3 hours after unilateral castration, but had recovered to concentrations observed in sham operated rats at 6, 12, and 24 hours. The recovery of serum androgen after castration was apparently due to increased testicular secretion of androgen, seen as a significant increase in testicular vein androgen. Systemic concentrations of bioactive and immunoactive LH were significantly increased only at 6 hours after castration. The authors next examined whether the increase in LH was necessary for the compensatory secretion of androgen seen after castration. This was accomplished by examining the response to castration when circulating LH was prevented from changing by suppressing endogenous LH secretion with subcutaneous steroid implants and maintaining circulating LH with subcutaneous osmotic pumps containing ovine LH. The compensatory increase in testicular vein androgen was observed 1 and 7 days after castration in rats bearing sham implants. When circulating LH was prevented from changing by using the combination of steroids and LH, however, compensatory secretion of androgen did not occur 1 and 7 days after castration.(ABSTRACT TRUNCATED AT 250 WORDS)