Insulin gene expression pattern in rat pancreas during the estrous cycle.

Sex steroid hormones influence insulin homeostasis and glucose metabolism, estradiol (E2) and progesterone (P4) induce changes in both fasting and postprandial insulinemia in rodents, however, insulin gene expression during estrous cycle is unknown. The aim of the present study was to determine an insulin gene expression pattern during the estrous cycle in the rat. Groups of 6 adult rats in each day of the estrous cycle were used. Serum P4, E2, testosterone (T) and insulin concentrations were determined by radioimmunoassay (RIA). A Northern blot analysis was performed to assess insulin gene expression in pancreatic tissue. We found a marked variation in insulin gene expression during the estrous cycle. The highest insulin expression was observed during the proestrus day. Interestingly, E2 and P4 but not T levels were correlated with changes in insulin mRNA content. The variations in serum insulin during the cycle were correlated with its mRNA content in pancreas. The overall results showed variations in serum insulin and insulin gene expression during estrous cycle of the rat that correlated with circulating E2 and P4 levels.

Testosterone effect on insulin content, messenger ribonucleic acid levels, promoter activity, and secretion in the rat.

Coexistence of hyperinsulinemia and hyperandrogenism in women has been frequently described. Most of the studies addressing this issue have focused on the mechanisms by which insulin produces hyperandrogenism. In the present study, we analyzed the effects of testosterone in vivo and in vitro upon insulin gene expression and release in the rat. Our studies demonstrate that testosterone increases insulin messenger RNA (mRNA) levels in vitro as well as in vivo. In both prepuberal and intact adult rats, serum testosterone concentrations were positively correlated with insulin mRNA levels and insulin concentration in serum. Testosterone deprivation after gonadectomy decreased both insulin gene expression and serum insulin concentration. Insulin mRNA levels were partially restored after 3 days of testosterone administration and serum insulin was 80% and 27% above baseline values at 5 and 7 days posttreatment. Primary cultured pancreatic islets treated with the sexual steroid increased about 80% insulin mRNA, as well as protein, and release. In transfected islets, testosterone increased the activity of the -410 bp rat insulin promoter I by 154%. These data demonstrate that testosterone has a direct effect upon pancreatic islet function by favoring insulin gene expression and release.

Cyclic adenosine 3',5'-monophosphate increases pancreatic glucokinase activity and gene expression.

Comparison of the pancreatic and hepatic glucokinase gene transcripts reveals tissue-specific control of expression and the existence of two distinct promoters in a single glucokinase gene. The existence of alternate promoters suggests that separate factors regulate glucokinase transcription in the two tissues. Hepatic glucokinase expression has been shown to be repressed by cAMP; however, in the pancreatic beta-cell it is unlikely that cAMP represses glucokinase activity, as cAMP is known to positively affect glucose-induced insulin secretion, a process that in mature islets requires pancreatic glucokinase activity. In this work we demonstrate that cAMP indeed has a stimulatory effect on pancreatic glucokinase. The cyclic nucleotide stimulates pancreatic glucokinase activity after 3-h incubation, and maximal effects are observed after 6 and 12 h of treatment. Using the bDNA assay, a sensitive signal amplification technique, we detected relative increases in glucokinase messenger RNA levels of 40.5 +/- 7.5% after 3-h incubation with cAMP. This stimulatory effect was increased to 106.3 +/- 22% after 6-h incubation and sustained up to 12 h of incubation. Inhibition of gene transcription by actinomycin D abolishes cAMP-induced glucokinase activity. In transfected fetal islets, cAMP increased the activity of the -1000 bp rat glucokinase promoter by 60 +/- 6%. These data demonstrate that cAMP has a stimulatory effect on pancreatic glucokinase gene expression and that the nucleotide has opposite effects on pancreatic and hepatic glucokinase, supporting the concept that glucokinase transcription in the liver and that in the beta-cell differ.

Biotin regulation of pancreatic glucokinase and insulin in primary cultured rat islets and in biotin-deficient rats.

Biotin has been reported to affect glucose homeostasis; however, its role on pancreatic islets of Langerhans has not been assessed. In this report, we demonstrate that physiologic concentrations of biotin stimulate glucokinase activity in rat islets in culture. Using the branched DNA (bDNA) assay, a sensitive signal amplification technique, we detected relative increases in glucokinase mRNA levels of 41.5 +/- 13% and 81.3 +/- 19% at 12 and 24 h respectively in islets treated with [10(-6) M] biotin. Because glucokinase activity controls insulin secretion, we also investigated the effect of biotin on insulin release. Treatment with [10(-6) M] biotin for 24 h increased insulin secretion. We extended our studies by analyzing the effect of biotin deficiency on pancreatic islet glucokinase expression and activity, as well as insulin secretion. Our results show that islet glucokinase activity and mRNA are reduced by 50% in the biotin deficient rat. Insulin secretion in response to glucose was also impaired in islets isolated from the deficient rat. These data show that biotin affects pancreatic islet glucokinase activity and expression and insulin secretion in cultured islets.