Transcriptional induction of glucokinase gene by insulin in cultured liver cells and its repression by the glucagon-cAMP system.

Primary cultures of rat hepatocytes were used to investigate the regulation of glucokinase gene expression by insulin and glucagon. Insulin added in physiological concentrations to the culture medium causes de novo induction of glucokinase mRNA. The induced plateau is reached 4 to 8 h after insulin addition, and the mRNA level remains high as long as insulin is present. Comparison of the potencies of insulin, proinsulin, and insulin-like growth factor I in this system indicates that induction by insulin is mediated via the insulin receptor. The magnitude of the insulin effect is independent of the extracellular glucose concentration. Run-on transcription assays with isolated nuclei show that the mRNA build up depends primarily on a specific stimulation of glucokinase gene transcription. Glucagon added to hepatocytes together with a supramaximal concentration of insulin prevents induction of glucokinase mRNA in a dose-dependent manner. The inhibitory effect of glucagon is mimicked by 8-(4-chlorophenylthio)-cAMP. The effect of this agent has also been tested in hepatocytes first induced for maximal glucokinase gene transcription by culture with insulin alone for 12 h. The transcriptional activity of the gene as measured by run-on assay was completely turned off within 30 min after addition of the cyclic nucleotide. Under these conditions, glucokinase mRNA decays rapidly, with an apparent half-life of 45 min. The mRNA degradation rate was similarly rapid after insulin withdrawal from induced cells. Thus, a cAMP-mediated repression mechanism is a key aspect in the regulation of glucokinase gene transcription in the hepatocyte. Insulin may act by relieving the gene from repression.

Differential expression and regulation of the glucokinase gene in liver and islets of Langerhans.

Glucokinase, a key regulatory enzyme of glucose metabolism in mammals, provides an interesting model of tissue-specific gene expression. The single-copy gene is expressed principally in liver, where it gives rise to a 2.4-kilobase mRNA. The islets of Langerhans of the pancreas also contain glucokinase. Using a cDNA complementary to rat liver glucokinase mRNA, we show that normal pancreatic islets and tumoral islet cells contain a glucokinase mRNA species approximately 400 nucleotides longer than hepatic mRNA. Hybridization with synthetic oligonucleotides and primer-extension analysis show that the liver and islet glucokinase mRNAs differ in the 5' region. Glucokinase mRNA is absent from the livers of fasted rats and is strongly induced within hours by an oral glucose load. In contrast, islet glucokinase mRNA is expressed at a constant level during the fasting-refeeding cycle. The level of glucokinase protein in islets measured by immunoblotting is unaffected by fasting and refeeding, whereas a 3-fold increase in the amount of enzyme occurs in liver during the transition from fasting to refeeding. From these data, we conclude (i) that alternative splicing and/or the use of distinct tissue-specific promoters generate structurally distinct mRNA species in liver and islets of Langerhans and (ii) that tissue-specific transcription mechanisms result in inducible expression of the glucokinase gene in liver but not in islets during the fasting-refeeding transition.

Hypoglycemia following insulin and proinsulin. A comparison.

The counterregulatory hormonal response to proinsulin-induced hypoglycemia was investigated in eight volunteers. Proinsulin cleared slower from the circulation than insulin. Hypoglycemia occurred slower (2P less than 0.005) and was prolonged, while the overall hypoglycemic activities were comparable. The antilipolytic effect of proinsulin was also prolonged (2P less than 0.001). The response of epinephrine to hypoglycemia was less pronounced after proinsulin (2P less than 0.05). The amount of epinephrine was correlated to the rate of fall in plasma glucose (P less than 0.005). The production of lactate induced by beta-stimulation was also correlated to the fall of glucose (P less than 0.005). The responses of prolactin (2P less than 0.02), norepinephrine (2P less than 0.02), cortisol, and growth hormone were attenuated following proinsulin. The decreases of serum potassium and serum phosphate (2P less than 0.05) were less pronounced. Symptoms like sweating (2P less than 0.01) and dizziness (2P less than 0.01) were milder after proinsulin. It is concluded that the rate of fall in glucose concentration determines the differing counterregulatory responses. We don't relate the differing counterregulatory responses to special insulin-like properties of proinsulin, but to the slower kinetics which is emphasized by the intravenous bolus injection.

Insulin secretion in vitro and insulin binding to isolated hepatocytes in congenic mice with different H-2 complexes.

Congenic male mice with differences in the H-2 complex have been used to investigate insulin secretion in vitro, insulin binding to isolated hepatocytes, plasma glucose, and serum insulin. Plasma glucose and serum insulin did not show consistent differences in the B10.BR, B10.D2, B10.A, B10.G, B10.M, B10.S, C57/10SCSN, and C3H.OH strains. Isolated islets of Langerhans responded to stimulation with 400 mg/dl glucose with a 3-5-fold increase in insulin secretion rates (2P less than 0.01): B10.BR greater than B10.M greater than C57BL/10SCSN greater than B10.G greater than C3H.OH, B10.D2, B10.A, B10.S. The biphasic pattern of insulin secretion was less distinct in B10.M, B10.G, and C3H.OH mice. The high-affinity constants of insulin binding to isolated hepatocytes at 37 degrees C varied between 4.5 X 10(7) L X mol-1 and 4.5 X 10(8) L X mol-1 (2P less than 0.01): B10.A greater than B10.BR greater than C57BL/10SCSN, B10.S, B10.D2 greater than B10.M, B10.G. The glucose-stimulated insulin secretion from isolated islets of Langerhans and the binding of insulin to isolated hepatocytes correlate to the H-2 complex independently.