Alfons Valera

Evidence from transgenic mice that glucokinase is rate limiting for glucose utilization in the liver.

To study the role of glucokinase (CK) in the control of glucose metabolism in the liver, transgenic mice were generated in which GK was overexpressed under control of the P‐enolpyru‐vate carboxykinase gene promoter. Whereas the expression of the GK gene in starved control mice was blocked, this promoter was able to direct the expression of the enzyme to the liver of starved transgenic mice. Furthermore, starved transgenic mice showed levels of GK activity fourfold higher than those of starved control and similar to those of fed control. This activation of GK led to an increase in the intracellular concentration of glucose 6‐phos‐phate, which was also related to an induction of glycogen accumulation. In addition, L‐pyruvate kinase (L‐PK) activity increased in transgenic mice, which when starved showed similar levels of activity to control fed mice. The induction of L‐PK caused an increase in the hepatic lactate concentration. Furthermore, hepatocytes in primary culture from transgenic mice incubated with 20 mM glucose produced levels of lactate threefold higher than controls, but no difference was noted when the hepatocytes from control and transgenic mice were incubated with 2 mM glucose. These results demonstrated in vivo that the activation of GK is a rate‐limiting step in the induction of glycolysis and glycogen synthesis. These changes in liver glucose metabolism led to a marked reduction in blood glucose (30%) and insulin (40%) concentrations. Furthermore, transgenic mice showed lower levels of blood glucose after an intraperitoneal glucose tolerance test, indicating that GK overexpression caused an increase in blood glucose disposal by the liver. All these findings show the key role of liver GK in the control of whole‐body glucose homeostasis.—Ferre, T., Riu, E., Bosch, F., Valera, A. Evidence from transgenic ‐mice that glucokinase is rate limiting for glucose utilization in the liver. FASEB J. 10, 1213‐1218 (1996)

Regulated expression of human insulin in the liver of transgenic mice corrects diabetic alterations.

Transgenic mice expressing the P‐enolpyruvate carboxykinase (PEPCK)/human insulin chimeric gene have been obtained as a model to study the feasibility of gene therapy for diabetes. These transgenic animals were healthy and normoglycemic and expressed human insulin in a physiologically regulated manner, mainly in the liver. Streptozotocin‐treated transgenic mice had high levels of human insulin immunoreactivity in serum and showed a significant decrease (up to 40%) in glycemia compared with streptozotocin‐treated control mice. The expression of genes involved in liver glucose metabolism, such as glucokinase, pyruvate kinase, and PEPCK, which is markedly altered by diabetes, was significantly recovered in transgenic mice treated with streptozotocin. In addition, the activity of both glucokinase and glycogen synthase, and the content of glucose 6‐phosphate and glycogen, were normal in the liver, even when transgenic animals were treated with diabetogenic doses of streptozotocin. These results constitute an indication in vivo that diabetes gene therapy is possible, by means of the production of insulin in extrapancreatic tissues.—Valera, A., Fillat, C., Costa, C., Sabater, J., Visa, J., Pujol, A., Bosch, F. Regulated expression of human insulin in the liver of transgenic mice corrects diabetic alterations. FASEB J. 8: 440‐447; 1994.

Vanadate treatment restores the expression of genes for key enzymes in the glucose and ketone bodies metabolism in the liver of diabetic rats.

Oral administration of vanadate to diabetic streptozotocin-treated rats decreased the high blood glucose and D-3-hydroxybutyrate levels related to diabetes. The increase in the expression of the P-enolpyruvate carboxykinase (PEPCK) gene, the main regulatory enzyme of gluconeogenesis, was counteracted in the liver and the kidney after vanadate administration to diabetic rats. Vanadate also counteracted the induction in tyrosine aminotransferase gene expression due to diabetes and was able to increase the expression of the glucokinase gene to levels even higher than those found in healthy animals. Similarly, an induction in pyruvate kinase mRNA transcripts was observed in diabetic vanadate-treated rats. These effects were correlated with changes on glucokinase and pyruvate kinase activities. Vanadate treatment caused a decrease in the expression of the liver-specific glucose transporter, GLUT-2. Thus, vanadate was able to restore liver glucose utilization and block glucose production in diabetic rats. The increase in the expression of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCoAS) gene, the key regulatory enzyme in the ketone bodies production pathway, observed in diabetic rats was also blocked by vanadate. Furthermore, a similar pattern in the expression of PEPCK, GLUT-2, HMGCoAS, and the transcription factor CCAAT/enhancer-binding protein alpha genes has been observed. All of these results suggest that the regulation of the expression of genes involved in the glucose and ketone bodies metabolism could be a key step in the normalization process induced by vanadate administration to diabetic rats.

Evidence from transgenic mice that myc regulates hepatic glycolysis.

The product of the c‐myc proto‐oncogene (c‐Myc) is involved in the control of cell proliferation, differentiation, and apoptosis. It acts as a transcription factor that recognizes the CACGTG motif. This sequence has also been found in the glucose‐responsive elements of genes involved in the control of liver glycolysis and lipogenesis. To determine whether c‐Myc can regulate hepatic carbohydrate metabolism in vivo, transgenic mice that overexpress c‐myc under control of the P‐enolpyruvate carboxykinase (PEPCK) gene promoter have been generated. These mice showed a threefold increase in c‐Myc protein in liver nuclei. Hepatocytes from transgenic mice were normal and did not acquire the fetal phenotype. However, transgenic mice showed higher levels (threefold) of L‐type pyruvate kinase mRNA and enzyme activity than control mice. The increase in pyruvate kinase activity led to a three to fivefold increase in liver lactate content and a fivefold induction of lactate production by hepatocytes in primary culture. The expression of the 6‐ phosphofructo‐2‐kinase gene was also increased in the liver of these transgenic mice. The induction of hepatic glycolysis was related with an increase in the expression (about fourfold) and activity (about three‐ fold) of liver glucokinase, whereas no change was noted in hexokinase‐I. This change in glucokinase activity led to an increase in both glucose 6‐phosphate and glycogen contents in the liver of transgenic mice. The expression of the liver‐specific glucose transporter GLUT2 was also increased in transgenic mice, whereas no change was noted in the mRNA concentration of GLUT1. Furthermore, the changes of liver glucose metabolism led to a marked reduction of blood glucose (25%) and insulin (40%) concentrations in starvation, whereas the fall in both was only 10% in fed mice. Thus, liver glucose metabolism could determine the blood glucose and insulin set points in the transgenic mice. All these results indicated that the increase in c‐Myc protein was able to induce liver glucose utilization and accumulation, and suggested that c‐Myc transcription factor is involved in the control in vivo of liver carbohydrate metabolism.—Valera, A., Pujol, A., Gregori, X., Riu, E., Visa, J., Bosch, F. Evidence from transgenic mice that myc regulates hepatic glycolysis. FASEB J. 9, 1067‐1078 (1995)

Evidence from Transgenic Mice That Interferon-β May Be Involved in the Onset of Diabetes Mellitus

A number of cytokines have been shown to alter the function of pancreatic β-cells and thus might be involved in the development of type 1 diabetes. Interferon-β (IFN-β) expression is induced in epithelial cells by several viruses, and it has been detected in islets of type 1 diabetic patients. Here we show that treatment of isolated mouse islets with this cytokine was able to alter insulin secretion in vitro. To study whether IFN-β alters β-cell function in vivo and leads to diabetes, we have developed transgenic mice (C57BL6/SJL) expressing IFN-β in β-cells. These mice showed functional alterations in islets and impaired glucose-stimulated insulin secretion. Transgenic animals presented mild hyperglycemia, hypoinsulinemia, hypertriglyceridemia, and altered glucose tolerance test, all features of a prediabetic state. However, they developed overt diabetes, with lymphocytic infiltration of the islets, when treated with low doses of streptozotocin, which did not induce diabetes in control mice. In addition, about 9% of the transgenic mice obtained from the N3 back-cross to outbred albino CD-1 mice spontaneously developed severe hyperglycemia and hypoinsulinemia and showed mononuclear infiltration of the islets. These results suggest that IFN-β may be involved in the onset of type 1 diabetes when combined with either an additional factor or a susceptible genetic background.

Glucose metabolism in transgenic mice containing a chimeric P-enolpyruvate carboxykinase/bovine growth hormone gene.

Transgenic mice, containing the chimeric gene obtained by linking the promoter‐regulatory region of P‐enolpyruvate carboxykinase (PEPCK) gene to the bovine growth hormone structural gene (bGH), were used to investigate the long‐term effects of bGH on glucose metabolism. Expression of the PEPCK/bGH gene was markedly enhanced by feeding a diet high in protein and inhibited by a high carbohydrate diet. All transgenic mice had normal levels of blood glucose but were hyperinsulinemic, indicating that they were insulin resistant. The glycogen synthase activity ratios in the muscle and liver of transgenic mice were lower than noted for control animals, and remained unchanged in liver after feeding a standard high carbohydrate or a high protein diet. Similar effects were detected in the activity of glycogen phosphorylase, except that a high carbohydrate diet activated this enzyme in the liver. The activation of glycogen phosphorylase in both muscle and liver correlated with the expression of their genes. These animals had a significant content of glycogen and glucose 6‐phosphate, which was related to the levels of glucokinase mRNA in the liver. The concentration of fructose 2,6‐bisphosphate in the liver of all fed transgenic mice was lower than noted in livers from fed animals. In addition, a decrease in the hepatic expression of the endogenous genes for PEPCK, tyrosine aminotransferase (TAT), and the glucose transporter GLUT‐2 was observed and directly correlated with the expression of bGH. Thus, bGH can control glucose metabolism in vivo, at least in part, by modifying the expression of several genes coding for proteins of importance in carbohydrate metabolism. Taken together, these results indicate a state of insulin resistance caused by chronic exposure of the animals to an elevated concentration of bGH.—Valera, A., Rodriguez‐Gil, J. E., Yun, J. S., Hanson, R. W., Bosch, F. Glucose metabolism in transgenic mice containing a chimeric P‐enolpyruvate carboxykinase/bovine growth hormone gene. FASEB J. 7: 791‐800; 1993.

Insulin Inhibits Liver Expression of the CCAAT/Enhancer-Binding Protein β

The CCAAT/enhancer-binding protein β (C/EBPβ) is a transcription factor that is abundant in the liver. The concentration of C/EBPβ mRNA in the liver of mice and rats fed a high-carbohydrate diet, which causes a rise in blood insulin levels, was lower (80 and 65%, respectively) than that detected in animals fed a standard diet. Similarly, the expression of the human insulin gene in the liver of transgenic mice led to a decrease in the concentration of C/EBPβ mRNA. However, no change was detected in the mRNA levels of C/EBα or cAMP regulatory element-binding protein transcription factors in the livers of these mice. Furthermore, the expression of the C/EBPβ gene increased in the liver of diabetic rats and decreased in the liver of diabetic animals treated with vanadate, an insulin mimetic agent. In addition, a decrease in C/EBPβ protein was observed in liver nuclei from mice after insulin injections, in mice fed a high-carbohydrate diet, and in transgenic mice expressing the insulin gene in the liver. These results suggest that insulin might control gene expression in vivo, at least in part, by a mechanism involving a decrease in the transcription factor C/EBPβ.

Epidermal growth factor inhibits phosphoenolpyruvate carboxykinase gene expression in rat hepatocytes in primary culture

Epidermal growth factor (EGF) decreased the basal, and blocked the dibutyryl cyclic AMP (Bt2cAMP)‐induced, expression of P‐enolpyruvate carboxykinase (GTP) (PEPCK) and tyrosine aminotransferase (TAT) genes in both rat hepatocytes in primary culture and the FTO‐2B hepatoma cell line. Treatment of hepatocytes with EGF in combination with phorbol ester (TPA) resulted in an additive decrease of PEPCK mRNA levels. Overnight pretreatment of hepatocytes with TPA, which is known to downregulate protein kinase C, abolished the TPA and reduced the EGF‐mediated inhibition of PEPCK gene expression. These results suggested that EGF caused its effect, at least in part, through protein kinase C.

Calcium-mobilizing effectors inhibit P-enolpyruvate carboxykinase gene expression in cultured rat hepatocytes

Incubation of primary cultures of hepatocytes from fed and fasted rats with calcium ionophore strongly decreased glucose production from pyruvate. Like insulin, calcium ionophore A23187, phenylephrine, vasopressin, and prostaglandins E2 and F2α caused a significant reduction (50–60%) in basal concentrations of mRNA for P‐enolpyruvate carboxykinase (PEPCK), the main regulatory enzyme of gluconeogenesis. Phenylephrine, prostaglandin E2 and calcium ionophore A23187 were also able to counteract the induction of PEPCK gene expression by Bt2cAMP. These effects were similar to those exerted by both vanadate and phorbol ester TPA. The decrease in extracellular calcium by the addition of the calcium‐chelating agent EGTA to the incubation medium caused an increase in PEPCK mRNA levels. This effect was additive to that of Bt2cAMP and was counteracted by vanadate.