Anna Pujol

Adipose Tissue Overexpression of Vascular Endothelial Growth Factor Protects Against Diet-Induced Obesity and Insulin Resistance

During the expansion of fat mass in obesity, vascularization of adipose tissue is insufficient to maintain tissue normoxia. Local hypoxia develops and may result in altered adipokine expression, proinflammatory macrophage recruitment, and insulin resistance. We investigated whether an increase in adipose tissue angiogenesis could protect against obesity-induced hypoxia and, consequently, insulin resistance. Transgenic mice overexpressing vascular endothelial growth factor (VEGF) in brown adipose tissue (BAT) and white adipose tissue (WAT) were generated. Vessel formation, metabolism, and inflammation were studied in VEGF transgenic mice and wild-type littermates fed chow or a high-fat diet. Overexpression of VEGF resulted in increased blood vessel number and size in both WAT and BAT and protection against high-fat diet–induced hypoxia and obesity, with no differences in food intake. This was associated with increased thermogenesis and energy expenditure. Moreover, whole-body insulin sensitivity and glucose tolerance were improved. Transgenic mice presented increased macrophage infiltration, with a higher number of M2 anti-inflammatory and fewer M1 proinflammatory macrophages than wild-type littermates, thus maintaining an anti-inflammatory milieu that could avoid insulin resistance. These studies suggest that overexpression of VEGF in adipose tissue is a potential therapeutic strategy for the prevention of obesity and insulin resistance.

Transgenic mice overexpressing insulin-like growth factor-II in β cells develop type 2 diabetes

During embryonic development, insulin-like growth factor-II (IGF-II) participates in the regulation of islet growth and differentiation. We generated transgenic mice (C57BL6/SJL) expressing IGF-II in β cells under control of the rat Insulin I promoter in order to study the role of islet hyperplasia and hyperinsulinemia in the development of type 2 diabetes. In contrast to islets from control mice, islets from transgenic mice displayed high levels of IGF-II mRNA and protein. Pancreases from transgenic mice showed an increase in β-cell mass (about 3-fold) and in insulin mRNA levels. However, the organization of cells within transgenic islets was disrupted, with glucagon-producing cells randomly distributed throughout the core. We also observed enhanced glucose-stimulated insulin secretion and glucose utilization in islets from transgenic mice. These mice displayed hyperinsulinemia, mild hyperglycemia, and altered glucose and insulin tolerance tests, and about 30% of these animals developed overt diabetes when fed a high-fat diet. Furthermore, transgenic mice obtained from the N1 backcross to C57KsJ mice showed high islet hyperplasia and insulin resistance, but they also developed fatty liver and obesity. These results indicate that local overexpression of IGF-II in islets might lead to type 2 diabetes and that islet hyperplasia and hypersecretion of insulin might occur early in the pathogenesis of this disease.

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.

Transgenic mice overexpressing α2A-adrenoceptors in pancreatic beta-cells show altered regulation of glucose homeostasis

Aims/hypothesis. To study the role of the human α2A-adrenoceptor in the regulation of insulin secretion and the maintenance of glucose homeostasis in transgenic mice overexpressing this receptor in pancreatic beta cells.¶Methods. A human insulin promoter/human α2C10-adrenoceptor chimeric gene was microinjected into mouse embryos and transgenic mice were obtained.¶Results. Analysis by RT-PCR showed that the expression of the transgene was restricted to pancreatic islets. Study of the binding of the α2-antagonist [3H]RX821 002 to membrane preparations showed that islets from transgenic mice had ninefold higher α2-adrenoceptor density than those from controls. Immunohistological analysis showed, however, no change in the number or size of islets between control and transgenic mice. Transgenic animals had normal glycaemia and insulinaemia in basal conditions but greater hyperglycaemic and hypoinsulinaemic responses after injection of the α2-agonist, UK14 304. The lower blood insulin concentration detected in transgenic mice was a reflection of a stronger inhibitory effect of the α2-agonist on glucose-stimulated insulin secretion in transgenic islets than in controls. Furthermore, transgenic mice did not have lower glycaemia to basal values after an intraperitoneal glucose tolerance test. This defect was abolished by treatment with the α2-adrenoceptor antagonist, RX821 002.¶Conclusion/interpretation. These results provide evidence in vivo that overexpression of α2-adrenoceptors in beta cells can lead to impaired insulin secretion and glucose intolerance. [Diabetologia (2000) 43: 899–906]

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.

Vascular Endothelial Growth Factor–Mediated Islet Hypervascularization and Inflammation Contribute to Progressive Reduction of β-Cell Mass

Type 2 diabetes (T2D) results from insulin resistance and inadequate insulin secretion. Insulin resistance initially causes compensatory islet hyperplasia that progresses to islet disorganization and altered vascularization, inflammation, and, finally, decreased functional β-cell mass and hyperglycemia. The precise mechanism(s) underlying β-cell failure remain to be elucidated. In this study, we show that in insulin-resistant high-fat diet-fed mice, the enhanced islet vascularization and inflammation was parallel to an increased expression of vascular endothelial growth factor A (VEGF). To elucidate the role of VEGF in these processes, we have genetically engineered β-cells to overexpress VEGF (in transgenic mice or after adeno-associated viral vector-mediated gene transfer). We found that sustained increases in β-cell VEGF levels led to disorganized, hypervascularized, and fibrotic islets, progressive macrophage infiltration, and proinflammatory cytokine production, including tumor necrosis factor-α and interleukin-1β. This resulted in impaired insulin secretion, decreased β-cell mass, and hyperglycemia with age. These results indicate that sustained VEGF upregulation may participate in the initiation of a process leading to β-cell failure and further suggest that compensatory islet hyperplasia and hypervascularization may contribute to progressive inflammation and β-cell mass loss during T2D.

PED/PEA-15 Regulates Glucose-Induced Insulin Secretion by Restraining Potassium Channel Expression in Pancreatic β-Cells

The phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (ped/pea-15) gene is overexpressed in human diabetes and causes this abnormality in mice. Transgenic mice with β-cell–specific overexpression of ped/pea-15 (β-tg) exhibited decreased glucose tolerance but were not insulin resistant. However, they showed impaired insulin response to hyperglycemia. Islets from the β-tg also exhibited little response to glucose. mRNAs encoding the Sur1 and Kir6.2 potassium channel subunits and their upstream regulator Foxa2 were specifically reduced in these islets. Overexpression of PED/PEA-15 inhibited the induction of the atypical protein kinase C (PKC)-ζ by glucose in mouse islets and in β-cells of the MIN-6 and INS-1 lines. Rescue of PKC-ζ activity elicited recovery of the expression of the Sur1, Kir6.2, and Foxa2 genes and of glucose-induced insulin secretion in PED/PEA-15–overexpressing β-cells. Islets from ped/pea-15–null mice exhibited a twofold increased activation of PKC-ζ by glucose; increased abundance of the Sur1, Kir6.2, and Foxa2 mRNAs; and enhanced glucose effect on insulin secretion. In conclusion, PED/PEA-15 is an endogenous regulator of glucose-induced insulin secretion, which restrains potassium channel expression in pancreatic β-cells. Overexpression of PED/PEA-15 dysregulates β-cell function and is sufficient to impair glucose tolerance in mice.

Expression of glucokinase in skeletal muscle : A new approach to counteract diabetic hyperglycemia

Chronic hyperglycemia is responsible for diabetes-specific microvascular and macrovascular complications. To reduce hyperglycemia, key tissues may be engineered to take up glucose. To determine whether an increase in skeletal muscle glucose phosphorylation leads to increased glucose uptake and to normalization of diabetic alterations, the liver enzyme glucokinase (GK) was expressed in muscle of transgenic mice. GK has a high Km for glucose and its activity is not inhibited by glucose 6-phosphate. The presence of GK activity in skeletal muscle resulted in increased concentrations of glucose 6-phosphate and glycogen. These mice showed lower glycemia and insulinemia, increased serum lactate levels, and higher blood glucose disposal after an intraperitoneal glucose tolerance test. Furthermore, transgenic mice were more sensitive to injection of low doses of insulin, which led to increased blood glucose disposal. In addition, streptozotocin (STZ)-treated transgenic mice showed lower levels of blood glucose than STZ-treated controls and maintained body weight. Moreover, injection of insulin to STZ-treated transgenic mice led to normoglycemia, while STZ-treated control mice remained highly hyperglycemic. Thus, these results are consistent with a key role of glucose phosphorylation in regulating glucose metabolism in skeletal muscle. Furthermore, this study suggests that engineering skeletal muscle to express GK may be a new approach to the therapy of diabetes mellitus.

Phosphofructo-1-kinase deficiency leads to a severe cardiac and hematological disorder in addition to skeletal muscle glycogenosis.

Mutations in the gene for muscle phosphofructo-1-kinase (PFKM), a key regulatory enzyme of glycolysis, cause Type VII glycogen storage disease (GSDVII). Clinical manifestations of the disease span from the severe infantile form, leading to death during childhood, to the classical form, which presents mainly with exercise intolerance. PFKM deficiency is considered as a skeletal muscle glycogenosis, but the relative contribution of altered glucose metabolism in other tissues to the pathogenesis of the disease is not fully understood. To elucidate this issue, we have generated mice deficient for PFKM (Pfkm−/−). Here, we show that Pfkm−/− mice had high lethality around weaning and reduced lifespan, because of the metabolic alterations. In skeletal muscle, including respiratory muscles, the lack of PFK activity blocked glycolysis and resulted in considerable glycogen storage and low ATP content. Although erythrocytes of Pfkm−/− mice preserved 50% of PFK activity, they showed strong reduction of 2,3-biphosphoglycerate concentrations and hemolysis, which was associated with compensatory reticulocytosis and splenomegaly. As a consequence of these haematological alterations, and of reduced PFK activity in the heart, Pfkm−/− mice developed cardiac hypertrophy with age. Taken together, these alterations resulted in muscle hypoxia and hypervascularization, impaired oxidative metabolism, fiber necrosis, and exercise intolerance. These results indicate that, in GSDVII, marked alterations in muscle bioenergetics and erythrocyte metabolism interact to produce a complex systemic disorder. Therefore, GSDVII is not simply a muscle glycogenosis, and Pfkm−/− mice constitute a unique model of GSDVII which may be useful for the design and assessment of new therapies.