Efren Riu

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.

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)

Silencing of episomal transgene expression in liver by plasmid bacterial backbone DNA is independent of CpG methylation.

Minicircle DNA vectors devoid of plasmid bacterial backbone, (BB) DNAs, are transcriptionally persistent, whereas their parent plasmid counterparts are silenced in the liver. In this study we establish that circular plasmid BB provided in trans did not silence a transgene expression cassette in vivo, further confirming our previous conclusions that the covalent attachment of the plasmid BB to the expression cassette is required for DNA silencing. Given the high concentration of CpG dinucleotides in the plasmid BB, we investigated the role of DNA methylation on transgene silencing in vivo. The presence or absence of methylation in CpG motifs in routine plasmid BBs had no significant effect on transcriptional silencing. Furthermore, the removal of the CpG motifs from the BB did not ameliorate transcriptional silencing. Transgene silencing was partially inhibited when two tandem copies of the chicken cHS4 insulator at each end of a routine plasmid vector were used. These results are consistent with the idea that the transcriptional repression observed with plasmid DNA vectors in the liver is caused by formation of repressive heterochromatin on the plasmid DNA backbone, which then spreads and inactivates the transgene in cis, and that CpG content or methylation has little or no influence in the process.

Long-term overexpression of glucokinase in the liver of transgenic mice leads to insulin resistance

Aims/hypothesisGlucokinase overexpression in the liver increases glucose uptake and utilization, and improves glucose tolerance in young transgenic mice. Here, we examined the long-term effects of hepatic overexpression of glucokinase on glucose homeostasis. Moreover, we determined whether glucokinase overexpression counteracted high-fat diet-induced insulin resistance.MethodsTransgenic mice overexpressing glucokinase in liver under the control of the phosphoenolpyruvate carboxykinase promoter, fed either a standard diet or a high-fat diet, were studied. We used non-transgenic littermates as controls.ResultsTransgenic mice over 6 months old developed impaired glucose tolerance. In addition, at 12 months of age, transgenic mice showed mild hyperglycaemia, hyperinsulinaemia and hypertriglyceridaemia. In spite of increased glucokinase activity, the liver of these mice accumulated less glycogen and increased triglyceride deposition. When 2-month-old glucose-tolerant mice were fed a high-fat diet, transgenic mice gained more body weight and became hyperglycaemic and hyperinsulinaemic. This was concomitant to glucose intolerance, liver steatosis and whole-body insulin resistance.Conclusion/interpretationLong-term overexpression of glucokinase increases hepatic lipogenesis and circulating lipids, which lead to insulin resistance. Our results also suggest that the liver plays a key role in the onset of diabetes.

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)

Insulin Production by Engineered Muscle Cells

Type 1 diabetic patients depend dramatically on insulin replacement therapy, which involves the administration of intermediate- or long-acting insulin, together with short-acting insulin to mimic physiological insulin profiles. However, the delayed-action preparations available are not generally able to produce smooth background levels of insulin. Muscle cells were tested for long-term delivery of active human insulin as an approach to achieve a constant basal level of insulin. Thus, C2C12 mouse myoblast cells were stably transfected with a chimeric gene obtained by linking the myosin-light chain 1 (MLC1) promoter to the human proinsulin gene, containing genetically engineered furin endoprotease cleavage sites (MLC1/Insm). When differentiated, C2C12Insm myotube cells expressed high levels of insulin mRNA and protein, whereas no insulin was detected in myoblast cells. HPLC fractionation of culture medium and cell extracts from differentiated C2C12Insm cells revealed that about 90% of the proinsulin was processed to mature insulin. In addition, these cells released significant levels (about 100 microU/10(6) cells/hr) of mature insulin to the medium. The hormone was biologically active since it increased glucose consumption and utilization by the differentiated C2C12Insm cells and was able to block the expression of the endogenous phosphoenolpyruvate carboxykinase (PEPCK) gene in FTO-2B rat hepatoma cells. Furthermore, when C2C12Insm myoblast cells were transplanted into diabetic mice an increase in insulinemia and a decrease in hyperglycemia were observed. Thus, our results suggest that the use of engineered myotube cells continuously secreting a defined level of insulin might be a useful approach to improve the efficacy of insulin injection treatment.

Reversal of Type 1 Diabetes by Engineering a Glucose Sensor in Skeletal Muscle

Type 1 diabetic patients develop severe secondary complications because insulin treatment does not guarantee normoglycemia. Thus, efficient regulation of glucose homeostasis is a major challenge in diabetes therapy. Skeletal muscle is the most important tissue for glucose disposal after a meal. However, the lack of insulin during diabetes impairs glucose uptake. To increase glucose removal from blood, skeletal muscle of transgenic mice was engineered both to produce basal levels of insulin and to express the liver enzyme glucokinase. After streptozotozin (STZ) administration of double-transgenic mice, a synergic action in skeletal muscle between the insulin produced and the increased glucose phosphorylation by glucokinase was established, preventing hyperglycemia and metabolic alterations. These findings suggested that insulin and glucokinase might be expressed in skeletal muscle, using adeno-associated viral 1 (AAV1) vectors as a new gene therapy approach for diabetes. AAV1-Ins+GK–treated diabetic mice restored and maintained normoglycemia in fed and fasted conditions for >4 months after STZ administration. Furthermore, these mice showed normalization of metabolic parameters, glucose tolerance, and food and fluid intake. Therefore, the joint action of basal insulin production and glucokinase activity may generate a “glucose sensor” in skeletal muscle that allows proper regulation of glycemia in diabetic animals and thus prevents secondary complications.

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.

Overexpression of c-myc in the liver prevents obesity and insulin resistance

Alterations in hepatic glucose metabolism play a key role in the development of the hyperglycemia observed in type 2 diabetes. Because the transcription factor c‐Myc induces hepatic glucose uptake and utilization and blocks gluconeogenesis, we examined whether hepatic overexpression of c‐myc counteracts the insulin resistance induced by a high‐fat diet. After 3 months on this diet, control mice became obese, hyperglycemic, and hyperinsulinemic, indicating that they had developed insulin resistance. In contrast, transgenic mice remained lean and showed improved glucose disposal and normal levels of blood glucose and insulin, indicating that they had developed neither obesity nor insulin resistance. These findings were concomitant with normalization of hepatic glucokinase and pyruvate kinase gene expression and enzyme activity, which led to normalization of intrahepatic glucose‐6‐phosphate and glycogen content. In the liver of control mice fed a high‐fat diet, the expression of genes encoding proteins that control energy metabolism, such as sterol receptor element binding protein 1‐c, peroxisome proliferator activated receptor α, and uncoupling protein‐2, was altered. In contrast, in the liver of transgenic mice fed a high‐fat diet, the expression of these genes was normal. These results suggest that c‐myc overexpression counteracted the obesity and insulin resistance induced by a high‐fat diet by modulating the expression of genes that regulate hepatic metabolism.