Sylvie Franckhauser

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.

Overexpression of Il6 leads to hyperinsulinaemia, liver inflammation and reduced body weight in mice

Aims/hypothesisIL-6 is released by the adipose tissue and increased circulating levels in obesity are associated with hyperinsulinaemia and insulin resistance. Short-term experiments suggest that increased IL-6 release by the skeletal muscle following exercise may improve insulin sensitivity.MethodsIn order to examine the effect of chronically elevated IL-6 levels, we overexpressed Il6 in skeletal muscle in mice using an electro-transfer procedure.ResultsCirculating IL-6 levels were increased and the animals rapidly lost both weight and body fat, but food intake was unchanged, which is consistent with the finding that IL-6 increased energy expenditure. Insulin levels were inappropriately elevated and combined with hypoglycaemia in spite of reduced 2-deoxy-d-glucose uptake by skeletal muscle. Insulin-stimulated glucose uptake by skeletal muscles ex vivo was reduced, probably due to the decreased amounts of glucose transporter (GLUT)-4. Beta cell insulin content was increased, while apparent beta cell mass was unchanged. Circulating serum amyloid A cluster levels were increased tenfold due to a pronounced proinflammatory state in the liver with infiltration of inflammatory cells. However, no liver steatosis was found, which may be accounted for by concomitant AMP kinase activation.Conclusions/interpretationChronically elevated IL-6 levels lead to inappropriate hyperinsulinaemia, reduced body weight, impaired insulin-stimulated glucose uptake by the skeletal muscles and marked inflammation in the liver. Thus, the pleiotrophic effects of chronically elevated IL-6 levels preclude any obvious usefulness in treating obesity or its associated metabolic complications in man, despite the fact that weight reduction may be expected.

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.

Expression of IGF-I in Pancreatic Islets Prevents Lymphocytic Infiltration and Protects Mice From Type 1 Diabetes

Type 1 diabetic patients are diagnosed when β-cell destruction is almost complete. Reversal of type 1 diabetes will require β-cell regeneration from islet cell precursors and prevention of recurring autoimmunity. IGF-I expression in β-cells of streptozotocin (STZ)-treated transgenic mice regenerates the endocrine pancreas by increasing β-cell replication and neogenesis. Here, we examined whether IGF-I also protects islets from autoimmune destruction. Expression of interferon (IFN)-β in β-cells of transgenic mice led to islet β2-microglobulin and Fas hyperexpression and increased lymphocytic infiltration. Pancreatic islets showed high insulitis, and these mice developed overt diabetes when treated with very-low doses of STZ, which did not affect control mice. IGF-I expression in IFN-β–expressing β-cells of double-transgenic mice reduced β2-microglobulin, blocked Fas expression, and counteracted islet infiltration. This was parallel to a decrease in β-cell death by apoptosis in islets of STZ-treated IGF-I+IFN-β–expressing mice. These mice were normoglycemic, normoinsulinemic, and showed normal glucose tolerance. They also presented similar pancreatic insulin content and β-cell mass to healthy mice. Thus, local expression of IGF-I prevented islet infiltration and β-cell death in mice with increased susceptibility to diabetes. These results indicate that pancreatic expression of IGF-I may regenerate and protect β-cell mass in type 1 diabetes.

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.

In Vivo Adeno-Associated Viral Vector–Mediated Genetic Engineering of White and Brown Adipose Tissue in Adult Mice

Adipose tissue is pivotal in the regulation of energy homeostasis through the balance of energy storage and expenditure and as an endocrine organ. An inadequate mass and/or alterations in the metabolic and endocrine functions of adipose tissue underlie the development of obesity, insulin resistance, and type 2 diabetes. To fully understand the metabolic and molecular mechanism(s) involved in adipose dysfunction, in vivo genetic modification of adipocytes holds great potential. Here, we demonstrate that adeno-associated viral (AAV) vectors, especially serotypes 8 and 9, mediated efficient transduction of white (WAT) and brown adipose tissue (BAT) in adult lean and obese diabetic mice. The use of short versions of the adipocyte protein 2 or uncoupling protein-1 promoters or micro-RNA target sequences enabled highly specific, long-term AAV-mediated transgene expression in white or brown adipocytes. As proof of concept, delivery of AAV vectors encoding for hexokinase or vascular endothelial growth factor to WAT or BAT resulted in increased glucose uptake or increased vessel density in targeted depots. This method of gene transfer also enabled the secretion of stable high levels of the alkaline phosphatase marker protein into the bloodstream by transduced WAT. Therefore, AAV-mediated genetic engineering of adipose tissue represents a useful tool for the study of adipose pathophysiology and, likely, for the future development of new therapeutic strategies for obesity and diabetes.

Chronically increased glucose uptake by adipose tissue leads to lactate production and improved insulin sensitivity rather than obesity in the mouse

Aims/hypothesisIn adipocytes, triacylglycerol synthesis depends on the formation of glycerol 3-phosphate, which originates either from glucose, through glycolysis, or from lactate, through glyceroneogenesis. However, glucose is traditionally viewed as the main precursor of the glycerol backbone and thus, enhanced glucose uptake would be expected to result in increased triacylglycerol synthesis and contribute to obesity.MethodsTo further explore this issue, we generated a mouse model with chronically increased glucose uptake in adipose tissue by expressing Gck, which encodes the glucokinase enzyme.ResultsHere we show that the production of high levels of glucokinase led to increased adipose tissue glucose uptake and lactate production, improved glucose tolerance and higher whole-body and skeletal muscle insulin sensitivity. There was no parallel increase in glycerol 3-phosphate synthesis in vivo, fat accumulation or obesity. Moreover, at high glucose concentrations, in cultured fat cells overproducing glucokinase, glycerol 3-phosphate synthesis from pyruvate decreased, while glyceroneogenesis increased in fat cells overproducing hexokinase II.Conclusions/interpretationsThese findings indicate that the absence of glucokinase inhibition by glucose 6-phosphate probably led to increased glycolysis and blocked glyceroneogenesis in the mouse model. Furthermore, this study suggests that under physiological conditions, when blood glucose increases, glyceroneogenesis may prevail over glycolysis for triacylglycerol formation because of the inhibition of hexokinase II by glucose 6-phosphate. Together these results point to the indirect pathway (glucose to lactate to glycerol 3-phosphate) being key for fat deposition in adipose tissue.

New insights into adipose tissue VEGF-A actions in the control of obesity and insulin resistance

Vascular endothelial growth factor A (VEGF-A) is classically viewed as a key factor in angiogenesis and tissue remodeling. However, recent evidence suggests a potential role of this growth factor in the control of energy metabolism and adipose tissue function. In this regard, we and others have described the effects of the up and downregulation of VEGF-A in adipose tissue on the control of energy homeostasis. VEGF-A overexpression protects against diet-induced obesity and insulin resistance. The observation that VEGF-A overexpression leads to an increase in brown adipose tissue (BAT) thermogenesis and also promotes a “BAT-like” phenotype in white adipose tissue depots is of particular relevance for the understanding of the mechanisms underlying obesity development. In addition, VEGF-A may not only have pro-inflammatory but also anti-inflammatory properties, with a chemotactic activity specific for M2 anti-inflammatory macrophages. This new scientific evidence highlights the importance that VEGF-A actions on metabolism could have on the design of new treatments for obesity, insulin resistance and obesity-related disorders.

ALOX5AP Overexpression in Adipose Tissue Leads to LXA4 Production and Protection Against Diet-Induced Obesity and Insulin Resistance.

Eicosanoids, such as leukotriene B4 (LTB4) and lipoxin A4 (LXA4), may play a key role during obesity. While LTB4 is involved in adipose tissue inflammation and insulin resistance, LXA4 may exert anti-inflammatory effects and alleviate hepatic steatosis. Both lipid mediators derive from the same pathway, in which arachidonate 5-lipoxygenase (ALOX5) and its partner, arachidonate 5-lipoxygenase–activating protein (ALOX5AP), are involved. ALOX5 and ALOX5AP expression is increased in humans and rodents with obesity and insulin resistance. We found that transgenic mice overexpressing ALOX5AP in adipose tissue had higher LXA4 rather than higher LTB4 levels, were leaner, and showed increased energy expenditure, partly due to browning of white adipose tissue (WAT). Upregulation of hepatic LXR and Cyp7a1 led to higher bile acid synthesis, which may have contributed to increased thermogenesis. In addition, transgenic mice were protected against diet-induced obesity, insulin resistance, and inflammation. Finally, treatment of C57BL/6J mice with LXA4, which showed browning of WAT, strongly suggests that LXA4 is responsible for the transgenic mice phenotype. Thus, our data support that LXA4 may hold great potential for the future development of therapeutic strategies for obesity and related diseases.

Enforced expression of protein kinase C in skeletal muscle causes physical inactivity, fatty liver and insulin resistance in the brain

Among the multitude of dysregulated signalling mechanisms that comprise insulin resistance in divergent organs, the primary events in the development of type 2 diabetes are not well established. As protein kinase C (PKC) activation is consistently present in skeletal muscle of obese and insulin resistant subjects, we generated a transgenic mouse model that overexpresses constitutively active PKC‐β2 in skeletal muscle to test whether activation of PKC is sufficient to cause an aversive whole‐body phenotype. Upon this genetic modification, increased serine phosphorylation in Irs1 was observed and followed by impaired 3H‐deoxy‐glucose uptake and muscle glycogen content, and transgenic mice exhibited insulin and glucose intolerance as they age. Muscle histochemistry revealed an increase in lipid deposition (intramyocellular lipids), and transgenic mice displayed impaired expression of transcriptional regulators of genes involved in fatty acid oxidation (peroxisome proliferator‐activated receptor‐γ, PGC‐1β, acyl‐CoA oxidase) and lipolysis (hormone‐sensitive lipase). In this regard, muscle of transgenic mice exhibited a reduced capacity to oxidize palmitate and contained less mitochondria as determined by citrate synthase activity. Moreover, the phenotype included a profound decrease in the daily running distance, intra‐abdominal and hepatic fat accumulation and impaired insulin action in the brain. Together, our data suggest that activation of a classical PKC in skeletal muscle as present in the pre‐diabetic state is sufficient to cause disturbances in whole‐body glucose and lipid metabolism followed by profound alterations in oxidative capacity, ectopic fat deposition and physical activity.