AAV-mediated BMP7 gene therapy counteracts insulin resistance and obesity.

Type 2 diabetes, insulin resistance, and obesity are strongly associated and are a major health problem worldwide. Obesity largely results from a sustained imbalance between energy intake and expenditure. Therapeutic approaches targeting metabolic rate may counteract body weight gain and insulin resistance. Bone morphogenic protein 7 (BMP7) has proven to enhance energy expenditure by inducing non-shivering thermogenesis in short-term studies in mice treated with the recombinant protein or adenoviral vectors encoding BMP7. To achieve long-term BMP7 effects, the use of adeno-associated viral (AAV) vectors would provide sustained production of the protein after a single administration. Here, we demonstrated that treatment of high-fat-diet-fed mice and ob/ob mice with liver-directed AAV-BMP7 vectors enabled a long-lasting increase in circulating levels of this factor. This rise in BMP7 concentration induced browning of white adipose tissue (WAT) and activation of brown adipose tissue, which enhanced energy expenditure, and reversed WAT hypertrophy, hepatic steatosis, and WAT and liver inflammation, ultimately resulting in normalization of body weight and insulin resistance. This study highlights the potential of AAV-BMP7-mediated gene therapy for the treatment of insulin resistance, type 2 diabetes, and obesity.

Vitamin D Receptor Overexpression in ß-Cells Ameliorates Diabetes in Mice.

Vitamin D deficiency has been associated with increased incidence of diabetes, both in humans and in animal models. In addition, an association between vitamin D receptor (VDR) gene polymorphisms and diabetes has also been described. However, the involvement of VDR in the development of diabetes, specifically in pancreatic ß-cells, has not been elucidated yet. Here, we aimed to study the role of VDR in ß-cells in the pathophysiology of diabetes. Our results indicate that Vdr expression was modulated by glucose in healthy islets and decreased in islets from both type 1 diabetes and type 2 diabetes mouse models. In addition, transgenic mice overexpressing VDR in ß-cells were protected against streptozotocin-induced diabetes and presented a preserved ß-cell mass and a reduction in islet inflammation. Altogether, these results suggest that sustained VDR levels in ß-cells may preserve ß-cell mass and ß-cell function and protect against diabetes.

FGF21 gene therapy as treatment for obesity and insulin resistance.

Prevalence of type 2 diabetes (T2D) and obesity is increasing worldwide. Currently available therapies are not suited for all patients in the heterogeneous obese/T2D population, hence the need for novel treatments. Fibroblast growth factor 21 (FGF21) is considered a promising therapeutic agent for T2D/obesity. Native FGF21 has, however, poor pharmacokinetic properties, making gene therapy an attractive strategy to achieve sustained circulating levels of this protein. Here, adeno-associated viral vectors (AAV) were used to genetically engineer liver, adipose tissue, or skeletal muscle to secrete FGF21. Treatment of animals under long-term high-fat diet feeding or of ob/ob mice resulted in marked reductions in body weight, adipose tissue hypertrophy and inflammation, hepatic steatosis, inflammation and fibrosis, and insulin resistance for > 1 year. This therapeutic effect was achieved in the absence of side effects despite continuously elevated serum FGF21. Furthermore, FGF21 overproduction in healthy animals fed a standard diet prevented the increase in weight and insulin resistance associated with aging. Our study underscores the potential of FGF21 gene therapy to treat obesity, insulin resistance, and T2D.

Long-Term Efficacy and Safety of Insulin and Glucokinase Gene Therapy for Diabetes: 8-Year Follow-Up in Dogs.

Diabetes is a complex metabolic disease that exposes patients to the deleterious effects of hyperglycemia on various organs. Achievement of normoglycemia with exogenous insulin treatment requires the use of high doses of hormone, which increases the risk of life-threatening hypoglycemic episodes. We developed a gene therapy approach to control diabetic hyperglycemia based on co-expression of the insulin and glucokinase genes in skeletal muscle. Previous studies proved the feasibility of gene delivery to large diabetic animals with adeno-associated viral (AAV) vectors. Here, we report the long-term (∼8 years) follow-up after a single administration of therapeutic vectors to diabetic dogs. Successful, multi-year control of glycemia was achieved without the need of supplementation with exogenous insulin. Metabolic correction was demonstrated through normalization of serum levels of fructosamine, triglycerides, and cholesterol and remarkable improvement in the response to an oral glucose challenge. The persistence of vector genomes and therapeutic transgene expression years after vector delivery was documented in multiple samples from treated muscles, which showed normal morphology. Thus, this study demonstrates the long-term efficacy and safety of insulin and glucokinase gene transfer in large animals and especially the ability of the system to respond to the changes in metabolic needs as animals grow older.

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.

HMGA1 overexpression in adipose tissue impairs adipogenesis and prevents diet-induced obesity and insulin resistance.

High-Mobility-Group-A1 (HMGA1) proteins are non-histone proteins that regulate chromatin structure and gene expression during embryogenesis, tumourigenesis and immune responses. In vitro studies suggest that HMGA1 proteins may be required to regulate adipogenesis. To examine the role of HMGA1 in vivo, we generated transgenic mice overexpressing HMGA1 in adipose tissues. HMGA1 transgenic mice showed a marked reduction in white and brown adipose tissue mass that was associated with downregulation of genes involved in adipogenesis and concomitant upregulation of preadipocyte markers. Reduced adipogenesis and decreased fat mass were not associated with altered glucose homeostasis since HMGA1 transgenic mice fed a regular-chow diet exhibited normal glucose tolerance and insulin sensitivity. However, when fed a high-fat diet, overexpression of HMGA1 resulted in decreased body-weight gain, reduced fat mass, but improved insulin sensitivity and glucose tolerance. Although HMGA1 transgenic mice exhibited impaired glucose uptake in adipose tissue due to impaired adipogenesis, the increased glucose uptake observed in skeletal muscle may account for the improved glucose homeostasis. Our results indicate that HMGA1 plays an important function in the regulation of white and brown adipogenesis in vivo and suggests that impaired adipocyte differentiation and decreased fat mass is not always associated with impaired whole-body glucose homeostasis.

AAV8-mediated Sirt1 gene transfer to the liver prevents high carbohydrate diet-induced nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is the most common hepatic disease worldwide, and evidence suggests that it promotes insulin resistance and type 2 diabetes. Caloric restriction (CR) is the only available strategy for NAFLD treatment. The protein deacetylase Sirtuin1 (SIRT1), which is activated by CR, increases catabolic metabolism and decreases lipogenesis and inflammation, both involved in the development of NAFLD. Here we show that adeno-associated viral vectors of serotype 8 (AAV8)-mediated liver-specific Sirt1 gene transfer prevents the development of NAFLD induced by a high carbohydrate (HC) diet. Long-term hepatic SIRT1 overexpression led to upregulation of key hepatic genes involved in ß-oxidation, prevented HC diet-induced lipid accumulation and reduced liver inflammation. AAV8-Sirt1-treated mice showed improved insulin sensitivity, increased oxidative capacity in skeletal muscle and reduced white adipose tissue inflammation. Moreover, HC feeding induced leptin resistance, which was also attenuated in AAV8-Sirt1-treated mice. Therefore, AAV-mediated gene transfer to overexpress SIRT1 specifically in the liver may represent a new gene therapy strategy to counteract NAFLD and related diseases such as type 2 diabetes.

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.

Treatment of diabetes and long-term survival after insulin and glucokinase gene therapy.

Diabetes is associated with severe secondary complications, largely caused by poor glycemic control. Treatment with exogenous insulin fails to prevent these complications completely, leading to significant morbidity and mortality. We previously demonstrated that it is possible to generate a "glucose sensor" in skeletal muscle through coexpression of glucokinase and insulin, increasing glucose uptake and correcting hyperglycemia in diabetic mice. Here, we demonstrate long-term efficacy of this approach in a large animal model of diabetes. A one-time intramuscular administration of adeno-associated viral vectors of serotype 1 encoding for glucokinase and insulin in diabetic dogs resulted in normalization of fasting glycemia, accelerated disposal of glucose after oral challenge, and no episodes of hypoglycemia during exercise for >4 years after gene transfer. This was associated with recovery of body weight, reduced glycosylated plasma proteins levels, and long-term survival without secondary complications. Conversely, exogenous insulin or gene transfer for insulin or glucokinase alone failed to achieve complete correction of diabetes, indicating that the synergistic action of insulin and glucokinase is needed for full therapeutic effect. This study provides the first proof-of-concept in a large animal model for a gene transfer approach to treat diabetes.

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.

Acute activation of central GLP-1 receptors enhances hepatic insulin action and insulin secretion in high-fat-fed, insulin resistant mice.

Glucagon-like peptide-1 (GLP-1) receptor knockout (Glp1r(-/-)) mice exhibit impaired hepatic insulin action. High fat (HF)-fed Glp1r(-/-) mice exhibit improved, rather than the expected impaired, hepatic insulin action. This is due to decreased lipogenic gene expression and triglyceride accumulation. The present studies overcome these secondary adaptations by acutely modulating GLP-1R action in HF-fed wild-type mice. The central GLP-1R was targeted given its role as a regulator of hepatic insulin action. We hypothesized that acute inhibition of the central GLP-1R impairs hepatic insulin action beyond the effects of HF feeding. We further hypothesized that activation of the central GLP-1R improves hepatic insulin action in HF-fed mice. Insulin action was assessed in conscious, unrestrained mice using the hyperinsulinemic euglycemic clamp. Mice received intracerebroventricular (icv) infusions of artificial cerebrospinal fluid, GLP-1, or the GLP-1R antagonist exendin-9 (Ex-9) during the clamp. Intracerebroventricular Ex-9 impaired the suppression of hepatic glucose production by insulin, whereas icv GLP-1 improved it. Neither treatment affected tissue glucose uptake. Intracerebroventricular GLP-1 enhanced activation of hepatic Akt and suppressed hypothalamic AMP-activated protein kinase. Central GLP-1R activation resulted in lower hepatic triglyceride levels but did not affect muscle, white adipose tissue, or plasma triglyceride levels during hyperinsulinemia. In response to oral but not intravenous glucose challenges, activation of the central GLP-1R improved glucose tolerance. This was associated with higher insulin levels. Inhibition of the central GLP-1R had no effect on oral or intravenous glucose tolerance. These results show that inhibition of the central GLP-1R deteriorates hepatic insulin action in HF-fed mice but does not affect whole body glucose homeostasis. Contrasting this, activation of the central GLP-1R improves glucose homeostasis in HF-fed mice by increasing insulin levels and enhancing hepatic insulin action.

Adipose overexpression of phosphoenolpyruvate carboxykinase leads to high susceptibility to diet-induced insulin resistance and obesity.

Obesity and insulin resistance are associated with increased serum free fatty acids (FFAs). Thus, a reduction in circulating FFAs may increase insulin sensitivity. This could be achieved by increasing FFA reesterification in adipose tissue. Transgenic mice with increased adipose tissue glyceroneogenesis, caused by overexpression of phosphoenolpyruvate carboxykinase (PEPCK), show increased FFA reesterification and develop obesity but are insulin sensitive. Here, we examined whether these transgenic mice were protected from diet-induced insulin resistance. Surprisingly, when fed a high-fat diet for a short period (6 weeks), transgenic mice developed severe obesity and were more hyperinsulinemic, glucose intolerant, and insulin resistant than controls. The high triglyceride accumulation prevented white adipose tissue from buffering the flux of lipids in circulation and led to increased serum triglyceride levels and fat deposition in liver. Furthermore, circulating leptin and FFA concentrations increased to similar levels in transgenic and control mice, while adiponectin levels decreased in transgenic mice compared with controls. In addition, transgenic mice showed fat accumulation in brown adipose tissue, which decreased uncoupling protein-1 expression, suggesting that these mice had impaired diet-induced thermogenesis. These results indicate that increased PEPCK expression in the presence of high-fat feeding may have deleterious effects and lead to severe insulin resistance and type 2 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 alpha, 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.

Prevention of obesity and insulin resistance by glucokinase expression in skeletal muscle of transgenic mice.

In type 2 diabetes, glucose phosphorylation, a regulatory step in glucose utilization by skeletal muscle, is impaired. Since glucokinase expression in skeletal muscle of transgenic mice increases glucose phosphorylation, we examined whether such mice counteract the obesity and insulin resistance induced by 12 wk of a high-fat diet. When fed this diet, control mice became obese, whereas transgenic mice remained lean. Furthermore, high-fat fed control mice developed hyperglycemia and hyperinsulinemia (a 3-fold increase), indicating that they were insulin resistant. In contrast, transgenic mice were normoglycemic and showed only a mild increase in insulinemia (1.5-fold). They also showed improved whole body glucose tolerance and insulin sensitivity and increased intramuscular concentrations of glucose 6-phosphate and glycogen. A parallel increase in uncoupling protein 3 mRNA levels in skeletal muscle of glucokinase-expressing transgenic mice was also observed. These results suggest that the rise in glucose phosphorylation by glucokinase expression in skeletal muscle leads to increased glucose utilization and energy expenditure that counteracts weight gain and maintains insulin sensitivity.

Overexpression of c-myc in diabetic mice restores altered expression of the transcription factor genes that regulate liver metabolism.

Overexpression of the c-Myc transcription factor in liver induces glucose uptake and utilization. Here we examined the effects of c- myc overexpression on the expression of hepatocyte-specific transcription factor genes which regulate the expression of genes controlling hepatic metabolism. At 4 months after streptozotocin (STZ) treatment, most diabetic control mice were highly hyperglycaemic and died, whereas in STZ-treated transgenic mice hyperglycaemia was markedly lower, the serum levels of beta-hydroxybutyrate, triacylglycerols and non-esterified fatty acids were normal, and they had greater viability in the absence of insulin. Furthermore, long-term STZ-treated transgenic mice showed similar glucose utilization and storage to healthy controls. This was consistent with the expression of glycolytic genes becoming normalized. In addition, restoration of gene expression of the transcription factor, sterol receptor element binding protein 1c, was observed in the livers of these transgenic mice. Further, in STZ-treated transgenic mice the expression of genes involved in the control of gluconeogenesis (phosphoenolpyruvate carbokykinase), ketogenesis (3-hydroxy-3-methylglutaryl-CoA synthase) and energy metabolism (uncoupling protein 2) had returned to normal. These findings were correlated with decreased expression of genes encoding the transcription factors hepatocyte nuclear factor 3gamma, peroxisome proliferator-activated receptor alpha and retinoid X receptor. These results indicate that c- myc overexpression may counteract diabetic changes by controlling hepatic glucose metabolism, both directly by altering the expression of metabolic genes and through the expression of key transcription factor genes.

Counteraction of type 1 diabetic alterations by engineering skeletal muscle to produce insulin: insights from transgenic mice.

Insulin replacement therapy in type 1 diabetes is imperfect because proper glycemic control is not always achieved. Most patients develop microvascular, macrovascular, and neurological complications, which increase with the degree of hyperglycemia. Engineered muscle cells continuously secreting basal levels of insulin might be used to improve the efficacy of insulin treatment. Here we examined the control of glucose homeostasis in healthy and diabetic transgenic mice constitutively expressing mature human insulin in skeletal muscle. Fed transgenic mice were normoglycemic and normoinsulinemic and, after an intraperitoneal glucose tolerance test, showed increased glucose disposal. When treated with streptozotocin (STZ), transgenic mice showed increased insulinemia and reduced hyperglycemia when fed and normoglycemia and normoinsulinemia when fasted. Injection of low doses of soluble insulin restored normoglycemia in fed STZ-treated transgenic mice, while STZ-treated controls remained highly hyperglycemic, indicating that diabetic transgenic mice were more sensitive to the hypoglycemic effects of insulin. Furthermore, STZ-treated transgenic mice presented normalization of both skeletal muscle and liver glucose metabolism. These results indicate that skeletal muscle may be a key target tissue for insulin production and suggest that muscle cells secreting basal levels of insulin, in conjunction with insulin therapy, may permit tight regulation of glycemia.

Glucose-regulated glucose uptake by transplanted muscle cells expressing glucokinase counteracts diabetic hyperglycemia.

Type 1 diabetic patients depend on insulin replacement therapy. However, chronic hyperglycemia due to failure to maintain proper glycemic control leads to microvascular, macrovascular, and neurological complications. Increased glucose disposal by tissues engineered to overexpress key regulatory genes in glucose transport or phosphorylation can reduce diabetic hyperglycemia. Here we report that differentiated myoblast cells expressing the glucose-phosphorylating enzyme glucokinase (GK) showed a glucose-dependent increase in glucose uptake and utilization in vitro. Transplantation of GK-expressing myotubes into healthy mice did not alter blood glucose levels and recipient mice maintained normoglycemia. After streptozotocin treatment, mice transplanted with GK-expressing myotubes counteracted hyperglycemia, polydipsia, and polyphagia, whereas mice transplanted with control myotubes developed diabetes. Similarly, diabetic mice transplanted with control myotubes remained hyperglycemic. In contrast, transplantation of GK-expressing myotubes into diabetic mice lowered hyperglycemia. These results suggest that the use of genetically engineered muscle cells to express glucokinase may provide a glucose-regulated approach to reduce diabetic hyperglycemia.