Sergio Muñoz

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.

Whole body correction of mucopolysaccharidosis IIIA by intracerebrospinal fluid gene therapy

For most lysosomal storage diseases (LSDs) affecting the CNS, there is currently no cure. The BBB, which limits the bioavailability of drugs administered systemically, and the short half-life of lysosomal enzymes, hamper the development of effective therapies. Mucopolysaccharidosis type IIIA (MPS IIIA) is an autosomic recessive LSD caused by a deficiency in sulfamidase, a sulfatase involved in the stepwise degradation of glycosaminoglycan (GAG) heparan sulfate. Here, we demonstrate that intracerebrospinal fluid (intra-CSF) administration of serotype 9 adenoassociated viral vectors (AAV9s) encoding sulfamidase corrects both CNS and somatic pathology in MPS IIIA mice. Following vector administration, enzymatic activity increased throughout the brain and in serum, leading to whole body correction of GAG accumulation and lysosomal pathology, normalization of behavioral deficits, and prolonged survival. To test this strategy in a larger animal, we treated beagle dogs using intracisternal or intracerebroventricular delivery. Administration of sulfamidase-encoding AAV9 resulted in transgenic expression throughout the CNS and liver and increased sulfamidase activity in CSF. High-titer serum antibodies against AAV9 only partially blocked CSF-mediated gene transfer to the brains of dogs. Consistently, anti-AAV antibody titers were lower in CSF than in serum collected from healthy and MPS IIIA-affected children. These results support the clinical translation of this approach for the treatment of MPS IIIA and other LSDs with CNS involvement.

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 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.

mTORC2 sustains thermogenesis via Akt-induced glucose uptake and glycolysis in brown adipose tissue

Activation of non‐shivering thermogenesis (NST) in brown adipose tissue (BAT) has been proposed as an anti‐obesity treatment. Moreover, cold‐induced glucose uptake could normalize blood glucose levels in insulin‐resistant patients. It is therefore important to identify novel regulators of NST and cold‐induced glucose uptake. Mammalian target of rapamycin complex 2 (mTORC2) mediates insulin‐stimulated glucose uptake in metabolic tissues, but its role in NST is unknown. We show that mTORC2 is activated in brown adipocytes upon β‐adrenergic stimulation. Furthermore, mice lacking mTORC2 specifically in adipose tissue (AdRiKO mice) are hypothermic, display increased sensitivity to cold, and show impaired cold‐induced glucose uptake and glycolysis. Restoration of glucose uptake in BAT by overexpression of hexokinase II or activated Akt2 was sufficient to increase body temperature and improve cold tolerance in AdRiKO mice. Thus, mTORC2 in BAT mediates temperature homeostasis via regulation of cold‐induced glucose uptake. Our findings demonstrate the importance of glucose metabolism in temperature regulation.

Biochemical, Histological and Functional Correction of Mucopolysaccharidosis Type IIIB by Intra-cerebrospinal Fluid Gene Therapy

Gene therapy is an attractive tool for the treatment of monogenic disorders, in particular for lysosomal storage diseases (LSD) caused by deficiencies in secretable lysosomal enzymes in which neither full restoration of normal enzymatic activity nor transduction of all affected cells are necessary. However, some LSD such as Mucopolysaccharidosis Type IIIB (MPSIIIB) are challenging because the diseases main target organ is the brain and enzymes do not efficiently cross the blood-brain barrier even if present at very high concentration in circulation. To overcome these limitations, we delivered AAV9 vectors encoding for α-N-acetylglucosaminidase (NAGLU) to the Cerebrospinal Fluid (CSF) of MPSIIIB mice with the disease already detectable at biochemical, histological and functional level. Restoration of enzymatic activity in Central Nervous System (CNS) resulted in normalization of glycosaminoglycan content and lysosomal physiology, resolved neuroinflammation and restored the pattern of gene expression in brain similar to that of healthy animals. Additionally, transduction of the liver due to passage of vectors to the circulation led to whole-body disease correction. Treated animals also showed reversal of behavioural deficits and extended lifespan. Importantly, when the levels of enzymatic activity were monitored in the CSF of dogs following administration of canine NAGLU-coding vectors to animals that were either naïve or had pre-existing immunity against AAV9, similar levels of activity were achieved, suggesting that CNS efficacy would not be compromised in patients seropositive for AAV9. Our studies provide a strong rationale for the clinical development of this novel therapeutic approach as the treatment for MPSIIIB.

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.

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.

Cleavage of NpzCH2 -OP and unexpected formation of bis(3,5-dimethyl-1H-pyrazol-1-yl)methane (dmbpm): X-ray crystal structure of [PdCl2(dmbpm)]

In the reaction of (3,5-dimethyl-1H-pyrazol-1-yl)methyldiphenylphosphinite (dmpmp) with [PdCl2(CH3CN)2], we have obtained hydrolysis and phosphorus oxidation products and the unexpected complex cis-[PdCl2(dmbpm)] (dmbpm = bis(3,5-dimethyl-1H-pyrazol-1-yl)methane). Modifications in the synthesis of dmpmp (high temperature, strong base, and the presence of Ph2P(O)Cl) show that dmbpm is a by-product from the synthetic route to dmpmp. The complex cis-[PdCl2(dmbpm)] is isolated and fully characterized by mass spectrometry, analytical, and spectroscopy techniques and the crystal structure is obtained by X-ray diffraction methods.

155. Non-viral gene therapy for type 1 diabetes by electrotransfer of insulin and glucokinase genes to skeletal muscle

Type 1 diabetes patients depend upon insulin replacement therapy. However, glycemic control is not always achieved and the resulting chronic hyperglycemia leads to microvascular, macrovascular and neurological complications. Therefore, there is a need for new treatment strategies for diabetes mellitus that would permit tight glucose regulation. Transgenic mice expressing insulin in skeletal muscle counteract type 1 diabetic alterations which indicates that muscle cells constitutively secreting low insulin levels could be used in gene therapy for diabetes. Furthermore, expression of glucokinase in skeletal muscle of transgenic mice reduced diabetic hyperglycemia without inducing hypoglycemia. However neither of these two transgenic mice showed complete reversion of the diabetic phenotype. Here, we examined the effect of co-expression of insulin and glucokinase in skeletal muscle on diabetic alterations and glycemia. Double transgenic mice expressing both insulin and glucokinase in skeletal muscle and skeletal muscle from diabetic mice electrotransferred with insulin and glucokinase genes were obtained. Blood glucose levels, skeletal muscle glucose uptake, glucose tolerance and insulin sensitivity were analyzed after streptozotozin (Stz) treatment. Double transgenic and electrotransferred mice counteracted hyperglycemia and restored fluid and food intake after treatment with Stz. In contrast, control mice presented polydipsia and polyphagia and developed severe diabetes. No hypogluycemia was detected in the Stz-treated mice expressing both insulin and glucokinase genes. In addition, these mice normalised both hepatic and skeletal muscle glucose metabolism and showed increased glucose disposal after an intraperitoneal glucose tolerance test. These results suggest that secretion of basal levels of insulin, in conjunction with increased glucose uptake by the skeletal muscle, might permit tight regulation of glycemia. Insulin and glucokinase genes cooperated by increasing glucose transport into the muscle cell and phosphorylation. Thus, this study provides evidence of a feasible non-viral gene therapy approach for type 1 diabetes involving the cooperative action of two genes, glucokinase and insulin, which leads to tight regulation of glucose homeostasis.