Pedro J. Otaegui

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.

In vivo gene transfer to pancreatic beta cells by systemic delivery of adenoviral vectors.

Type 1 diabetes results from autoimmune destruction of pancreatic beta cells. This process might be reversed by genetically engineering the endocrine pancreas in vivo to express factors that induce beta cell replication and neogenesis and counteract the immune response. However, the pancreas is difficult to manipulate and pancreatitis is a serious concern, which has made effective gene transfer to this organ elusive. Thus, new approaches for gene delivery to the pancreas in vivo are required. Here we show that pancreatic beta cells were efficiently transduced to express beta-galactosidase after systemic injection of adenovirus into mice with clamped hepatic circulation. Seven days after vector administration about 70% of pancreatic islets showed beta-galactosidase expression, with an average of about 20% of the cells within positive islets being transduced. In addition, scattered acinar cells expressing beta-galactosidase were also observed. Thus, this approach may be used to transfer genes of interest to mouse islets and beta cells, both for the study of islet biology and gene therapy of diabetes and other pancreatic disorders.

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.

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.

The presence of a high-Km hexokinase activity in dog, but not in boar, sperm

The presence of a high‐K m hexokinase activity was tested in both dog and boar spermatozoa. Hexokinase kinetics from dog extracts showed the presence of a specific activity (dog‐sperm glucokinase‐like protein, DSGLP), in the range of glucose concentrations of 4–10 mM, whereas boar sperm did not show any DSGLP activity. Furthermore, dog‐sperm cells, but not those of boar, showed the presence of a protein which specifically reacted against a rat‐liver anti‐glucokinase antibody. This protein also had a molecular weight equal to that observed in rat‐liver extracts, suggesting a close similarity between both the proteins. This glucokinase‐like protein was distributed in the peri‐ and post‐acrosomal zones of the head, and the midpiece and principal piece of tail of dog spermatozoa. These results indicate that dog spermatozoa have functional high‐K m hexokinase activity, which could contribute to a very fine regulation of their hexose metabolism. This strict regulation could ultimately be very important in optimizing dog‐sperm function along its life‐time.

Molecular signature of the immune and tissue response to non-coding plasmid DNA in skeletal muscle after electrotransfer

Electrotransfer of plasmid DNA in skeletal muscle is a common non-viral delivery method for both therapeutic genes and DNA vaccines. Yet, despite the similar approaches, an immune response is detrimental in gene therapy, but desirable for vaccines. However, the full nature of the immune and tissue responses to nucleic acids and electrotransfer in skeletal muscle has not been addressed. Here we used microarray analysis, fluorescence-activated cell sorting and quantitative polymerase chain reaction to obtain the molecular and cellular signature of the tissue and immune response to electrotransfer of saline and non-coding plasmid DNA. Saline electrotransfer resulted in limited infiltration and induction of a moderate damage–repair gene expression pattern not involving innate immune activation. However, plasmid electrotransfer augmented expression of the same genes in addition to inducing a strong innate immune response associated with pro-inflammatory infiltration. In particular, the inflammasome, Toll-like receptor 9 and other pattern recognition receptors able to respond to cytoplasmic DNA were upregulated. Several key differences in the nature of the inflammatory infiltrate and the kinetics of gene expression were also identified when comparing electrotransfer of conventional and CpG-free plasmids. Our data provide insights into the mechanisms of DNA detection and response in muscle that has relevance for non-viral gene therapy and DNA vaccination.

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.

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.

Experimental diabetes in neonatal mice induces early peripheral sensorimotor neuropathy

Animal models of diabetes do not reach the severity of human diabetic neuropathy but relatively mild neurophysiological deficits and minor morphometric changes. The lack of degenerative neuropathy in diabetic rodent models seems to be a consequence of the shorter length of the axons or the shorter animal life span. Diabetes-induced demyelination needs many weeks or even months before it can be evident by morphometrical analysis. In mice myelination of the peripheral nervous system starts at the prenatal period and it is complete several days after birth. Here we induced experimental diabetes to neonatal mice and we evaluated its effect on the peripheral nerve 4 and 8 weeks after diabetes induction. Neurophysiological values showed a decline in sensory nerve conduction velocity at both time-points. Morphometrical analysis of the tibial nerve demonstrated a decrease in the number of myelinated fibers, fiber size and myelin thickness at both time-points studied. Moreover, aldose reductase and poly(ADP-ribose) polymerase activities were increased even if the amount of the enzyme was not affected. Thus, type 1 diabetes in newborn mice induces early peripheral neuropathy and may be a good model to assay pharmacological or gene therapy strategies to treat diabetic neuropathy.

Conservation of Phenotypes in the Roman High- and Low-Avoidance Rat Strains After Embryo Transfer

The Roman high- (RHA-I) and low-avoidance (RLA-I) rat strains are bi-directionally bred for their good versus non-acquisition of two-way active avoidance, respectively. They have recently been re-derived through embryo transfer (ET) to Sprague–Dawley females to generate specific pathogen free (SPF) RHA-I/RLA-I rats. Offspring were phenotyped at generations 1 (G1, born from Sprague–Dawley females), 3 and 5 (G3 and G5, born from RHA-I and RLA-I from G2–G4, respectively), and compared with generation 60 from our non-SPF colony. Phenotyping included two-way avoidance acquisition, context-conditioned fear, open-field behaviour, novelty-seeking, baseline startle, pre-pulse inhibition (PPI) and stress-induced increase in plasma corticosterone concentration. Post-ET between-strain differences in avoidance acquisition, context-conditioned freezing and novelty-induced self-grooming are conserved. Other behavioural traits (i.e. hole-board head-dipping, novel object exploration, open-field activity, startle, PPI) differentiate the strains at G3–G5 but not at G1, suggesting that the pre-/post-natal environment may have influenced these co-segregated traits at G1, though further selection pressure along the subsequent generations (G1–G5) rescues the typical strain-related differences.