Pedro Otaegui - Academia.edu (original) (raw)

Papers by Pedro Otaegui

Lab animal, 2014

Repeated, low-dose administration of streptozotocin (STZ) is widely used to induce insulin-depend... more Repeated, low-dose administration of streptozotocin (STZ) is widely used to induce insulin-dependent diabetes mellitus in mice. The authors adapted this method using neonatal mice and determined the long-term effects of STZ injection in the mice. After receiving intraperitoneal injections of STZ at postnatal day 3 (P3), P4 and P8, male and female mice were hyperglycemic by week 4. A clear sex difference was found, with blood glucose levels in STZ-treated males remaining higher than those in STZ-treated females until week 23. Whereas STZ-treated males remained hyperglycemic until week 23, STZ-treated females did not have significantly higher glucose levels than control mice after week 18. Additionally, STZ-treated mice had neoplastic lesions in their livers by week 4, with a progression in the severity of these lesions until week 24. The results confirm that, in addition to pancreatic beta cell toxicity, STZ has an oncogenic effect on the liver when administered to neonates.

The FASEB Journal, 2003

In type 2 diabetes, glucose phosphorylation, a regulatory step in glucose utilization by skeletal... more 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 glucokinaseexpressing 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.

The FASEB Journal, 2003

Alterations in hepatic glucose metabolism play a key role in the development of the hyperglycemia... more 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.

Reproduction, 2002

Incubation of dog spermatozoa with 10 mmol l -1 glucose or fructose rapidly increased the intrace... more Incubation of dog spermatozoa with 10 mmol l -1 glucose or fructose rapidly increased the intracellular content of glucose 6-phosphate and fructose 6-phosphate, although the effect of fructose was greater. These effects were correlated with increases in ATP, ribose 5-phosphate and glycogen contents, and in the rates of formation of Llactate and CO 2 . In all cases, except for ATP and glycogen, the effect of fructose was greater than that of glucose. The total hexokinase activity of the crude extracts of dog spermatozoa was more sensitive to fructose than to glucose at lower concentrations (0.1-3.0 mmol l -1 ). Both monosaccharides induced a fast and intense increase in the overall tyrosine phosphorylation of dog spermatozoa, although their specific induced-phosphorylation patterns differed slightly. Glut 3 and Glut 5 hexose transporters were the main hexose transporters in dog spermatozoa; however, other possible SGLT family-related hexose transporters were also localized. These data indicate that, at concentrations from 1 mmol l -1 to 10 mmol l -1 , fructose has a stronger effect than glucose on hexose metabolism of dog spermatozoa. These differences appear to be related to variations in the sensitivity of hexokinase activity. Moreover, the differential hexose metabolism induced by the two sugars had distinct effects on the function of dog spermatozoa, as revealed by the diverse patterns of tyrosine phosphorylation.

Molecular Therapy - MOL THER, 2004

Type 1 diabetes results from autoimmune destruction of pancreatic β cells. This process might be ... more Type 1 diabetes results from autoimmune destruction of pancreatic β cells. This process might be reverted by genetically engineering endocrine pancreas in vivo to express factors that induce β-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 pancreas in vivo are required. Here we show that mouse pancreatic β-cells were efficiently transduced, and they expressed β-galactosidase after systemic injection of adenovirus with clamped hepatic circulation. Seven days after vector administration about 70% of pancreatic islets showed β-gal expression. In these islets, about 20% of the β-cells were transduced by the adenoviruses. Furthermore, scattered acinar cells expressing β-gal were also observed. In addition to mouse, we also examined the ability of adenovirus to transduce dog panc...

Molecular Therapy, 2006

Gene therapy may provide new treatments for severe pancreatic disorders. However, gene transfer t... more Gene therapy may provide new treatments for severe pancreatic disorders. However, gene transfer to the pancreas is difficult because of its anatomic location and structure, and pancreatitis is a serious concern. Like the human pancreas, the canine pancreas is compact, with similar vascularization and lobular structure. It is therefore a suitable model in which to assess gene transfer strategies. Here we examined the ability of adenoviral vectors to transfer genes into the pancreas of dogs in which pancreatic circulation had been clamped. Adenoviruses carrying the B-galactosidase (B-gal) gene were injected into the pancreatic-duodenal vein and the clamp was released 10 min later. These dogs showed B-gal-positive cells throughout the pancreas, with no evidence of pancreatic damage. B-Gal was expressed mainly in acinar cells, but also in ducts and islets. Moreover, transduction was prominent in connective tissue of the lobe septa. B-Gal expression in the exocrine pancreas of a diabetic dog was also found to be similar to that observed in healthy dogs. Thus, efficient gene transfer to canine pancreas in vivo may be achieved by adenovirus injection after clamping pancreatic circulation. This technique may be used to assay new gene therapy approaches for diabetes mellitus and other pancreatic disorders.

Human Gene Therapy, 2004

Type 1 diabetes results from autoimmune destruction of pancreatic beta cells. This process might ... more 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 ␤-galactosidase after systemic injection of adenovirus into mice with clamped hepatic circulation. Seven days after vector administration about 70% of pancreatic islets showed ␤-galactosidase expression, with an average of about 20% of the cells within positive islets being transduced. In addition, scattered acinar cells expressing ␤-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. 805

Human Gene Therapy, 2002

Type 1 diabetic patients depend on insulin replacement therapy. However, chronic hyperglycemia du... more 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.

FEBS Letters, 2004

The presence of a high-Km hexokinase activity was tested in both dog and boar spermatozoa. Hexoki... more The presence of a high-Km 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-Km 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.

Diabetes, 2002

Insulin replacement therapy in type 1 diabetes is imperfect because proper glycemic control is no... more 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.

Diabetes, 2002

Adipose tissue glyceroneogenesis generates glycerol 3-phosphate, which could be used for fatty ac... more Adipose tissue glyceroneogenesis generates glycerol 3-phosphate, which could be used for fatty acid esterification during starvation. To determine whether increased glyceroneogenesis leads to increased fat mass and to explore the role of obesity in the development of insulin resistance, we overexpressed PEPCK, a regulatory enzyme of glyceroneogenesis in adipose tissue. Transgenic mice showed a chronic increase in PEPCK activity, which led to increased glyceroneogenesis, re-esterification of free fatty acids (FFAs), increased adipocyte size and fat mass, and higher body weight. In spite of increased fat mass, transgenic mice showed decreased circulating FFAs and normal leptin levels. Moreover, glucose tolerance and whole-body insulin sensitivity were preserved. Skeletal muscle basal and insulin-stimulated glucose uptake and glycogen content were not affected, suggesting that skeletal muscle insulin sensitivity is normal in transgenic obese mice. Our results indicate the key role of PEPCK in the control of FFA re-esterification in adipose tissue and, thus, the contribution of glyceroneogenesis to fat accumulation. Moreover, they suggest that higher fat mass without increased circulating FFAs does not lead to insulin resistance or type 2 diabetes in these mice.

Diabetes, 2006

Type 1 diabetic patients develop severe secondary complications because insulin treatment does no... more 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.

Biochemical and Biophysical Research Communications, 2002

Intra-testicular inoculation of an adenoviral vector carrying the fusion gene Aequorea victoria g... more Intra-testicular inoculation of an adenoviral vector carrying the fusion gene Aequorea victoria green fluorescence protein/rat-liver glycogen synthase (GFP/LGS) resulted in the presence of GFP/GLS in spermatozoa from 7 days to, at least, 16 days after inoculation. The GFP/LGS was detected in the sperm heads after an ''in vitro'' fertilization procedure, either before or after the oocyte penetration. Our results indicate that spermatozoa carrying GFP/LGS protein conserved their fertilizing ability and were also detectable after oocyte penetration. This technique will allow to develop an easy system to follow the fate of mature sperm proteins.

Human Gene Therapy, 2000

Chronic hyperglycemia is responsible for diabetes-specific microvascular and macrovascular compli... more 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.

Lab animal, 2014

Repeated, low-dose administration of streptozotocin (STZ) is widely used to induce insulin-depend... more Repeated, low-dose administration of streptozotocin (STZ) is widely used to induce insulin-dependent diabetes mellitus in mice. The authors adapted this method using neonatal mice and determined the long-term effects of STZ injection in the mice. After receiving intraperitoneal injections of STZ at postnatal day 3 (P3), P4 and P8, male and female mice were hyperglycemic by week 4. A clear sex difference was found, with blood glucose levels in STZ-treated males remaining higher than those in STZ-treated females until week 23. Whereas STZ-treated males remained hyperglycemic until week 23, STZ-treated females did not have significantly higher glucose levels than control mice after week 18. Additionally, STZ-treated mice had neoplastic lesions in their livers by week 4, with a progression in the severity of these lesions until week 24. The results confirm that, in addition to pancreatic beta cell toxicity, STZ has an oncogenic effect on the liver when administered to neonates.

The FASEB Journal, 2003

In type 2 diabetes, glucose phosphorylation, a regulatory step in glucose utilization by skeletal... more 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 glucokinaseexpressing 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.

The FASEB Journal, 2003

Alterations in hepatic glucose metabolism play a key role in the development of the hyperglycemia... more 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.

Reproduction, 2002

Incubation of dog spermatozoa with 10 mmol l -1 glucose or fructose rapidly increased the intrace... more Incubation of dog spermatozoa with 10 mmol l -1 glucose or fructose rapidly increased the intracellular content of glucose 6-phosphate and fructose 6-phosphate, although the effect of fructose was greater. These effects were correlated with increases in ATP, ribose 5-phosphate and glycogen contents, and in the rates of formation of Llactate and CO 2 . In all cases, except for ATP and glycogen, the effect of fructose was greater than that of glucose. The total hexokinase activity of the crude extracts of dog spermatozoa was more sensitive to fructose than to glucose at lower concentrations (0.1-3.0 mmol l -1 ). Both monosaccharides induced a fast and intense increase in the overall tyrosine phosphorylation of dog spermatozoa, although their specific induced-phosphorylation patterns differed slightly. Glut 3 and Glut 5 hexose transporters were the main hexose transporters in dog spermatozoa; however, other possible SGLT family-related hexose transporters were also localized. These data indicate that, at concentrations from 1 mmol l -1 to 10 mmol l -1 , fructose has a stronger effect than glucose on hexose metabolism of dog spermatozoa. These differences appear to be related to variations in the sensitivity of hexokinase activity. Moreover, the differential hexose metabolism induced by the two sugars had distinct effects on the function of dog spermatozoa, as revealed by the diverse patterns of tyrosine phosphorylation.

Molecular Therapy - MOL THER, 2004

Type 1 diabetes results from autoimmune destruction of pancreatic β cells. This process might be ... more Type 1 diabetes results from autoimmune destruction of pancreatic β cells. This process might be reverted by genetically engineering endocrine pancreas in vivo to express factors that induce β-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 pancreas in vivo are required. Here we show that mouse pancreatic β-cells were efficiently transduced, and they expressed β-galactosidase after systemic injection of adenovirus with clamped hepatic circulation. Seven days after vector administration about 70% of pancreatic islets showed β-gal expression. In these islets, about 20% of the β-cells were transduced by the adenoviruses. Furthermore, scattered acinar cells expressing β-gal were also observed. In addition to mouse, we also examined the ability of adenovirus to transduce dog panc...

Molecular Therapy, 2006

Gene therapy may provide new treatments for severe pancreatic disorders. However, gene transfer t... more Gene therapy may provide new treatments for severe pancreatic disorders. However, gene transfer to the pancreas is difficult because of its anatomic location and structure, and pancreatitis is a serious concern. Like the human pancreas, the canine pancreas is compact, with similar vascularization and lobular structure. It is therefore a suitable model in which to assess gene transfer strategies. Here we examined the ability of adenoviral vectors to transfer genes into the pancreas of dogs in which pancreatic circulation had been clamped. Adenoviruses carrying the B-galactosidase (B-gal) gene were injected into the pancreatic-duodenal vein and the clamp was released 10 min later. These dogs showed B-gal-positive cells throughout the pancreas, with no evidence of pancreatic damage. B-Gal was expressed mainly in acinar cells, but also in ducts and islets. Moreover, transduction was prominent in connective tissue of the lobe septa. B-Gal expression in the exocrine pancreas of a diabetic dog was also found to be similar to that observed in healthy dogs. Thus, efficient gene transfer to canine pancreas in vivo may be achieved by adenovirus injection after clamping pancreatic circulation. This technique may be used to assay new gene therapy approaches for diabetes mellitus and other pancreatic disorders.

Human Gene Therapy, 2004

Type 1 diabetes results from autoimmune destruction of pancreatic beta cells. This process might ... more 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 ␤-galactosidase after systemic injection of adenovirus into mice with clamped hepatic circulation. Seven days after vector administration about 70% of pancreatic islets showed ␤-galactosidase expression, with an average of about 20% of the cells within positive islets being transduced. In addition, scattered acinar cells expressing ␤-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. 805

Human Gene Therapy, 2002

Type 1 diabetic patients depend on insulin replacement therapy. However, chronic hyperglycemia du... more 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.

FEBS Letters, 2004

The presence of a high-Km hexokinase activity was tested in both dog and boar spermatozoa. Hexoki... more The presence of a high-Km 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-Km 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.

Diabetes, 2002

Insulin replacement therapy in type 1 diabetes is imperfect because proper glycemic control is no... more 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.

Diabetes, 2002

Adipose tissue glyceroneogenesis generates glycerol 3-phosphate, which could be used for fatty ac... more Adipose tissue glyceroneogenesis generates glycerol 3-phosphate, which could be used for fatty acid esterification during starvation. To determine whether increased glyceroneogenesis leads to increased fat mass and to explore the role of obesity in the development of insulin resistance, we overexpressed PEPCK, a regulatory enzyme of glyceroneogenesis in adipose tissue. Transgenic mice showed a chronic increase in PEPCK activity, which led to increased glyceroneogenesis, re-esterification of free fatty acids (FFAs), increased adipocyte size and fat mass, and higher body weight. In spite of increased fat mass, transgenic mice showed decreased circulating FFAs and normal leptin levels. Moreover, glucose tolerance and whole-body insulin sensitivity were preserved. Skeletal muscle basal and insulin-stimulated glucose uptake and glycogen content were not affected, suggesting that skeletal muscle insulin sensitivity is normal in transgenic obese mice. Our results indicate the key role of PEPCK in the control of FFA re-esterification in adipose tissue and, thus, the contribution of glyceroneogenesis to fat accumulation. Moreover, they suggest that higher fat mass without increased circulating FFAs does not lead to insulin resistance or type 2 diabetes in these mice.

Diabetes, 2006

Type 1 diabetic patients develop severe secondary complications because insulin treatment does no... more 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.

Biochemical and Biophysical Research Communications, 2002

Intra-testicular inoculation of an adenoviral vector carrying the fusion gene Aequorea victoria g... more Intra-testicular inoculation of an adenoviral vector carrying the fusion gene Aequorea victoria green fluorescence protein/rat-liver glycogen synthase (GFP/LGS) resulted in the presence of GFP/GLS in spermatozoa from 7 days to, at least, 16 days after inoculation. The GFP/LGS was detected in the sperm heads after an ''in vitro'' fertilization procedure, either before or after the oocyte penetration. Our results indicate that spermatozoa carrying GFP/LGS protein conserved their fertilizing ability and were also detectable after oocyte penetration. This technique will allow to develop an easy system to follow the fate of mature sperm proteins.

Human Gene Therapy, 2000

Chronic hyperglycemia is responsible for diabetes-specific microvascular and macrovascular compli... more 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.