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.     

ATP-sensitive K+ channel signaling in glucokinase-deficient diabetes

As the rate-limiting controller of glucose metabolism, glucokinase represents the primary beta-cell "glucose sensor." Inactivation of both glucokinase (GK) alleles results in permanent neonatal diabetes; inactivation of a single allele causes maturity-onset diabetes of the young type 2 (MODY-2). Similarly, mice lacking both alleles (GK(-/-)) exhibit severe neonatal diabetes and die within a week, whereas heterozygous GK(+/-) mice exhibit markedly impaired glucose tolerance and diabetes, resembling MODY-2. Glucose metabolism increases the cytosolic [ATP]-to-[ADP] ratio, which closes ATP-sensitive K(+) channels (K(ATP) channels), leading to membrane depolarization, Ca(2+) entry, and insulin exocytosis. Glucokinase insufficiency causes defective K(ATP) channel regulation, which may underlie the impaired secretion. To test this prediction, we crossed mice lacking neuroendocrine glucokinase (nGK(+/-)) with mice lacking K(ATP) channels (Kir6.2(-/-)). Kir6.2 knockout rescues perinatal lethality of nGK(-/-), although nGK(-/-)Kir6.2(-/-) animals are postnatally diabetic and still die prematurely. nGK(+/-) animals are diabetic on the Kir6.2(+/+) background but only mildly glucose intolerant on the Kir6.2(-/-) background. In the presence of glutamine, isolated nGK(+/-)Kir6.2(-/-) islets show improved insulin secretion compared with nGK(+/-)Kir6.2(+/+). The significant abrogation of nGK(-/-) and nGK(+/-) phenotypes in the absence of K(ATP) demonstrate that a major factor in glucokinase deficiency is indeed altered K(ATP) signaling. The results have implications for understanding and therapy of glucokinase-related diabetes.     

Restoration of glucokinase expression in the liver normalizes postprandial glucose disposal in mice with hepatic deficiency of PDK1

Phosphoinositide-dependent kinase-1 (PDK1) is implicated in the metabolic effects of insulin as a key mediator of phosphoinositide 3-kinase-dependent signaling. Here we show that mice with liver-specific PDK1 deficiency manifest various defects in the metabolic actions of insulin in the liver as well as a type 2 diabetes-like phenotype characterized by marked hyperinsulinemia and postprandial hyperglycemia. The hepatic abundance of glucokinase, an important determinant of glucose flux and glucose-evoked signaling in hepatocytes, was substantially reduced in these mice. Restoration of hepatic glucokinase expression, with the use of an adenoviral vector, induced insulin-like effects in the liver and almost completely normalized the fasting hyperinsulinemia and postprandial hyperglycemia in these animals. These results indicate that, if the hepatic abundance of glucokinase is maintained, ingested glucose is normally disposed of even in the absence of acute activation of proximal insulin signaling, such as the activation of Akt, in the liver.     

Metabolic impact of glucokinase overexpression in liver: lowering of blood glucose in fed rats is accompanied by hyperlipidemia.

The balance between hepatic glucose uptake and production is perturbed in both major forms of diabetes. It has been suggested that pharmacologic or genetic methods for enhancing glucokinase (GK) enzymatic activity in liver might be a means of increasing glucose disposal and lowering blood glucose in diabetic patients. To better evaluate this possibility, we used a recombinant adenovirus containing the cDNA encoding GK (AdCMV-GKL) to achieve overexpression of the enzyme at different levels in liver of normal rats. In a first set of experiments, in rats fasted for 18 h, AdCMV-GKL infusion caused a 211% increase in hepatic GK activity relative to animals infused with a control virus (AdCMV-betaGAL). AdCMV-GKL-treated fasted rats exhibited no significant changes in circulating glucose, free fatty acids (FFAs), lactate, beta-hydroxybutyrate, or insulin levels relative to controls, whereas triglyceride (TG) levels were slightly increased (53%). In a second set of studies, in rats fed ad libitum, GK was overexpressed in liver by 3- and 6.4-fold. Animals with the lower degree of GK overexpression exhibited no significant changes in circulating glucose, FFAs, insulin, TG, or lactate levels relative to controls that received a virus encoding a catalytically inactive mutant GK (AdCMV-GK203), but did show a modest increase in lactate (58%) relative to AdCMV-betaGAL-infused controls. In contrast, the higher level of GK overexpression caused a 38% decrease in blood glucose levels and a 67% decrease in circulating insulin levels relative to AdCMV-GK203-infused animals. The decline in glucose levels was accompanied by a 190% increase in circulating TG and a 310% increase in circulating FFAs; total plasma cholesterol was unaffected. Finally, fasted animals treated with AdCMV-GKL had 5.4 times as much liver glycogen as AdCMV-betaGAL-treated controls; no significant increases in liver glycogen were observed at either level of GK overexpression in ad libitum-fed rats relative to fed controls. In sum, levels of hepatic GK overexpression associated with a decline in blood glucose are accompanied by equally dramatic increases in FFAs and TG, raising concerns about manipulation of liver GK activity as a viable strategy for treatment of diabetes.     

Altered hepatic and muscle substrate utilization provoked by GLUT4 ablation

Studies were conducted to explore altered substrate utilization and metabolism in GLUT4 null mice. Liver fatty acid synthase mRNA and fatty acid synthesis rates were dramatically increased in GLUT4 null mice compared with control mice and were supported by increased rates of the pentose phosphate pathway oxidative phase and sterol regulatory binding protein mRNA expression. Increased GLUT2 protein content, glucokinase mRNA, and glucose-6-phosphate in GLUT4 null mice may provide substrate for the enhanced fatty acid synthesis. Increased fatty acid synthesis, however, did not lead to hepatic triglyceride accumulation in GLUT4 null mice because of increased hepatic triglyceride secretion rates. GLUT4 null mice rapidly cleared orally administered olive oil, had reduced serum triglyceride concentrations in the fed and the fasted state, and increased skeletal muscle lipoprotein lipase when compared with controls. Oleate oxidation rates were increased in GLUT4 null skeletal muscle in association with mitochondrial hyperplasia/hypertrophy. This study demonstrated that GLUT4 null mice had increased hepatic glucose uptake and conversion into triglyceride for subsequent use by muscle. The ability of GLUT4 null mice to alter hepatic carbohydrate and lipid metabolism to provide proper nutrients for peripheral tissues may explain (in part) their ability to resist diabetes when fed a normal diet.     

Intracerebroventricular neuropeptide Y infusion precludes inhibition of glucose and VLDL production by insulin

Recent evidence demonstrates that hypothalamic insulin signaling is required for inhibition of endogenous glucose production. The downstream mechanisms that are responsible for the effects of hypothalamic insulin receptor activation on hepatic fuel flux remain to be determined. To establish whether downregulation of neuropeptide Y (NPY) release by insulin is mandatory for its capacity to suppress glucose production, we examined the effects of a continuous intracerebroventricular (ICV) infusion of NPY (10 microg/h for 3-5 h) on glucose flux during a hyperinsulinemic-euglycemic clamp in mice. We also evaluated the effects of ICV NPY administration on free fatty acid and glycerol flux and VLDL production in this experimental context. In basal conditions, none of the metabolic parameters was affected by NPY infusion. In hyperinsulinemic conditions, peripheral glucose disposal was not different between vehicle- and NPY-infused animals. In contrast, hyperinsulinemia suppressed endogenous glucose production by approximately 8% vs. 30% in NPY- vs. vehicle-infused mice, respectively (P < 0.05). Also, VLDL production was significantly higher during hyperinsulinemia in NPY- compared with vehicle-infused mice (97.5 +/- 18.0 vs. 54.7 +/- 14.9 micromol. kg(-1). h(-1); P < 0.01). These data suggest that the neurophysiological action of insulin to downregulate hypothalamic NPY release is a prerequisite for its ability to suppress hepatic fuel production, whereas it is not mandatory for its capacity to modulate glucose disposal or lipolysis.     

The effect of pancreatic polypeptide on glucose disposal after surgical alterations of the pancreas.

Surgical alterations of the pancreas result in anatomic changes that can affect postoperative glucose metabolism. Pancreas transplantation results in reduction of beta-cell mass, systemic release of insulin, and denervation. The authors hypothesized that such alterations affect peripheral glucose disposal to induce an "insensitivity" to endogenously (systemically) released insulin. Additionally, they hypothesized that surgically induced deficiency of the postprandial hormone, pancreatic polypeptide, might contribute to altered glucose disposal. The authors studied two surgical models in dogs known to be devoid of pancreatic polypeptide--70% proximal pancreatectomy (PPx) and PPx plus distal pancreas autotransplantation (PAT/B). Oral glucose challenge and euglycemic hyperinsulinemic clamp studies were performed before and after a 16-day "pulsed" infusion of pancreatic polypeptide. Both surgical procedures resulted in elevations in the integrated glucose response after oral glucose, which was not affected by pancreatic polypeptide infusion. Euglycemic clamp studies showed decreased hepatic glucose output (Ra) and overall glucose disposal (Rd) in the fasted state for both surgical groups. The transplant animals demonstrated significant decreases in Rd during the hyperinsulinemic challenge (3.2 +/- 0.01 versus 5.7 +/- 0.01 mg/kg/minute at 60 to 120 minutes for PAT/B versus control). After 16 days of pancreatic polypeptide infusion, however, basal Ra, as well as basal and 60- to 120-minute Rd values, were returned to control values in the transplant group. The authors conclude that pancreas transplantation results in altered glucose disposal, possibly due to an altered effectiveness of systemically released insulin. They conclude that pancreatic polypeptide is an important modulator of peripheral insulin action. Therefore, the role of pancreatic polypeptide must be taken into account when evaluating postoperative glucose metabolism in canine models of pancreas transplantation.     

Lipin deficiency impairs diurnal metabolic fuel switching

Fatty liver is a common feature of both obesity and lipodystrophy, reflecting compromised adipose tissue function. The lipin-deficient fatty liver dystrophy (fld) mouse is an exception, as there is lipodystrophy without a fatty liver. Using a combination of indirect calorimetry and stable-isotope flux phenotyping, we determined that fld mice exhibit abnormal fuel utilization throughout the diurnal cycle, with increased glucose oxidation near the end of the fasting period and increased fatty acid oxidation during the feeding period. The mechanisms underlying these alterations include a twofold increase compared with wild-type mice in tissue glycogen storage during the fed state, a 40% reduction in hepatic glucose production in the fasted state, and a 27-fold increase in de novo fatty acid synthesis in liver during the fed state. Thus, the inability to store energy in adipose tissue in the fld mouse leads to a compensatory increase in glycogen storage for use during the fasting period and reliance upon hepatic fatty acid synthesis to provide fuel for peripheral tissues during the fed state. The increase in hepatic fatty acid synthesis and peripheral utilization provides a potential mechanism to ameliorate fatty liver in the fld that would otherwise occur as a consequence of adipose tissue dysfunction.     

Reduction of hepatic and adipose tissue glucocorticoid receptor expression with antisense oligonucleotides improves hyperglycemia and hyperlipidemia in diabetic rodents without causing systemic glucocorticoid antagonism

Glucocorticoids (GCs) increase hepatic gluconeogenesis and play an important role in the regulation of hepatic glucose output. Whereas systemic GC inhibition can alleviate hyperglycemia in rodents and humans, it results in adrenal insufficiency and stimulation of the hypothalamic-pituitary-adrenal axis. In the present study, we used optimized antisense oligonucleotides (ASOs) to cause selective reduction of the glucocorticoid receptor (GCCR) in liver and white adipose tissue (WAT) and evaluated the resultant changes in glucose and lipid metabolism in several rodent models of diabetes. Treatment of ob/ob mice with GCCR ASOs for 4 weeks resulted in approximately 75 and approximately 40% reduction in GCCR mRNA expression in liver and WAT, respectively. This was accompanied by approximately 65% decrease in fed and approximately 30% decrease in fasted glucose levels, a 60% decrease in plasma insulin concentration, and approximately 20 and 35% decrease in plasma resistin and tumor necrosis factor-alpha levels, respectively. Furthermore, GCCR ASO reduced hepatic glucose production and inhibited hepatic gluconeogenesis in liver slices from basal and dexamethasone-treated animals. In db/db mice, a similar reduction in GCCR expression caused approximately 40% decrease in fed and fasted glucose levels and approximately 50% reduction in plasma triglycerides. In ZDF and high-fat diet-fed streptozotocin-treated (HFD-STZ) rats, GCCR ASO treatment caused approximately 60% reduction in GCCR expression in the liver and WAT, which was accompanied by a 40-70% decrease in fasted glucose levels and a robust reduction in plasma triglyceride, cholesterol, and free fatty acids. No change in circulating corticosterone levels was seen in any model after GCCR ASO treatment. To further demonstrate that GCCR ASO does not cause systemic GC antagonism, normal Sprague-Dawley rats were challenged with dexamethasone after treating with GCCR ASO. Dexamethasone increased the expression of GC-responsive genes such as PEPCK in the liver and decreased circulating lymphocytes. GCCR ASO treatment completely inhibited the increase in dexamethasone-induced PEPCK expression in the liver without causing any change in the dexamethasone-induced lymphopenia. These studies demonstrate that tissue-selective GCCR antagonism with ASOs may be a viable therapeutic strategy for the treatment of the metabolic syndrome.     

Impact of the liver-specific expression of SHIP2 (SH2-containing inositol 5'-phosphatase 2) on insulin signaling and glucose metabolism in mice

We investigated the role of hepatic SH2-containing inositol 5'-phosphatase 2 (SHIP2) in glucose metabolism in mice. Adenoviral vectors encoding wild-type SHIP2 (WT-SHIP2) and a dominant-negative SHIP2 (DeltaIP-SHIP2) were injected via the tail vein into db/+m and db/db mice, respectively. Four days later, amounts of hepatic SHIP2 protein were increased by fivefold. Insulin-induced phosphorylation of Akt in liver was impaired in WT-SHIP2-expressing db/+m mice, whereas the reduced phosphorylation was restored in DeltaIP-SHIP2-expressing db/db mice. The abundance of mRNA for glucose-6-phosphatase (G6Pase) and PEPCK was increased, that for glucokinase (GK) was unchanged, and that for sterol regulatory element-binding protein 1 (SREBP)-1 was decreased in hepatic WT-SHIP2-overexpressing db/+m mice. The increased expression of mRNA for G6Pase and PEPCK was partly suppressed, that for GK was further enhanced, and that for SREBP1 was unaltered by the expression of DeltaIP-SHIP2 in db/db mice. The hepatic expression did not affect insulin signaling in skeletal muscle and fat tissue in both mice. After oral glucose intake, blood glucose levels and plasma insulin concentrations were elevated in WT-SHIP2-expressing db/+m mice, while elevated values were decreased by the expression of DeltaIP-SHIP2 in db/db mice. These results indicate that hepatic SHIP2 has an impact in vivo on the glucose metabolism in both physiological and diabetic states possibly by regulating hepatic gene expression.     

Glucose metabolism in mice lacking muscle glycogen synthase

Glycogen is an important component of whole-body glucose metabolism. MGSKO mice lack skeletal muscle glycogen due to disruption of the GYS1 gene, which encodes muscle glycogen synthase. MGSKO mice were 5-10% smaller than wild-type littermates with less body fat. They have more oxidative muscle fibers and, based on the activation state of AMP-activated protein kinase, more capacity to oxidize fatty acids. Blood glucose in fed and fasted MGSKO mice was comparable to wild-type littermates. Serum insulin was lower in fed but not in fasted MGSKO animals. In a glucose tolerance test, MGSKO mice disposed of glucose more effectively than wild-type animals and had a more sustained elevation of serum insulin. This result was not explained by increased conversion to serum lactate or by enhanced storage of glucose in the liver. However, glucose infusion rate in a euglycemic-hyperinsulinemic clamp was normal in MGSKO mice despite diminished muscle glucose uptake. During the clamp, MGSKO animals accumulated significantly higher levels of liver glycogen as compared with wild-type littermates. Although disruption of the GYS1 gene negatively affects muscle glucose uptake, overall glucose tolerance is actually improved, possibly because of a role for GYS1 in tissues other than muscle.     

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.     

Insulin-independent induction of sterol regulatory element-binding protein-1c expression in the livers of streptozotocin-treated mice

Insulin and glucose together have been previously shown to regulate hepatic sterol regulatory element-binding protein (SREBP)-1c expression. We sought to explore the nutritional regulation of lipogenesis through SREBP-1c induction in a setting where effects of sugars versus insulin could be distinguished. To do so, mice were insulin depleted by streptozotocin (STZ) administration and subjected to a fasting-refeeding protocol with glucose, fructose, or sucrose. Unexpectedly, the insulin-depleted mice exhibited a marked induction of SREBP-1c on all sugars, and this increase in SREBP-1c was even more dramatic than in the non-STZ-administered controls. The time course of changes in SREBP-1 induction varied depending on the type of sugars in both control and STZ-administered mice. Glucose refeeding gave a peak of SREBP-1c induction, whereas fructose refeeding caused slow and gradual increments, and sucrose refeeding fell between these two responses. Expression of various lipogenic enzymes were also gradually increased over time, irrespective of the types of sugars, with greater intensities in STZ-administered than in nontreated mice. In contrast, induction of hepatic glucokinase and suppression of phoshoenolpyruvate carboxykinase were insulin dependent in an early refed state. These data clearly demonstrate that nutritional regulation of SREBP-1c and lipogenic genes may be completely independent of insulin as long as sufficient carbohydrates are available.     

Phenotypic correction of diabetic mice by adenovirus-mediated glucokinase expression

Hyperglycemia of diabetes is caused in part by perturbation of hepatic glucose metabolism. Hepatic glucokinase (GK) is an important regulator of glucose storage and disposal in the liver. GK levels are lowered in patients with maturity-onset diabetes of the young and in some diabetic animal models. Here, we explored the adenoviral vector-mediated overexpression of GK in a diet-induced murine model of type 2 diabetes as a treatment for diabetes. Diabetic mice were treated by intravenous administration with an E1/E2a/E3-deleted adenoviral vector encoding human hepatic GK (Av3hGK). Two weeks posttreatment, the Av3hGK-treated diabetic mice displayed normalized fasting blood glucose levels (95 +/- 4.8 mg/dl; P < 0.001) when compared with Av3Null (135 +/- 5.9 mg/dl), an analogous vector lacking a transgene, and vehicle-treated diabetic mice (134 +/- 8 mg/dl). GK treatment also resulted in lowered insulin levels (632 +/- 399 pg/ml; P < 0.01) compared with the control groups (Av3Null, 1,803 +/- 291 pg/ml; vehicle, 1,861 +/- 392 pg/ml), and the glucose tolerance of the Av3hGK-treated diabetic mice was normalized. No significant increase in plasma or hepatic triglycerides, or plasma free fatty acids was observed in the Av3hGK-treated mice. These data suggest that overexpression of GK may have a therapeutic potential for the treatment of type 2 diabetes.     

Improved glycemic control in C57Bl/KsJ (db/db) mice after treatment with the thermogenic beta-adrenoceptor agonist, BRL 26830

BRL 26830, (R*,R*)-(+/-)-methyl-4-(2-[(2-hydroxy-2-phenylethyl)amino]propyl)-benzoa te, is a new type of beta-adrenoceptor receptor agonist that combines antihyperglycemic and thermogenic properties. In C57Bl/KsJ db/db mice, treatment with BRL 26830 (50 mg of the hemifumarate salt/kg diet) decreased blood glucose concentration and normalized water intake. As judged by the normalization of polydipsia, BRL 26830 was effective within 2 days and the effect was maintained throughout a treatment period of up to 11 wk. Treatment of db/db mice with BRL 26830 resulted in an increase in both plasma and pancreatic insulin concentrations and a partial restoration of first-phase insulin secretion by the isolated, perfused pancreas in response to a high (16.7 mM) glucose pulse. Given acutely, BRL 26830 increased energy expenditure in both fed and fasted db/db mice. When given chronically, BRL 26830 increased significantly the dietary and thermoregulatory component of metabolic rate. It is suggested that the antidiabetic and thermogenic properties of BRL 26830 are linked and that blood glucose acts either directly or indirectly as a substrate for thermogenesis.     

Hormone-sensitive lipase has a role in lipid signaling for insulin secretion but is nonessential for the incretin action of glucagon-like peptide 1

We previously reported decreased glucose-stimulated insulin secretion (GSIS) in hormone-sensitive lipase-null mice (HSL(-/-)), both in vivo and in vitro. The focus of the current study was to gain further insight into the signaling role and regulation of lipolysis in islet tissue. The effect of glucagon-like peptide 1 (GLP-1) on GSIS was also studied, as GLP-1 could augment GSIS via protein kinase A activation of HSL and lipolysis. Freshly isolated islets from fasted and fed male HSL(-/-) and wild-type (HSL(+/+)) mice were studied at ages 4 and 7 months. Neutral cholesteryl ester hydrolase activity was markedly reduced in islets from both 4- and 7-month-old male HSL(-/-) mice, whereas a marked deficiency in triglyceride lipase activity became evident only in the older mice. The deficiencies in lipase activities were associated with higher islet triglyceride content and reduced lipolysis at basal glucose levels. Lipolysis was stimulated by high glucose in islets of both wild-type and HSL-null mice. Severe deficiencies in GSIS were found, but only in islets from 7-month-old, fasted, male HSL(-/-) mice. GSIS was less affected in 4-month-old fasted male HSL(-/-) mice and not reduced in female mice. Exogenous delivery of free fatty acids (FFAs) rescued GSIS, supporting the view that the lack of endogenous FFA supply for lipid-signaling processes in HSL(-/-) mice was responsible for the loss of GSIS. GLP-1 also rescued GSIS in HSL(-/-) mice, indicating that signaling via HSL is not a major pathway for its incretin effect. Thus, the secretory phenotype of HSL-null mice is gender dependent, increases with age, and is influenced by the nutritional state. Under most circumstances, the major determinant of lipolytic flux in the beta-cell involves an enzyme(s) other than HSL that is acutely activated by glucose. Our results support the view that the availability of endogenous FFA through HSL and an additional enzyme(s) is involved in providing lipid moieties for beta-cell signaling for secretion in response to glucose.