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.     

Glucose toxicity is responsible for the development of impaired regulation of endogenous glucose production and hepatic glucokinase in Zucker diabetic fatty rats

The effect of restoration of normoglycemia by a novel sodium-dependent glucose transporter inhibitor (T-1095) on impaired hepatic glucose uptake was examined in 14-week-old Zucker diabetic fatty (ZDF) rats. The nontreated group exhibited persistent endogenous glucose production (EGP) despite marked hyperglycemia. Gluconeogenesis and glucose cycling (GC) were responsible for 46 and 51% of glucose-6-phosphatase (G6Pase) flux, respectively. Net incorporation of plasma glucose into hepatic glycogen was negligible. Glucokinase (GK) and its inhibitory protein, GK regulatory protein (GKRP), were colocalized in the cytoplasm of hepatocytes. At day 7 of drug administration, EGP was slightly reduced, but G6Pase flux and GC were markedly lower compared with the nontreated group. In this case, GK and GKRP were colocalized in the nuclei of hepatocytes. When plasma glucose and insulin levels were raised during a clamp, EGP was completely suppressed and GC, glycogen synthesis from plasma glucose, and the fractional contribution of plasma glucose to uridine diphosphoglucose flux were markedly increased. GK, but not GKRP, was translocated from the nucleus to the cytoplasm. Glucotoxicity may result in the blunted response of hepatic glucose flux to elevated plasma glucose and/or insulin associated with impaired regulation of GK by GKRP in ZDF rats.     

Stimulation of hepatocyte glucose metabolism by novel small molecule glucokinase activators

Glucokinase (GK) has a major role in the control of blood glucose homeostasis and is a strong potential target for the pharmacological treatment of type 2 diabetes. We report here the mechanism of action of two novel and potent direct activators of GK: 6-[(3-isobutoxy-5-isopropoxybenzoyl)amino]nicotinic acid(GKA1) and 5-([3-isopropoxy-5-[2-(3-thienyl)ethoxy]benzoyl]amino)-1,3,4-thiadiazole-2-carboxylic acid(GKA2), which increase the affinity of GK for glucose by 4- and 11-fold, respectively. GKA1 increased the affinity of GK for the competitive inhibitor mannoheptulose but did not affect the affinity for the inhibitors palmitoyl-CoA and the endogenous 68-kDa regulator (GK regulatory protein [GKRP]), which bind to allosteric sites or to N-acetylglucosamine, which binds to the catalytic site. In hepatocytes, GKA1 and GKA2 stimulated glucose phosphorylation, glycolysis, and glycogen synthesis to a similar extent as sorbitol, a precursor of fructose 1-phosphate, which indirectly activates GK through promoting its dissociation from GKRP. Consistent with their effects on isolated GK, these compounds also increased the affinity of hepatocyte metabolism for glucose. GKA1 and GKA2 caused translocation of GK from the nucleus to the cytoplasm. This effect was additive with the effect of sorbitol and is best explained by a "glucose-like" effect of the GK activators in translocating GK to the cytoplasm. In conclusion, GK activators are potential antihyperglycemic agents for the treatment of type 2 diabetes through the stimulation of hepatic glucose metabolism by a mechanism independent of GKRP.     

The effect of global SSTR5 gene ablation on the endocrine pancreas and glucose regulation in aging mice

INTRODUCTION: The purpose of this study was to examine the effect of global gene ablation of SSTR5 on the endocrine pancreas, insulin secretion, and glucose tolerance in aging mice, as SSTR5 is a primary regulator of insulin secretion in the mouse pancreas. METHODS: Global SSTR5-/- mice were generated and genotypes were verified using Southern blot and RT-PCR. Glucose tolerance and in vivo insulin secretion in SSTR5-/- and WT mice were examined using intraperitoneal glucose tolerance test (IPGTT;1.2-2.0 mg/kg) at 3 and 12 months of age (n = 8 per group). Basal and glucose-stimulated insulin secretion in vitro was studied using the isolated perfused mouse pancreas model at 3 and 12 months. Pancreata were removed and levels of insulin, glucagon, somatostatin, and SSTR1 were studied using immunohistochemical analysis along with H&E staining of the pancreata. RESULTS: Genotyping verified the absence of SSTR5 in SSTR5-/- mice. IPGTT demonstrated that 3-month-old SSTR5-/- mice were glucose intolerant despite similar insulin secretion both in vivo and in vitro and enlarged islets. At 12 months of age, SSTR5-/- mice had basal hypoglycemia and improved glucose intolerance associated with hyperinsulinemia in vivo and in vitro and enlarged islets. SSTR5-/- mice had increased insulin clearance at 3 and 12 months of age. SSTR1 expression was significantly increased in islets at 3 months of age, but was nearly absent in islets at 12 months of age, as was somatostatin staining in SSTR5-/- mice. CONCLUSIONS: These results suggest that both SSTR5 and SSTR1 play a pivotal role in insulin secretion and glucose regulation in mice and that their regulatory effects are age-related.     

Muscarinic stimulation of pancreatic insulin and glucagon release is abolished in m3 muscarinic acetylcholine receptor-deficient mice

Pancreatic muscarinic acetylcholine receptors play an important role in stimulating insulin and glucagon secretion from islet cells. To study the potential role of the M(3) muscarinic receptor subtype in cholinergic stimulation of insulin release, we initially examined the effect of the muscarinic agonist, oxotremorine-M (Oxo-M), on insulin secretion from isolated pancreatic islets prepared from wild-type (WT) and M(3) receptor-deficient mice (M3(+/-) and M3(-/-) mice). At a stimulatory glucose level (16.7 mmol/l), Oxo-M strongly potentiated insulin output from islets of WT mice. Strikingly, this effect was completely abolished in islets from M3(-/-) mice and significantly reduced in islets from M3(+/-) mice. Additional in vitro studies showed that Oxo-M-mediated glucagon release was also virtually abolished in islets from M3(-/-) mice. Consistent with the in vitro data, in vivo studies showed that M3(-/-) mice displayed reduced serum insulin and plasma glucagon levels and a significantly blunted increase in serum insulin after an oral glucose load. Despite the observed impairments in insulin release, M3(-/-) mice showed significantly reduced blood glucose levels and even improved glucose tolerance, probably due to the reduction in plasma glucagon levels and the fact that M3(-/-) mice are hypophagic and lean. These findings provide important new insights into the metabolic roles of the M(3) muscarinic receptor subtype.     

Differential effects of hyperlipidemia on insulin secretion in islets of langerhans from hyperglycemic versus normoglycemic rats

Chronic elevations in plasma levels of fatty acids (FAs) adversely affect pancreatic beta-cell function in type 2 diabetes. In vitro, we have previously shown that deleterious effects of prolonged exposure of isolated islets to FAs were dependent on the presence of elevated glucose concentration. This led us to hypothesize that both hyperlipidemia and hyperglycemia must be present simultaneously for FAs to affect beta-cell function. To test this hypothesis in vivo, we administered a high-fat diet for 6 weeks to Goto-Kakizaki (GK) rats. High-fat feeding had no effect on insulin secretion, insulin content, or insulin mRNA levels in islets from normoglycemic Wistar rats. In contrast, high-fat feeding markedly impaired glucose-induced insulin secretion in islets from GK rats. High-fat feeding did not affect triglyceride (TG) content or the rate of glucose oxidation in islets. It was, however, accompanied by a twofold increase in uncoupling protein (UCP)-2 levels in GK rat islets. Insulin treatment completely normalized glucose-induced insulin secretion and prevented the increase in UCP-2 expression in islets from high-fat-fed GK rats. We conclude that hyperlipidemia induced by high-fat feeding affects insulin secretion in islets from hyperglycemic GK rats only, by a mechanism which may involve, at least in part, modulation of UCP-2 expression.     

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.     

Cell biology assessment of glucokinase mutations V62M and G72R in pancreatic beta-cells: evidence for cellular instability of catalytic activity

Mutations in the glucokinase (GK) gene cause defects in blood glucose homeostasis. In some cases (V62M and G72R), the phenotype cannot be explained by altered enzyme kinetics or protein instability. We used transient and stable expression of green fluorescent protein (GFP) GK chimaeras in MIN6 beta-cells to study the phenotype defect of V62M and G72R. GK activity in lysates of MIN6 cell lines stably expressing wild-type or mutant GFP GK showed the expected affinity for glucose and response to pharmacological activators, indicating the expression of catalytically active enzymes. MIN6 cells stably expressing GFP V62M or GFP G72R had a lower GK activity-to-GK immunoreactivity ratio and GK activity-to-GK mRNA ratio but not GK immunoreactivity-to-GK mRNA ratio than wild-type GFP GK. Heterologous expression of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2/FDP2) in cell lines increased GK activity for wild-type GK and V62M but not for G72R, whereas expression of liver GK regulatory protein (GKRP) increased GK activity for wild type but not V62M or G72R. Lack of interaction of these mutants with GKRP was also evident in hepatocyte transfections from the lack of nuclear accumulation. These results suggest that cellular loss of GK catalytic activity rather than impaired translation or enhanced protein degradation may account for the hyperglycemia in subjects with V62M and G72R mutations.     

Beta-cell insensitivity to glucose in the GK rat, a spontaneous nonobese model for type II diabetes

In early 1988, a colony of GK rats was started in Paris with progenitors issued from F35 of the original colony reported by Goto and Kakisaki. When studied longitudinally up to 8 mo, GK rats showed as early as 1 mo (weaning) significantly higher basal plasma glucose (9 mM) and insulin levels (doubled), altered glucose tolerance (intravenous glucose), and a very poor insulin secretory response to glucose in vivo compared with Wistar controls. Males and females were similarly affected. Studies of in vitro pancreatic function were carried out with the isolated perfused pancreas preparation. Compared with nondiabetic Wistar rats, GK rats at 2 mo showed a significantly increased basal insulin release, no insulin response to 16 mM glucose, and hyperresponse to 19 mM arginine. Pancreatic insulin stores were only 50% of that in Wistar rats. Perfusion of GK pancreases for 50 or 90 min with buffer containing no glucose partially improved the insulin response to 16 mM glucose and markedly diminished the response to 19 mM arginine, whereas the responses by Wistar pancreases were unchanged. These findings are similar to those reported in rats with non-insulin-dependent diabetes induced by neonatal streptozocin administration and support the concept that chronic elevation in plasma glucose may be responsible, at least in part, for the beta-cell desensitization to glucose in this model. The GK rat seems to be a valuable model for identifying the etiology of beta-cell desensitization to glucose.     

Persistent improvement of type 2 diabetes in the Goto-Kakizaki rat model by expansion of the beta-cell mass during the prediabetic period with glucagon-like peptide-1 or exendin-4

In the Goto-Kakizaki (GK) rat, a genetic model of type 2 diabetes, the neonatal beta-cell mass deficit is considered to be the primary defect leading to basal hyperglycemia, which is detectable for the first time 3 weeks after birth. We investigated in GK females the short- and the long-term effects of a treatment with glucagon-like peptide-1 (GLP-1) or its long-acting analog exendin-4 (Ex-4) during the first postnatal week (during the prediabetic period). GK rats were treated with daily injections of glucagon-like peptide-1 (400 microg x kg(-1) x day(-1)) or Ex-4 (3 microg x kg(-1) x day(-1)) from day 2 to day 6 after birth and were evaluated against Wistar and untreated GK rats. Under these conditions, on day 7 both treatments enhanced pancreatic insulin content and total beta-cell mass by stimulating beta-cell neogenesis and regeneration. Follow-up of biological characteristics from day 7 to adult age (2 months) showed that such a GLP-1 or Ex-4 treatment exerted long-term favorable influences on beta-cell mass and glycemic control at adult age. As compared to untreated GK rats, 2-month-old treated rats exhibited significantly decreased basal plasma glucose. Their glucose-stimulated insulin secretion, in vivo after intravenous glucose load or in vitro using isolated perfused pancreas, was slightly improved. This contributed at least partly to improve the in vivo plasma glucose disappearance rate, which was found to be increased in both treated GK groups compared to the untreated GK group. These findings in the GK model indicated, for the first time, that GLP-1 or Ex-4 treatment limited to the prediabetic period delays the installation and limits the severity of type 2 diabetes. Under these conditions, GLP-1 represents a unique tool because of its beta-cell replenishing effect in spontaneously diabetic rodents. It may prove to be an invaluable agent for the prevention of human type 2 diabetes.     

Platelet-derived growth factor stimulates glucose transport in skeletal muscles of transgenic mice specifically expressing platelet-derived growth factor receptor in the muscle, but it does not affect blood glucose levels

Insulin stimulates the disposal of blood glucose into skeletal muscle and adipose tissues by the translocation of GLUT4 from intracellular pools to the plasma membrane, and consequently the concentration of blood glucose levels decreases rapidly in vivo. Phosphatidylinositol (PI) 3-kinase and Akt play a pivotal role in the stimulation of glucose transport by insulin, but detailed mechanisms are unknown. We and others reported that not only insulin but also platelet-derived growth factor (PDGF) and epidermal growth factor facilitate glucose uptake through GLUT4 translocation by activation of PI 3-kinase and Akt in cultured cells. However, opposite results were also reported. We generated transgenic mice that specifically express the PDGF receptor in skeletal muscle. In these mice, PDGF stimulated glucose transport into skeletal muscle in vitro and in vivo. Thus, PDGF apparently shares with insulin some of the signaling molecules needed for the stimulation of glucose transport. The degree of glucose uptake in vivo reached approximately 60% of that by insulin injection in skeletal muscle, but blood glucose levels were not decreased by PDGF in these mice. Therefore, PDGF-induced disposal of blood glucose into skeletal muscle is insufficient for rapid decrease of blood glucose levels.     

Two generations of insulinotropic imidazoline compounds

The imidazoline RX871024 increased basal- and glucose-stimulated insulin release in vitro and in vivo. The compound inhibited activity of ATP-sensitive K(+) channels as well as voltage-gated K(+) channels, which led to membrane depolarization, an increase in the cytosolic Ca(2+) concentration ([Ca(2+)](i)), and insulin release. Importantly, RX871024 also enhanced the insulinotropic effect of glucose in cells with clamped [Ca(2+)](i) but in the presence of high ATP and Ca(2+)concentration inside the cell. We believe that the latter effect on insulin exocytosis was at least in part mediated by a rise in diacylglycerol, which then activated protein kinase C (PKC) and increased the generation of arachidonic acid (AA) metabolites. Activation of both the PKC and AA pathways resulted in potentiation of glucose effects on insulin secretion. Unlike RX871024, the novel imidazoline BL11282 did not block ATP-dependent K(+) channels, but similarly to RX871024, it stimulated insulin secretion in depolarized or permeabilized islets. Accordingly, BL11282 did not influence glucose and insulin levels under basal conditions either in vitro or in vivo, but it markedly enhanced the insulinotropic effects of glucose. BL11282 restored the impaired insulin response to glucose in islets from spontaneously diabetic GK rats. We conclude that BL11282 belongs to a new class of insulinotropic compounds that demonstrate a strong glucose-dependent effect on insulin exocytosis.     

Muscle fiber type-specific defects in insulin signal transduction to glucose transport in diabetic GK rats

To determine whether defects in the insulin signal transduction pathway to glucose transport occur in a muscle fiber type-specific manner, post-receptor insulin-signaling events were assessed in oxidative (soleus) and glycolytic (extensor digitorum longus [EDL]) skeletal muscle from Wistar or diabetic GK rats. In soleus muscle from GK rats, maximal insulin-stimulated (120 nmol/l) glucose transport was significantly decreased, compared with that of Wistar rats. In EDL muscle from GK rats, maximal insulin-stimulated glucose transport was normal, while the submaximal response was reduced compared with that of Wistar rats. We next treated diabetic GK rats with phlorizin for 4 weeks to determine whether restoration of glycemia would lead to improved insulin signal transduction. Phlorizin treatment of GK rats resulted in full restoration of insulin-stimulated glucose transport in soleus and EDL muscle. In soleus muscle from GK rats, submaximal and maximal insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation and IRS-1-associated phosphatidylinositol (PI) 3-kinase activity were markedly reduced, compared with that of Wistar rats, but only submaximal insulin-stimulated PI 3-kinase was restored after phlorizin treatment. In EDL muscle, insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1-associated PI-3 kinase were not altered between GK and Wistar rats. Maximal insulin-stimulated Akt (protein kinase B) kinase activity is decreased in soleus muscle from GK rats and restored upon normalization of glycemia (Krook et al., Diabetes 46:2100-2114, 1997). Here, we show that in EDL muscle from GK rats, maximal insulin-stimulated Akt kinase activity is also impaired and restored to Wistar rat levels after phlorizin treatment. In conclusion, functional defects in IRS-1 and PI 3-kinase in skeletal muscle from diabetic GK rats are fiber-type-specific, with alterations observed in oxidative, but not glycolytic, muscle. Furthermore, regardless of muscle fiber type, downstream steps to PI 3-kinase (i.e., Akt and glucose transport) are sensitive to changes in the level of glycemia.     

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.     

Age-related impairment in insulin release: the essential role of β(2)-adrenergic receptor

In this study, we investigated the significance of β(2)-adrenergic receptor (β(2)AR) in age-related impaired insulin secretion and glucose homeostasis. We characterized the metabolic phenotype of β(2)AR-null C57Bl/6N mice (β(2)AR(-/-)) by performing in vivo and ex vivo experiments. In vitro assays in cultured INS-1E β-cells were carried out in order to clarify the mechanism by which β(2)AR deficiency affects glucose metabolism. Adult β(2)AR(-/-) mice featured glucose intolerance, and pancreatic islets isolated from these animals displayed impaired glucose-induced insulin release, accompanied by reduced expression of peroxisome proliferator-activated receptor (PPAR)γ, pancreatic duodenal homeobox-1 (PDX-1), and GLUT2. Adenovirus-mediated gene transfer of human β(2)AR rescued these defects. Consistent effects were evoked in vitro both upon β(2)AR knockdown and pharmacologic treatment. Interestingly, with aging, wild-type (β(2)AR(+/+)) littermates developed impaired insulin secretion and glucose tolerance. Moreover, islets from 20-month-old β(2)AR(+/+) mice exhibited reduced density of β(2)AR compared with those from younger animals, paralleled by decreased levels of PPARγ, PDX-1, and GLUT2. Overexpression of β(2)AR in aged mice rescued glucose intolerance and insulin release both in vivo and ex vivo, restoring PPARγ/PDX-1/GLUT2 levels. Our data indicate that reduced β(2)AR expression contributes to the age-related decline of glucose tolerance in mice.     

Restoration of hepatic glucokinase expression corrects hepatic glucose flux and normalizes plasma glucose in zucker diabetic fatty rats

OBJECTIVE—We examined in 20-week-old Zucker diabetic fatty (ZDF) rats whether restoration of hepatic glucokinase (GK) expression would alter hepatic glucose flux and improve hyperglycemia.RESEARCH DESIGN AND METHODS—ZDF rats were treated at various doses with an adenovirus that directs the expression of rat liver GK (AdvCMV-GKL) dose dependently, and various metabolic parameters were compared with those of nondiabetic lean littermates (ZCL rats) before and during a hyperglycemic clamp. Viral infection per se did not affect hepatic GK activity, since expression of a catalytically inactive form of GK did not alter endogenous hepatic GK activity.RESULTS—ZDF rats compared with ZCL rats have lower hepatic GK activity (11.6 ± 1.9 vs. 32.5 ± 3.2 mU/mg protein), marked hyperglycemia (23.9 ± 1.2 vs. 7.4 ± 0.3 mmol/l), higher endogenous glucose production (80 ± 3 vs. 38 ± 3 μmol · kg⁻¹ · min⁻¹), increased glucose-6-phosphatase flux (150 ± 11 vs. 58 ± 8 μmol · kg⁻¹ · min⁻¹), and during a hyperglycemic clamp, a failure to suppress endogenous glucose production (80 ± 7 vs. −7 ± 4 μmol · kg⁻¹ · min⁻¹) and promote glucose incorporation into glycogen (15 ± 5 vs. 43 ± 3 μmol/g liver). Treatment of ZDF rats with different doses of AdvCMV-GKL, which restored hepatic GK activity to one to two times that of ZCL rats, normalized plasma glucose levels and endogenous glucose production. During a hyperglycemic clamp, glucose production was suppressed and glucose incorporation into glycogen was normal.CONCLUSIONS—Alteration of hepatic GK activity in ZDF rats has profound effects on plasma glucose and hepatic glucose flux.In Western society, obesity is generally considered a primary risk for individuals with type 2 diabetes, since 60–90% of type 2 diabetic subjects are also obese (1). Initially, obese individuals have normal fasting glycemia with elevated plasma insulin, but become progressively more hyperglycemic and insulin resistant with a decline in plasma insulin levels associated with pancreatic β-cell dysfunction (2). Diabetic hyperglycemia results from a failure of insulin and elevated plasma glucose to increase glucose utilization and suppress endogenous glucose production (2). With >90% of endogenous glucose production derived from liver (3,4) and as much as 40% of alimentary glucose taken up by liver (5–7), for storage as glycogen (8–10), a progressive loss in these liver functions is associated with the deterioration of glycemic control and the eventual development of diabetes.Whether liver uses or produces glucose is mostly determined by the activity of the first and last enzymes of hepatic glucose utilization and production, respectively. Net hepatic glucose flux is therefore the balance between the rate of glucose phosphorylation catalyzed by glucokinase (GK), the first step of hepatic glucose utilization, and the rate of glucose-6-phosphate (G-6-P) dephosphorylation catalyzed by glucose-6-phosphatase (G-6-Pase), the last step of hepatic glucose production. In studies of nondiabetic rats, glucose-induced suppression of net hepatic glucose production was associated with increased glucose phosphorylation (11) and GK activity was required for a rise in plasma glucose to suppress hepatic glucose production (12).Male Zucker diabetic fatty (ZDF) rats are a widely used genetic model of obese type 2 diabetes, since many characteristic features of this model are common with human obese type 2 diabetes (13). Young ZDF rats exhibit normal fasting glycemia with slightly elevated plasma insulin levels and become progressively more hyperglycemic and insulin resistant as plasma insulin levels are decreased with pancreatic β-cell dysfunction (14). Previously, we reported that blunted hepatic glucose flux in response to a rise in plasma glucose and insulin, as seen in the early (10 weeks of age) and middle (14 weeks of age) phases of diabetes development in ZDF rats, is associated with impaired regulation of GK by GK regulatory protein (GKRP) (15–17). In our current study, we investigated GK expression in liver during the progressive development of diabetes in ZDF rats and examined what correlation altered GK expression may have with the development of defective hepatic glucose metabolism and hyperglycemia. We report here that GK expression in liver is progressively reduced with the development of hyperglycemia in ZDF rats and that normalizing liver GK expression restores plasma glucose to nearly normal levels by improving the responsiveness of hepatic glucose metabolism to alterations in blood glucose during this later phase of diabetes development in ZDF rats.     

Pathophysiological and genetic characterization of the major diabetes locus in GK rats

Genetic studies of the type 2 diabetes-like GK rat have revealed several susceptibility loci for the compound diabetes phenotype. Congenic strains were established for Niddm1, the major quantitative trait locus (QTL) for postprandial glucose levels, by transfer of GK alleles onto the genome of the normoglycemic F344 rat. Despite the polygenic nature of diabetes in GK, the locus-specific diabetes phenotype was retained in the congenic strain Niddmla, containing a GK-derived genomic fragment of 52 cM from the Niddm1 locus. Furthermore, Niddm1 was divided into two non-overlapping loci, physically separated in the two congenic strains Niddmlb and Niddm1i with distinct metabolic phenotypes. Both strains displayed postprandial hyperglycemia and reduced insulin action in isolated adipose cells. Furthermore, Niddm1i already exhibits a pronounced in vivo insulin secretion defect at 65 days, while Niddm1b develops a relative insulin secretory defect at 95 days. This suggests that Niddm1i impairs mechanisms common to insulin secretion in pancreatic B-cells and insulin action in adipocytes. Niddm1b rats show signs of increasing insulin resistance with age associated with obesity, hyperinsulinemia, and dyslipidemia. Moreover, the data indicated nonallelic interaction (epistasis) between Niddm1b and Niddm1i on the postprandial glucose levels. These data emphasize the pathophysiological complexity of diabetes, even within an apparently single QTL, and demonstrate the potential of the GK model in transforming the multifactorial diabetes phenotype into single traits, suitable for positional cloning.     

Hyperglycemia contributes to impaired insulin response in GK rat islets

Insulin secretion and glucose metabolism were compared in pancreatic islets from type 2 diabetic GK rats treated with phlorizin or vehicle. Treatment of control and GK rats with phlorizin for 30 days did not affect body weight, islet glucose utilization, or islet glucose oxidation. In phlorizin-treated GK rats, glucose-induced insulin release was about twofold higher at 11.0 and 16.7 mmol/l glucose compared with vehicle, treated GK rats, whereas phlorizin had no effect on control Wistar rats. However, also in phlorizin-treated GK rats, the amount of insulin released by the islets was significantly less than that from control rats (5.29+/-0.33 vs. 7.50+/-1.31 pmol x min(-1) islet(-1) at 16.7 mmol/l glucose; P<0.001). Islet glucose-6-phosphatase activity was significantly higher in GK rats than in control rats; phlorizin treatment significantly decreased this activity. These findings demonstrate that hyperglycemia per se constitutes an important factor for impaired insulin release in GK rats. Correction of hyperglycemia normalizes islet glucose-6-phosphatase activity, which may be an underlying factor for the partial improvement of glucose-induced insulin release.