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An adaptation to continuous total parenteral nutrition (TPN; 75% of nonprotein calories as glucose) is the liver becomes a major consumer of glucose with lactate release as a by-product. The liver is able to further increase liver glucose uptake when a small dose of fructose is acutely infused via the portal system. Glucagon, commonly elevated during inflammatory stress, is a potent inhibitor of glucose uptake by the liver during TPN. The aim was to determine if continuous fructose infusion could overcome the glucagon-mediated decrease in hepatic glucose uptake. Studies were performed in conscious, insulin-treated, chronically catheterized, pancreatectomized dogs that adapted to TPN for 33 hours. They were then assigned to 1 of 4 groups: TPN (C), TPN + fructose (4.4 μmol kg(-1) min(-1); F), TPN + glucagon (0.2 pmol kg(-1) min(-1); GGN), or TPN + fructose and glucagon (F + GGN) for an additional 63 hours (33-96 hours). Insulin, fructose, and glucagon were infused into the portal vein. During that period, all animals received a fixed insulin infusion of 0.4 mU·kg(-1)·min(-1) (33-96 hours); and the glucose infusion rates were adjusted to maintain euglycemia (6.6 mmol/L). Continuous fructose infusion was unable to further enhance net hepatic glucose uptake (in micromoles per kilogram per minute) (31.1 ± 2.8 vs 36.1 ± 5.0; C vs F), nor was it able to overcome glucagon-mediated decrease in net hepatic glucose uptake (10.0 ± 4.4 vs 12.2 ± 3.9; GGN vs F + GGN). In summary, continuous fructose infusion cannot augment liver glucose uptake during TPN; nor can it overcome the inhibitory effects of glucagon.
Copyright © 2011 Elsevier Inc. All rights reserved.
Glucagon is released from alpha-cells present in intact pancreatic islets at glucose concentrations below 4 mm, whereas higher glucose levels inhibit its secretion. The mechanisms underlying the suppression of alpha-cell secretory activity are poorly understood, but two general types of models have been proposed as follows: direct inhibition by glucose or paracrine inhibition from non-alpha-cells within the islet of Langerhans. To identify alpha-cells for analysis, we utilized transgenic mice expressing fluorescent proteins targeted specifically to these cells. Measurements of glucagon secretion from pure populations of flow-sorted alpha-cells show that contrary to its effect on intact islets, glucose does stimulate glucagon secretion from isolated alpha-cells. This observation argues against a direct inhibition of glucagon secretion by glucose and supports the paracrine inhibition model. Imaging of cellular metabolism by two-photon excitation of NAD(P)H autofluorescence indicates that glucose is metabolized in alpha-cells and that glucokinase is the likely rate-limiting step in this process. Imaging calcium dynamics of alpha-cells in intact islets reveals that inhibiting concentrations of glucose increase the intracellular calcium concentration and the frequency of alpha-cell calcium oscillations. Application of candidate paracrine inhibitors leads to reduced glucagon secretion but did not decrease the alpha-cell calcium activity. Taken together, the data suggest that suppression occurs downstream from alpha-cell calcium signaling, presumably at the level of vesicle trafficking or exocytotic machinery.
Glucose, fat, and glucagon availability are increased in diabetes. The normal response of the liver to chronic increases in glucose availability is to adapt to become a marked consumer of glucose. Yet this fails to occur in diabetes. The aim was to determine whether increased glucagon and lipid interact to impair the adaptation to increased glucose availability. Chronically catheterized well controlled depancreatized conscious dogs (n = 21) received 3 days of continuous parenteral nutrition (TPN) that was either high in glucose [C; 75% nonprotein calories (NPC)] or in lipid (HL; 75% NPC) in the presence or absence of a low dose (one-third basal) chronic intraportal infusion of glucagon (GN; 0.25 ng.kg(-1).min(-1)). During the 3 days of TPN, all groups received the same insulin algorithm; the total amount of glucose infused (GIR) was varied to maintain isoglycemia ( approximately 120 mg/dl). On day 3 of TPN, hepatic metabolism was assessed. Glucose and insulin levels were similar in all groups. GIR was decreased in HL and C + GN ( approximately 30%) and was further decreased in HL + GN (55%). Net hepatic glucose uptake was decreased approximately 15% in C + GN, and HL and was decreased approximately 50% in HL + GN. Lipid alone or combined with glucagon decreased glucose uptake by peripheral tissues. Despite impairing whole body glucose utilization, HL did not limit whole body energy disposal. In contrast, glucagon suppressed whole body energy disposal irrespective of the diet composition. In summary, failure to appropriately suppress glucagon secretion adds to the dietary fat-induced impairment in both hepatic and peripheral glucose disposal. In addition, unlike increasing the percentage of calories as fat, inappropriate glucagon secretion in the absence of compensatory hyperinsulinemia limits whole body nutrient disposition.
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 micromol x kg(-1) x min(-1)), increased glucose-6-phosphatase flux (150 +/- 11 vs. 58 +/- 8 micromol x kg(-1) x min(-1)), and during a hyperglycemic clamp, a failure to suppress endogenous glucose production (80 +/- 7 vs. -7 +/- 4 micromol x kg(-1) x min(-1)) and promote glucose incorporation into glycogen (15 +/- 5 vs. 43 +/- 3 micromol/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.
Tryptophan fluorescence was used to study GK (glucokinase), an enzyme that plays a prominent role in glucose homoeostasis which, when inactivated or activated by mutations, causes diabetes mellitus or hypoglycaemia in humans. GK has three tryptophan residues, and binding of D-glucose increases their fluorescence. To assess the contribution of individual tryptophan residues to this effect, we generated GST-GK [GK conjugated to GST (glutathione transferase)] and also pure GK with one, two or three of the tryptophan residues of GK replaced with other amino acids (i.e. W99C, W99R, W167A, W167F, W257F, W99R/W167F, W99R/W257F, W167F/W257F and W99R/W167F/W257F). Enzyme kinetics, binding constants for glucose and several other sugars and fluorescence quantum yields (varphi) were determined and compared with those of wild-type GK retaining its three tryptophan residues. Replacement of all three tryptophan residues resulted in an enzyme that retained all characteristic features of GK, thereby demonstrating the unique usefulness of tryptophan fluorescence as an indicator of GK conformation. Curves of glucose binding to wild-type and mutant GK or GST-GK were hyperbolic, whereas catalysis of wild-type and most mutants exhibited co-operativity with D-glucose. Binding studies showed the following order of affinities for the enzyme variants: N-acetyl-D-glucosamine>D-glucose>D-mannose>D-mannoheptulose>2-deoxy-D-glucose>L-glucose. GK activators increased sugar binding of most enzymes, but not of the mutants Y214A/V452A and C252Y. Contributions to the fluorescence increase from Trp(99) and Trp(167) were large compared with that from Trp(257) and are probably based on distinct mechanisms. The average quantum efficiency of tryptophan fluorescence in the basal and glucose-bound state was modified by activating (Y214A/V452A) or inactivating (C213R and C252Y) mutations and was interpreted as a manifestation of distinct conformational states.
OBJECTIVE - Type 2 diabetes is often accompanied by abnormal blood lipid and lipoprotein levels, but most studies on the link between hyperlipidemia and diabetes have focused on free fatty acids (FFAs). In this study, we examined the relationship between cholesterol and insulin secretion from pancreatic beta-cells that is independent of the effects of FFAs.
RESEARCH DESIGN AND METHODS - Several methods were used to modulate cholesterol levels in intact islets and cultured beta-cells, including a recently developed mouse model that exhibits elevated cholesterol but normal FFA levels. Acute and metabolic alteration of cholesterol was done using pharmacological reagents.
RESULTS - We found a direct link between elevated serum cholesterol and reduced insulin secretion, with normal secretion restored by cholesterol depletion. We further demonstrate that excess cholesterol inhibits secretion by downregulation of metabolism through increased neuronal nitric oxide synthase dimerization.
CONCLUSIONS - This direct effect of cholesterol on beta-cell metabolism opens a novel set of mechanisms that may contribute to beta-cell dysfunction and the onset of diabetes in obese patients.
To better understand how glucokinase (GK) missense mutations associated with human glycemic diseases perturb glucose homeostasis, we generated and characterized mice with either an activating (A456V) or inactivating (K414E) mutation in the gk gene. Animals with these mutations exhibited alterations in their blood glucose concentration that were inversely related to the relative activity index of GK. Moreover, the threshold for glucose-stimulated insulin secretion from islets with either the activating or inactivating mutation were left- or right-shifted, respectively. However, we were surprised to find that mice with the activating mutation had markedly reduced amounts of hepatic GK activity. Further studies of bacterially expressed mutant enzymes revealed that GK(A456V) is as stable as the wild type enzyme, whereas GK(K414E) is thermolabile. However, the ability of GK regulatory protein to inhibit GK(A456V) was found to be less than that of the wild type enzyme, a finding consistent with impaired hepatic nuclear localization. Taken together, this study indicates that it is necessary to have knowledge of both thermolability and the interactions of mutant GK enzymes with GK regulatory protein when attempting to predict in vivo glycemic phenotypes based on the measurement of enzyme kinetics.
Glucokinase (GK) plays a key role in the regulation of glucose use and glucose-stimulated insulin secretion in pancreatic islet cells. Gene targeting of the IGF-I receptor down-regulated pancreatic islet GK activity. That finding prompted us to examine the potential mechanism that may control GK gene activity using an islet cell line, INS-1, known to express IGF-I receptor. Exposure of these cells to IGF-I induced GK protein expression and activity of the enzyme in a dose-dependent manner. In addition, IGF-I induced activity of a reporter construct containing the GK promoter in parallel with the effect on endogenous GK mRNA levels. The stimulatory effect of IGF-I on GK promoter activity was abrogated by wortmannin and LY294002, specific inhibitors of phosphatidylinositol 3-kinase. Exposure of cells to IGF-I elicited a rapid phosphorylation of Akt and FoxO1, a known target of Akt signaling. Constitutively active Akt stimulates the activity of the GK promoter, and a dominant-negative mutant of Akt or mutagenesis of a FoxO1 response element in the GK promoter abolished the ability of IGF-I to stimulate the promoter activity. Furthermore, cell knockdown of FoxO1 with small interfering RNA disrupted the effect of IGF-I on GK expression. These results demonstrate that the phosphatidylinositol 3-kinase/Akt/FoxO1 pathway contributes to the regulation of GK gene expression in response to IGF-I stimulation.
Effect of stimulation of glucokinase (GK) export from the nucleus by small amounts of sorbitol on hepatic glucose flux in response to elevated plasma glucose was examined in 6-h fasted Zucker diabetic fatty rats at 10 wk of age. Under basal conditions, plasma glucose, insulin, and glucagon were approximately 8 mM, 2,000 pmol/l, and 60 ng/l, respectively. Endogenous glucose production (EGP) was 44 +/- 4 micromol x kg(-1) x min(-1). When plasma glucose was raised to approximately 17 mM, GK was still predominantly localized with its inhibitory protein in the nucleus. EGP was not suppressed. When sorbitol was infused at 5.6 and 16.7 micromol x kg(-1) x min(-1), along with the increase in plasma glucose, GK was exported to the cytoplasm. EGP (23 +/- 19 and 12 +/- 5 micromol x kg(-1) x min(-1)) was suppressed without a decrease in glucose 6-phosphatase flux (145 +/- 23 and 126 +/- 16 vs. 122 +/- 10 micromol x kg(-1) x min(-1) without sorbitol) but increased in glucose phosphorylation as indicated by increases in glucose recycling (122 +/- 17 and 114 +/- 19 vs. 71 +/- 11 microl x kg(-1) x min(-1)), glucose-6-phosphate content (254 +/- 32 and 260 +/- 35 vs. 188 +/- 20 nmol/g liver), fractional contribution of plasma glucose to uridine 5'-diphosphate-glucose flux (43 +/- 8 and 42 +/- 8 vs. 27 +/- 6%), and glycogen synthesis from plasma glucose (20 +/- 4 and 22 +/- 5 vs. 9 +/- 4 mumol glucose/g liver). The decreased glucose effectiveness to suppress EGP and stimulate hepatic glucose uptake may result from failure of the sugar to activate GK by stimulating the translocation of the enzyme.
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.