Hepatic Glucose 6-phosphate Research Articles

Hepatic ChREBP orchestrates intrahepatic carbohydrate metabolism to limit hepatic glucose 6-phosphate and glycogen accumulation in a mouse model for acute Glycogen Storage Disease type Ib

ObjectiveCarbohydrate Response Element Binding Protein (ChREBP) is a glucose 6-phosphate (G6P)-sensitive transcription factor that acts as a metabolic switch to maintain intracellular glucose and phosphate homeostasis. Hepatic ChREBP is well-known for its regulatory role in glycolysis, the pentose phosphate pathway, and de novo lipogenesis. The physiological role of ChREBP in hepatic glycogen metabolism and blood glucose regulation has not been assessed in detail, and ChREBP's contribution to carbohydrate flux adaptations in hepatic Glycogen Storage Disease type 1 (GSD I) requires further investigation. MethodsThe current study aimed to investigate the role of ChREBP as a regulator of glycogen metabolism in response to hepatic G6P accumulation, using a model for acute hepatic GSD type Ib. The immediate biochemical and regulatory responses to hepatic G6P accumulation were evaluated upon G6P transporter inhibition by the chlorogenic acid S4048 in mice that were either treated with a short hairpin RNA (shRNA) directed against ChREBP (shChREBP) or a scrambled shRNA (shSCR). Complementary stable isotope experiments were performed to quantify hepatic carbohydrate fluxes in vivo. ResultsShChREBP treatment normalized the S4048-mediated induction of hepatic ChREBP target genes to levels observed in vehicle- and shSCR-treated controls. In parallel, hepatic shChREBP treatment in S4048-infused mice resulted in a more pronounced accumulation of hepatic glycogen and further reduction of blood glucose levels compared to shSCR treatment. Hepatic ChREBP knockdown modestly increased glucokinase (GCK) flux in S4048-treated mice while it enhanced UDP-glucose turnover as well as glycogen synthase and phosphorylase fluxes. Hepatic GCK mRNA and protein levels were induced by shChREBP treatment in both vehicle- and S4048-treated mice, while glycogen synthase 2 (GYS2) and glycogen phosphorylase (PYGL) mRNA and protein levels were reduced. Finally, knockdown of hepatic ChREBP expression reduced starch domain binding protein 1 (STBD1) mRNA and protein levels while it inhibited acid alpha-glucosidase (GAA) activity, suggesting reduced capacity for lysosomal glycogen breakdown. ConclusionsOur data show that ChREBP activation controls hepatic glycogen and blood glucose levels in acute hepatic GSD Ib through concomitant regulation of glucose phosphorylation, glycogenesis, and glycogenolysis. ChREBP-mediated control of GCK enzyme levels aligns with corresponding adaptations in GCK flux. In contrast, ChREBP activation in response to acute hepatic GSD Ib exerts opposite effects on GYS2/PYGL enzyme levels and their corresponding fluxes, indicating that GYS2/PYGL expression levels are not limiting to their respective fluxes under these conditions.

High Lipid Content of Prey Fish and n-3 PUFA Peroxidation Impair the Thiamine Status of Feeding-Migrating Atlantic Salmon (Salmo salar) and Is Reflected in Hepatic Biochemical Indices.

Signs of impaired thiamine (vitamin B1) status in feeding-migrating Atlantic salmon (Salmo salar) were studied in three Baltic Sea areas, which differ in the proportion and nutritional composition of prey fish sprat (Sprattus sprattus) and herring (Clupea harengus). The concentration of n−3 polyunsaturated fatty acids (n−3 PUFAs) increased in salmon with dietary lipids and n−3 PUFAs, and the hepatic peroxidation product malondialdehyde (MDA) concentration increased exponentially with increasing n−3 PUFA and docosahexaenoic acid (DHA, 22:6n−3) concentration, whereas hepatic total thiamine concentration, a sensitive indicator of thiamine status, decreased with the increase in both body lipid and n−3 PUFA or DHA concentration. The hepatic glucose 6-phosphate dehydrogenase activity was suppressed by high dietary lipids. In salmon muscle and in prey fish, the proportion of thiamine pyrophosphate increased, and that of free thiamine decreased, with increasing body lipid content or PUFAs, or merely DHA. The thiamine status of salmon was impaired mainly due to the peroxidation of n−3 PUFAs, whereas lipids as a source of metabolic energy had less effect. Organochlorines or general oxidative stress did not affect the thiamine status. The amount of lipids, and, specifically, their long-chain n−3 PUFAs, are thus responsible for generating thiamine deficiency, and not a prey fish species per se.

Survival strategies of Wuchang bream (Megalobrama amblycephala) juveniles for chronic ammonia exposure: Antioxidant defense and the synthesis of urea and glutamine

This study aimed to explore how Wuchang bream (Megalobrama amblycephala) survive and defend against the toxicity of ambient total ammonia nitrogen (0, 5, 10, 20 and 30 mg/L TA-N) during 30-day exposure. As a result, hepatic malondialdehyde and protein carbonylation as well as histopathological alterations increased with increasing TA-N level, which suggested that chronic ammonia exposure caused oxidative stress and damage in the liver of fish. Meanwhile, the activities of hepatic total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glucose 6-phosphate dehydrogenase (G6PD) as well as the mRNA expression of Cu/Zn sod, cat, gpx and g6pd were elevated significantly along with significant reduction of glutathione (GSH) and nicotinamide adenine dinucleotide phosphate (NADPH) (P < 0.05). These results indicated that hepatic antioxidant responses were activated to alleviate oxidative damages induced by ammonia, in which lower-concentration ammonia only initiate SOD-CAT-GR-G6PDH defense and higher ammonia activated the SOD-CAT-GPx-GSH-GR-G6PDH antioxidant response. In addition, significant increases of serum urea and hepatic ammonia, urea, glutamine, arginase as well as glutamine synthetase were detected with the increase of TA-N (P < 0.05), while serum ammonia levels kept stable (P > 0.05). The present findings further revealed that ammonia could be detoxified directly into glutamine and urea in Wuchang bream to cope with ammonia exposure. In conclusion, under chronic ammonia exposure, enhanced hepatic antioxidant responses as well as increased urea and glutamine synthesis worked in combination to allow Megalobrama amblycephala to defend against environmental ammonia toxicity.

Glucose-6-Phosphate Regulates Hepatic Bile Acid Synthesis in Mice.

It is well established that, besides facilitating lipid absorption, bile acids act as signaling molecules that modulate glucose and lipid metabolism. Bile acid metabolism, in turn, is controlled by several nutrient‐sensitive transcription factors. Altered intrahepatic glucose signaling in type 2 diabetes associates with perturbed bile acid synthesis. We aimed to characterize the regulatory role of the primary intracellular metabolite of glucose, glucose‐6‐phosphate (G6P), on bile acid metabolism. Hepatic gene expression patterns and bile acid composition were analyzed in mice that accumulate G6P in the liver, that is, liver‐specific glucose‐6‐phosphatase knockout (L‐G6pc−/−) mice, and mice treated with a pharmacological inhibitor of the G6P transporter. Hepatic G6P accumulation induces sterol 12α‐hydroxylase (Cyp8b1) expression, which is mediated by the major glucose‐sensitive transcription factor, carbohydrate response element‐binding protein (ChREBP). Activation of the G6P‐ChREBP‐CYP8B1 axis increases the relative abundance of cholic‐acid–derived bile acids and induces physiologically relevant shifts in bile composition. The G6P‐ChREBP–dependent change in bile acid hydrophobicity associates with elevated plasma campesterol/cholesterol ratio and reduced fecal neutral sterol loss, compatible with enhanced intestinal cholesterol absorption. Conclusion: We report that G6P, the primary intracellular metabolite of glucose, controls hepatic bile acid synthesis. Our work identifies hepatic G6P‐ChREBP‐CYP8B1 signaling as a regulatory axis in control of bile acid and cholesterol metabolism.

G6PC mRNA Therapy Positively Regulates Fasting Blood Glucose and Decreases Liver Abnormalities in a Mouse Model of Glycogen Storage Disease 1a.

Glycogen storage disease type Ia (GSD1a) is an inherited metabolic disorder caused by the deficiency of glucose-6-phosphatase (G6Pase). GSD1a is associated with life-threatening hypoglycemia and long-term liver and renal complications. We examined the efficacy of mRNA-encoding human G6Pase in a liver-specific G6Pase−/− mouse model (L-G6PC−/−) that exhibits the same hepatic biomarkers associated with GSD1a patients, such as fasting hypoglycemia, and elevated levels of hepatic glucose-6-phosphate (G6P), glycogen, and triglycerides. We show that a single systemic injection of wild-type or native human G6PC mRNA results in significant improvements in fasting blood glucose levels for up to 7 days post-dose. These changes were associated with significant reductions in liver mass, hepatic G6P, glycogen, and triglycerides. In addition, an engineered protein variant of human G6Pase, designed for increased duration of expression, showed superior efficacy to the wild-type sequence by maintaining improved fasting blood glucose levels and reductions in liver mass for up to 12 days post-dose. Our results demonstrate for the first time the effectiveness of mRNA therapy as a potential treatment in reversing the hepatic abnormalities associated with GSD1a.

Growth, biochemical, fatty acid composition, and mRNA levels of hepatic enzymes in genetically improved farmed tilapia (GIFT,Oreochromis niloticus) (Linnaeus, 1758) at different stocking densities

Investigated were the effects of stocking density on juvenile growth performance, serum biochemistry, fatty acid composition, and mRNA levels of enzymes involved in lipid metabolism in genetically improved farmed tilapia (GIFT), Oreochromis niloticus. Juvenile fish (n = 900) were distributed into 15 tanks at five initial stocking densities (75, 150, 225, 300, and 375 g/m3 per 800-L tank, represented as D1–D5, respectively). Temperature was maintained at 28 ± 0.3°C, and water flow rate into the tanks was about 8 L/min. The fish accepted floating pelleted feed (crude protein 28%, crude lipid 6%, and gross energy 149.2 KJ/g diet). The feed amount offered was 4%–8% of body weight. At the end of the 60-day experiment, the final stocking densities were 1,454.18, 2,010.60, 2,913.13, 3,819.73, and 4,380.80 g/m3, respectively. Growth and body mass coefficient of variation were significantly affected (p < .05). No differences in feed conversion rate or survival were found (p > .05). Muscle lipid contents decreased with stocking density from D1 to D5. Serum total protein and cholesterol levels were not affected by the stocking density but the D5 group produced the highest serum cortisol and lactate levels and the lowest glucose levels compared to those of the other groups. Lower muscle saturated fatty acid and n−3 polyunsaturated fatty acid contents were observed in the D4 group than in the other groups, whereas the reverse was found in the n−6 polyunsaturated fatty acid content. Hepatic glucose 6-phosphate dehydrogenase, fatty acid synthetase, hormone-sensitive triglyceride lipase, and lipoprotein lipase mRNA levels were significantly upregulated in response to a high density. These data suggest that the GIFT adapted to high-density conditions by altering the composition of muscle fatty acid and stimulating lipid metabolic enzymes. These mechanisms may help fish respond to high stocking density stress in intensive aquaculture systems.

Effects of the Combined Extracts of Grape Pomace and Omija Fruit on Hyperglycemia and Adiposity in Type 2 Diabetic Mice.

Grape products have been known to exert greater antioxidant and anti-obesity than anti-hyperglycemic effects in animals and humans. Omija is used as an ingredient in traditional medicine, and it is known to have an anti-hyperglycemic effect. We investigated whether the combined extracts of grape pomace and omija fruit (GE+OE) could reduce fat accumulation in adipose and hepatic tissues and provide beneficial effects against hyperglycemia and insulin resistance in type 2 diabetic mice. C57BL/KsJ-db/db mice were fed either a normal control diet or GE+OE (0.5% grape pomace extract and 0.05% omija fruit extract, w/w) for 7 weeks. GE+OE decreased plasma leptin and resistin levels while increasing adiponectin levels and reducing the total white adipose tissue weight. Furthermore, GE+OE lowered plasma free fatty acid (FFA), triglyceride, and total-cholesterol levels as well as hepatic FFA and cholesterol levels. Hepatic fatty acid synthase and glucose 6-phosphate dehydrogenase activities were decreased in the GE+OE group, whereas hepatic β-oxidation activity was increased. Furthermore, GE+OE supplementation not only reduced hyperglycemia and pancreatic β-cell failure but also lowered blood glycosylated hemoglobin and plasma insulin levels. The homeostasis model assessment of insulin resistance levels was also decreased and the decrease seems to be mediated by the lowered activities of hepatic glucose-6-phosphatase and phosphoenolpyruvate carboxykinases. The present data suggest that GE+OE may have the potential to reduce hyperglycemia, insulin resistance, and obesity in patients with type 2 diabetes.

Contribution of dietary starch to hepatic and systemic carbohydrate fluxes in European seabass (Dicentrarchus labraxL.)

In the present study, the effects of partial substitution of dietary protein by digestible starch on endogenous glucose production were evaluated in European seabass (Dicentrarchus labrax). The fractional contribution of dietary carbohydrates v. gluconeogenesis to blood glucose appearance and hepatic glycogen synthesis was quantified in two groups of seabass fed with a diet containing 30% digestible starch (DS) or without a carbohydrate supplement as the control (CTRL). Measurements were performed by transferring the fish to a tank containing water enriched with 5% (2)H2O over the last six feeding days, and quantifying the incorporation of (2)H into blood glucose and hepatic glycogen by (2)H NMR. For CTRL fish, gluconeogenesis accounted for the majority of circulating glucose while for the DS fish, this contribution was significantly lower (CTRL 85 (SEM 4) % v. DS 54 (SEM 2) %; P < 0.001). Hepatic glycogen synthesis via gluconeogenesis (indirect pathway) was also significantly reduced in the DS fish, in both relative (CTRL 100 (SEM 1) % v. DS 72 (SEM 1) %; P < 0.001) and absolute terms (CTRL 28 (SEM 1) v. DS 17 (sem 1) μmol/kg per h; P < 0.001). A major fraction of the dietary carbohydrates that contributed to blood glucose appearance (33 (sem 1) % of the total 47 (SEM 2) %) had undergone exchange with hepatic glucose 6-phosphate. This indicated the simultaneous activity of hepatic glucokinase and glucose 6-phosphatase. In conclusion, supplementation of digestible starch resulted in a significant reduction of gluconeogenic contributions to systemic glucose appearance and hepatic glycogen synthesis.

Hepatic anaplerotic outflow fluxes are redirected from gluconeogenesis to lactate synthesis in patients with Type 1a glycogen storage disease

Hepatic glucose production and relative Krebs cycle fluxes (indexed to a citrate synthase flux of 1.0) were evaluated with [U- 13C]glycerol tracer in 5 fed healthy controls and 5 Type 1a glycogen storage disease (GSD1a) patients. Plasma glucose, hepatic glucose-6-phosphate (G6P) and glutamine 13C-isotopomers were analyzed by 13C NMR via blood sampling and chemical biopsy. In healthy subjects, 35±14% of plasma glucose originated from hepatic G6P while GSD1a patients had no detectable G6P contribution. Compared to controls, GSD1a patients had an increased fraction of acetyl-CoA from pyruvate (0.5±0.2 vs. 0.3±0.1, p<0.01), and increased pyruvate recycling fluxes (14.4±3.8 vs. 8.7±2.8, p<0.05). Despite negligible gluconeogenic flux, net anaplerotic outflow was not significantly different from controls (2.2±0.8 vs. 1.3±0.5). The enrichment of lactate with 13C-isotopomers derived from the Krebs cycle suggests that lactate was the main anaplerotic product in GSD1a patients.

Lxrα Deficiency Hampers the Hepatic Adaptive Response to Fasting in Mice

Besides its well established role in control of cellular cholesterol homeostasis, the liver X receptor (LXR) has been implicated in the regulation of hepatic gluconeogenesis. We investigated the role of the major hepatic LXR isoform in hepatic glucose metabolism during the feeding-to-fasting transition in vivo. In addition, we explored hepatic glucose sensing by LXR during carbohydrate refeeding. Lxralpha(-/-) mice and their wild-type littermates were subjected to a fasting-refeeding protocol and hepatic carbohydrate fluxes as well as whole body insulin sensitivity were determined in vivo by stable isotope procedures. Lxralpha(-/-) mice showed an impaired response to fasting in terms of hepatic glycogen depletion and triglyceride accumulation. Hepatic glucose 6-phosphate turnover was reduced in 9-h fasted Lxralpha(-/-) mice as compared with controls. Although hepatic gluconeogenic gene expression was increased in 9-h fasted Lxralpha(-/-) mice compared with wild-type controls, the actual gluconeogenic flux was not affected by Lxralpha deficiency. Hepatic and peripheral insulin sensitivity were similar in Lxralpha(-/-) and wild-type mice. Compared with wild-type controls, the induction of hepatic lipogenic gene expression was blunted in carbohydrate-refed Lxralpha(-/-) mice, which was associated with lower plasma triglyceride concentrations. Yet, expression of "classic" LXR target genes Abca1, Abcg5, and Abcg8 was not affected by Lxralpha deficiency in carbohydrate-refed mice. In summary, these studies identify LXRalpha as a physiologically relevant mediator of the hepatic response to fasting. However, the data do not support a role for LXR in hepatic glucose sensing.

Quantification of hepatic transaldolase exchange activity and its effects on tracer measurements of indirect pathway flux in humans

Exchange of hepatic glucose-6-phosphate (G6P) and glyceraldehyde-3-phosphate via transaldolase modifies hepatic G6P enrichment from glucose or gluconeogenic tracers. Transaldolase exchange was quantified in five healthy, fed subjects following an oral bolus of [1,2,3-(13)C(3)]glycerol (25-30 mg/kg) and paracetamol (10-12 mg/kg). (13)C Isotopomers of hepatic G6P were quantified by (13)C NMR spectroscopy of urinary glucuronide. [1,2,3-(13)C(3)]- and [4,5,6-(13)C(3)]glucuronide isotopomers, representing the conversion of [1,2,3-(13)C(3)]glycerol to G6P via dihydroxyacetone phosphate, were resolved from [1,2-(13)C(2)]- and [5,6-(13)C(2)]glucuronide (13)C-isotopomers, derived from metabolism of [1,2,3-(13)C(3)]glycerol via pyruvate and phosphoenolpyruvate. Enrichment of [1,2,3-(13)C(3)]glucuronide was significantly less than that of [4,5,6-(13)C(3)]glucuronide (1.30 +/- 0.57% versus 1.67 +/- 0.42%, P < 0.05). Also, [1,2-(13)C(2)]glucuronide enrichment was significantly less than that of [5,6-(13)C(2)]glucuronide (0.28 +/- 0.08% versus 0.36 +/- 0.03%, P < 0.05). Transaldolase and triose phosphate isomerase exchange activities were estimated by applying the (13)C-isotopomer data to a model of hepatic sugar phosphate metabolism. Triose phosphate isomerase exchange was approximately 99% complete and did not contribute significantly to the unequal (13)C-isotopomer distributions of the glucuronide triose halves. Instead, this was attributable to 25 +/- 23% of hepatic G6P flux undergoing transaldolase exchange. This results in substantial overestimates of indirect pathway contributions to hepatic glycogen synthesis with tracers such as [5-(3)H]glucose and (2)H(2)O.

Specific Reduction of Hepatic Glucose 6-Phosphate Transporter-1 Ameliorates Diabetes while Avoiding Complications of Glycogen Storage Disease

D-Glucose-6-phosphatase is a key regulator of endogenous glucose production, and its inhibition may improve glucose control in type 2 diabetes. Herein, 2'-O-(2-methoxy)ethyl-modified phosphorothioate antisense oligonucleotides (ASOs) specific to the glucose 6-phosphate transporter-1 (G6PT1) enabled reduction of hepatic D-Glu-6-phosphatase activity in diabetic ob/ob mice. Treatment with G6PT1 ASOs decreased G6PT1 expression, reduced G6PT1 activity, blunted glucagon-stimulated glucose production, and lowered plasma glucose concentration in a dose-dependent manner. In contrast to G6PT1 knock-out mice and patients with glycogen storage disease, excess hepatic and renal glycogen accumulation, hyperlipidemia, neutropenia, and elevations in plasma lactate and uric acid did not occur. In addition, hypoglycemia was not observed in animals during extended periods of fasting, and the ability of G6PT1 ASO-treated mice to recover from an exogenous insulin challenge was not impaired. Together, these results demonstrate that effective glucose lowering by G6PT1 inhibitors can be achieved without adversely affecting carbohydrate and lipid metabolism.

Evidence for Impaired Gluconeogenesis in Very Long-chain Acyl-CoA Dehydrogenase-deficient Mice

Hypoketotic hypoglycaemia is a characteristic feature of fatty acid oxidation (FAO) defects. Although the underlying pathogenic mechanism is unknown, one hypothesis points to an impairment in gluconeogenesis. To study hepatic glucose production in FAO defects, we used the knockout mouse model of very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency presenting with stress-induced hypoglycaemia. We analysed metabolites of hepatic glucose production under non-stressed conditions and after stress in comparison to wildtype controls. Analysis included glycogen, glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), glycerol-3-phosphate (G3P) and dihydroxyacetone-phosphate (DHAP). We also measured the activity of the key enzyme glucose-6-phosphatase. Blood and liver glucose were found to be low after stress, and liver glycogen was depleted. In addition, hepatic G6P and F6P were significantly reduced, especially during hypoglycaemia. Importantly, the activity of the enzyme converting G6P into glucose was not impaired. These data indicate a reduced rate of gluconeogenesis. The levels of DHAP and G3P were significantly lower suggesting decreased availability of glucose precursors from glycerol. This study gives biochemical evidence of impaired gluconeogenesis as one of the causes for hypoglycaemia observed in VLCAD deficiency. Whether this is due to lack of a substrate, inhibitory effects on other gluconeogenic enzymes or impaired transcription of gluconeogenic enzymes needs to be resolved in the future.

Effects of some drugs on hepatic glucose 6-phosphate dehydrogenase activity in Lake Van Fish ( Chalcalburnus Tarischii Pallas, 1811)

Inhibitory effects of some drugs on hepatic glucose 6-phosphate dehydrogenase from Lake Van fish ( chalcalburnus tarischii pallas, 1811) were investigated. For this purpose, initially liver glucose 6-phosphate dehydrogenase was purified 899-fold in a yield of 46.24% by using 2′,5′-ADP Sepharose 4B affinity gel. In order to control the purification of enzyme was done SDS polyacrylamide gel electrophoresis. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. A constant temperature (+4 °C) was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. Vankomycine, sulfanylamide, sulfanylacetamide, nidazole, ciprofloxacin, amoxicillin and KMnO 4 were used as drugs. These drugs exhibited inhibitory effects on the enzyme. IC 50 values of vankomycine, sulfanylamide, sulfanylacetamide, nidazole, ciprofloxacin, amoxicillin and KMnO 4 were 1.88, 0.037, 0.032, 1.178, 2.26, 643.5 and 0.0002 mM, and the K i constants 1.18 ± 0.148, 0.119 ± 0.021, 0.075 ± 0.015, 1.15 ± 0.21, 7.69 ± 0.67, 1007 ± 69, and 0.001 ± 0.00022 mM, respectively. While vankomycine and nidazole showed competitive inhibition, others displayed noncompetitive inhibition. K i constants and IC 50 values for drugs were determined by Lineweaver–Burk graphs and plotting activity percentage versus [ I], respectively.

Glucose 6-phosphate regulates hepatic glycogenolysis through inactivation of phosphorylase.

High glucose concentration suppresses hepatic glycogenolysis by allosteric inhibition and dephosphorylation (inactivation) of phosphorylase-a. The latter effect is attributed to a direct effect of glucose on the conformation of phosphorylase-a. Although glucose-6-phosphate (G6P), like glucose, stimulates dephosphorylation of phosphorylase-a by phosphorylase phosphatase, its physiological role in regulating glycogenolysis in intact hepatocytes has not been tested. We show in this study that metabolic conditions associated with an increase in G6P, including glucokinase overexpression and incubation with octanoate or dihydroxyacetone, cause inactivation of phosphorylase. The latter conditions also inhibit glycogenolysis. The activity of phosphorylase-a correlated inversely with the G6P concentration within the physiological range. The inhibition of glycogenolysis and inactivation of phosphorylase-a caused by 10 mmol/l glucose can be at least in part counteracted by inhibition of glucokinase with 5-thioglucose, which lowers G6P. In conclusion, metabolic conditions that alter the hepatic G6P content affect glycogen metabolism not only through regulation of glycogen synthase but also through regulation of the activation state of phosphorylase. Dysregulation of G6P in diabetes by changes in activity of glucokinase or glucose 6-phosphatase may be a contributing factor to impaired suppression of glycogenolysis by hyperglycemia.

Kinetic properties of the glucose 6-phosphatase of the liver from arthritic rats

According to previous reports, adjuvant-induced arthritic rats present reduced activities of the hepatic glucose 6-phosphatase. A kinetic study was done in order to characterize this phenomenon. Microsomes were isolated from livers of arthritic and control rats (Holtzman strain) and the glucose 6-phosphatase was measured at various temperatures (13–37 °C) and glucose 6-phosphate concentrations. Irrespective of the temperature, the enzyme from arthritic rats presented a reduction of both V max and K M. Detergent treatment of liver microsomes from control rats increased the activity, but no increase was found when microsomes from arthritic rats were treated in the same way. The mannose 6-phosphatase activity of detergent-treated microsomes from arthritic rats was only 25% of the activity found with detergent-treated microsomes from control rats. Without detergent treatment, the mannose 6-phosphatase activities of both control and arthritic rats were minimal. The activation energy, derived from V max, was not changed by arthritis. In vivo arthritic rats presented higher hepatic glucose 6-phosphate concentrations, a phenomenon that is consistent with a reduced activity of glucose 6-phosphatase. It was concluded that in arthritic rats, the hydrolase is probably reduced, without a similar change in the translocase activity.

Free fatty acids increase basal hepatic glucose production and induce hepatic insulin resistance at different sites.

To investigate the sites of the free fatty acid (FFA) effects to increase basal hepatic glucose production and to impair hepatic insulin action, we performed 2-h and 7-h Intralipid + heparin (IH) and saline infusions in the basal fasting state and during hyperinsulinemic clamps in overnight-fasted rats. We measured endogenous glucose production (EGP), total glucose output (TGO, the flux through glucose-6-phosphatase), glucose cycling (GC, index of flux through glucokinase = TGO - EGP), hepatic glucose 6-phosphate (G-6-P) content, and hepatic glucose-6-phosphatase and glucokinase activities. Plasma FFA levels were elevated about threefold by IH. In the basal state, IH increased TGO, in vivo glucose-6-phosphatase activity (TGO/G-6-P), and EGP (P < 0.001). During the clamp compared with the basal experiments, 2-h insulin infusion increased GC and in vivo glucokinase activity (GC/TGO; P < 0.05) and suppressed EGP (P < 0.05) but failed to significantly affect TGO and in vivo glucose-6-phosphatase activity. IH decreased the ability of insulin to increase GC and in vivo glucokinase activity (P < 0.01), and at 7 h, it also decreased the ability of insulin to suppress EGP (P < 0.001). G-6-P content was comparable in all groups. In vivo glucose-6-phosphatase and glucokinase activities did not correspond to their in vitro activities as determined in liver tissue, suggesting that stable changes in enzyme activity were not responsible for the FFA effects. The data suggest that, in overnight-fasted rats, FFA increased basal EGP and induced hepatic insulin resistance at different sites. 1) FFA increased basal EGP through an increase in TGO and in vivo glucose-6-phosphatase activity, presumably due to a stimulatory allosteric effect of fatty acyl-CoA on glucose-6-phosphatase. 2) FFA induced hepatic insulin resistance (decreased the ability of insulin to suppress EGP) through an impairment of insulin's ability to increase GC and in vivo glucokinase activity, presumably due to an inhibitory allosteric effect of fatty acyl-CoA on glucokinase and/or an impairment in glucokinase translocation.

Disturbed lipid metabolism in glycogen storage disease type 1.

Glycogen storage disease type 1 (GSD1) is an inborn error of metabolism caused by deficiency of glucose-6-phosphatase, the enzyme catalysing the conversion of glucose-6-phosphate (G6P) to glucose. GSD1 is associated with severe hyperlipidaemia and hepatic steatosis. The underlying mechanisms responsible for these abnormalities in lipid metabolism are only partly known. This review summarises data available on hyperlipidaemia and steatosis in GSD1 and postulates new hypotheses for unresolved issues. Evidence indicates that lipid clearance from the blood compartment is decreased in GSD1. Furthermore, in two GSD1a patients synthesis of palmitate, an indicator of de novo lipogenesis, and cholesterol were found to be increased 40-fold and 7-fold, respectively. Elevated hepatic G6P levels may play a regulatory role in lipid synthesis via activation of transcription of lipogenic genes. In addition, accelerated glycolysis will supply acetyl-CoA molecules required for lipogenesis. It is as yet unclear whether hepatic secretion of lipids in the form of very low density lipoprotein-triglycerides (VLDL-TG) is altered in GSD1 patients: we recently found unaffected VLDL-TG secretion rates in an acute animal model of GSD1b. Hepatic steatosis, which is invariably present in GSD1 is probably mainly caused by an increased free fatty acid flux from adipose tissue to the liver and, to a limited extent, by increased de novo lipogenesis. future studies, using novel stable isotope methodologies, are warranted to further clarify the disturbances in lipid and lipoprotein metabolism in glycogen storage disease type 1 and the role of glucose-6-phosphate herein.

Acute inhibition of glucose-6-phosphate translocator activity leads to increased de novo lipogenesis and development of hepatic steatosis without affecting VLDL production in rats.

Glucose-6-phosphatase (G6Pase) is a key enzyme in hepatic glucose metabolism. Altered G6Pase activity in glycogen storage disease and diabetic states is associated with disturbances in lipid metabolism. We studied the effects of acute inhibition of G6Pase activity on hepatic lipid metabolism in nonanesthetized rats. Rats were infused with an inhibitor of the glucose-6-phosphate (G6P) translocator (S4048, 30 mg. kg(-1). h(-1)) for 8 h. Simultaneously, [1-(13)C]acetate was administered for determination of de novo lipogenesis and fractional cholesterol synthesis rates by mass isotopomer distribution analysis. In a separate group of rats, Triton WR 1339 was injected for determination of hepatic VLDL-triglyceride production. S4048 infusion significantly decreased plasma glucose (-11%) and insulin (-48%) levels and increased hepatic G6P (201%) and glycogen (182%) contents. Hepatic triglyceride contents increased from 5.8 +/- 1.4 micromol/g liver in controls to 20.6 +/- 5.5 micromol/g liver in S4048-treated animals. De novo lipogenesis was increased >10-fold in S4048-treated rats, without changes in cholesterol synthesis rates. Hepatic mRNA levels of acetyl-CoA carboxylase and fatty acid synthase were markedly induced. Plasma triglyceride levels increased fourfold, but no differences in plasma cholesterol levels were seen. Surprisingly, hepatic VLDL-triglyceride secretion was not increased in S4048-treated rats. These studies demonstrate that inhibition of the G6Pase system leads to acute stimulation of fat synthesis and development of hepatic steatosis, without affecting hepatic cholesterol synthesis and VLDL secretion. The results emphasize the strong interactions that exist between hepatic carbohydrate and fat metabolism.

Effects of arginine on pancreatic hormones and hepatic metabolism in rainbow trout.

Arginine (Arg), injected intraperitoneally into rainbow trout (Oncorhynchus mykiss), increases plasma concentrations of glucagon, glucagon-like peptide-1 (GLP-1), and insulin by three- to 10-fold. Resulting ratios of glucagon and GLP-1 over insulin are unchanged in 20-d food-deprived fish (saline, 1.28 vs. Arg, 0.93; not significant) while slightly increased in feeding trout (saline, 0.70 vs. Arg, 0.92; P<0.05). In food-deprived juveniles, Arg injection leads to significant decreases in plasma fatty acids (saline, 1.65 mM L(-1) vs. Arg, 1.09 mM L(-1); P<0.05) and increases in glycogen phosphorylase total activity (saline, 3.7 units g(-1) vs. Arg, 4.6 units g(-1); P<0.05) and degree of phosphorylation (saline, 1.7 units g(-1) vs. Arg, 2.33 units g(-1); P<0.05). Plasma and liver glucose and liver enzymes (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, pyruvate kinase, phosphoenolpyruvate carboxykinase, lactate dehydrogenase, and malic enzyme) are unaffected. Otherwise, fish show the changes in plasma metabolites expected with food deprivation. Arg injection into feeding fish results in decreases in plasma fatty acids, liver glycogen, and glucose, while liver glucose 6-phosphate concentrations increase. Hepatocytes isolated from feeding fish injected with Arg 2 h previously show significantly lower rates of lactate oxidation than controls (85% of control), while rates of gluconeogenesis and hormonal responses to mammalian glucagon and GLP-1 remain unchanged. Rates of lactate oxidation and gluconeogenesis are significantly decreased by 5%-10% on treatment with porcine insulin. Complete immunoneutralization of insulin with rabbit antisalmon insulin serum decreases hepatic glucose 6-phosphate concentrations and abolishes the Arg-dependent effects on glycogen phosphorylase. It appears that short-term increases in pancreatic hormones cause only minor metabolic readjustments in the relatively short time frame covered in these experiments. Surprisingly, complete removal of insulin does not have immediate altering or detrimental effects on key metabolites and metabolic pathways, even if glucagon and GLP-1 concentrations are concurrently several-fold higher than usual. Our data clearly show the dual role of Arg in fish metabolism.