Glucose production, gluconeogenesis, and hepatic tricarboxylic acid cycle fluxes measured by nuclear magnetic resonance analysis of a single glucose derivative

A triple-tracer method was developed to provide absolute fluxes contributing to endogenous glucose production and hepatic tricarboxylic acid (TCA) cycle fluxes in 24-h-fasted rats by (2)H and (13)C nuclear magnetic resonance (NMR) analysis of a single glucose derivative. A primed, intravenous [3,4-(13)C(2)]glucose infusion was used to measure endogenous glucose production; intraperitoneal (2)H(2)O (to enrich total body water) was used to quantify sources of glucose (TCA cycle, glycerol, and glycogen), and intraperitoneal [U-(13)C(3)] propionate was used to quantify hepatic anaplerosis, pyruvate cycling, and TCA cycle flux. Plasma glucose was converted to monoacetone glucose (MAG), and a single (2)H and (13)C NMR spectrum of MAG provided the following metabolic data (all in units of micromol/kg/min; n = 6): endogenous glucose production (40.4+/-2.9), gluconeogenesis from glycerol (11.5+/-3.5), gluconeogenesis from the TCA cycle (67.3+/-5.6), glycogenolysis (1.0+/-0.8), pyruvate cycling (154.4+/-43.4), PEPCK flux (221.7+/-47.6), and TCA cycle flux (49.1+/-16.8). In a separate group of rats, glucose production was not different in the absence of (2)H(2)O and [U-(13)C]propionate, demonstrating that these tracers do not alter the measurement of glucose turnover.     

Effect of liver glycogen content on glucose production in running rats

The influence of supranormal compared with normal hepatic glycogen levels on hepatic glucose production (Ra) during exercise was investigated in chronically catheterized rats. Supranormal hepatic glycogen levels were obtained by a 24-h fast-24-h refeeding regimen. During treadmill running for 35 min at a speed of 21 m/min, Ra and plasma glucose increased more (P less than 0.05) and liver glucogen breakdown was larger in fasted-refed compared with control rats, although the stimuli for Ra were higher in control rats, the plasma concentrations of insulin and glucose being lower (P less than 0.05) in control compared with fasted-refed rats. Also, plasma concentrations of glucagon and both catecholamines tended to be higher and muscle glycogenolysis lower in control compared with fasted-refed rats. Lipid metabolism was similar in the two groups. The results indicate that hepatic glycogenolysis during exercise is directly related to hepatic glycogen content. The smaller endocrine glycogenolytic signal in face of higher plasma glucose concentrations in fasted-refed compared with control rats is indicative of metabolic feedback control of glucose mobilization during exercise. However, the higher exercise-induced increase in Ra, plasma glucose, and liver glycogen breakdown in fasted-refed compared with control rats indicates that metabolic feedback mechanisms are not able to accurately match Ra to the metabolic needs of working muscles.     

An integrated (2)H and (13)C NMR study of gluconeogenesis and TCA cycle flux in humans

Hepatic glucose synthesis from glycogen, glycerol, and the tricarboxylic acid (TCA) cycle was measured in five overnight-fasted subjects by (1)H, (2)H, and (13)C NMR analysis of blood glucose, urinary acetaminophen glucuronide, and urinary phenylacetylglutamine after administration of [1,6-(13)C(2)]glucose, (2)H(2)O, and [U-(13)C(3)]propionate. This combination of tracers allows three separate elements of hepatic glucose production (GP) to be probed simultaneously in a single study: 1) endogenous GP, 2) the contribution of glycogen, phosphoenolpyruvate (PEP), and glycerol to GP, and 3) flux through PEP carboxykinase, pyruvate recycling, and the TCA cycle. Isotope-dilution measurements of [1,6-(13)C(2)] glucose by (1)H and (13)C NMR indicated that GP in 16-h-fasted humans was 10.7 +/- 0.9 micromol.kg(-1).min(-1). (2)H NMR spectra of monoacetone glucose (derived from plasma glucose) provided the relative (2)H enrichment at glucose H-2, H-5, and H-6S, which, in turn, reflects the contribution of glycogen, PEP, and glycerol to total GP (5.5 +/- 0.7, 4.8 +/- 1.0, and 0.4 +/- 0.3 micromol.kg(-1).min(-1), respectively). Interestingly, (13)C NMR isotopomer analysis of phenylacetylglutamine and acetaminophen glucuronide reported different values for PEP carboxykinase flux (68.8 +/- 9.8 vs. 37.5 +/- 7.9 micromol.kg(-1).min(-1)), PEP recycling flux (59.1 +/- 9.8 vs. 27.8 +/- 6.8 micromol.kg(-1).min(-1)), and TCA cycle flux (10.9 +/- 1.4 vs. 5.4 +/- 1.4 micromol.kg(-1).min(-1)). These differences may reflect zonation of propionate metabolism in the liver.     

Defect in glucokinase translocation in Zucker diabetic fatty rats

Hepatic glucose fluxes and intracellular movement of glucokinase (GK) in response to increased plasma glucose and insulin were examined in 10-wk-old, 6-h-fasted, conscious Zucker diabetic fatty (ZDF) rats and lean littermates. Under basal conditions, plasma glucose (mmol/l) and glucose turnover rate (GTR; micromol.kg(-1).min(-1)) were slightly higher in ZDF (8.4 +/- 0.3 and 53 +/- 7, respectively) than in lean rats (6.2 +/- 0.2 and 45 +/- 4, respectively), whereas plasma insulin (pmol/l) was higher in ZDF (1,800 +/- 350) than in lean rats (150 +/- 14). The ratio of hepatic uridine 5'-diphosphate-glucose 3H specific activity to plasma glucose 3H specific activity ([3H]UDP-G/[3H]G; %), total hepatic glucose output (micromol.kg(-1).min(-1)), and hepatic glucose cycling (micromol.kg(-1).min(-1)) were higher in ZDF (35 +/- 5, 87 +/- 16, and 33 +/- 10, respectively) compared with lean rats (18 +/- 3, 56 +/- 6, and 11 +/- 2, respectively). [3H]glucose incorporation into glycogen (micromol glucose/g liver) was similar in lean (1.0 +/- 0.7) and ZDF (1.6 +/- 0.8) rats. GK was predominantly located in the nucleus in both rats. With elevated plasma glucose and insulin, GTR (micromol.kg(-1).min(-1)), [3H]UDP-G/[3H]G (%), and [3H]glucose incorporation into glycogen (micromol glucose/g liver) were markedly higher in lean (191 +/- 22, 62 +/- 3, and 5.0 +/- 1.4, respectively) but similar in ZDF rats (100 +/- 6, 37 +/- 3, and 1.4 +/- 0.4, respectively) compared with basal conditions. GK translocation from the nucleus to the cytoplasm occurred in lean but not in ZDF rats. The unresponsiveness of hepatic glucose flux to the rise in plasma glucose and insulin seen in prediabetic ZDF rats was associated with impaired GK translocation.     

Impaired tricarboxylic acid cycle activity in mouse livers lacking cytosolic phosphoenolpyruvate carboxykinase

Liver-specific phosphoenolpyruvate carboxykinase (PEPCK) null mice, when fasted, maintain normal whole body glucose kinetics but develop dramatic hepatic steatosis. To identify the abnormalities of hepatic energy generation that lead to steatosis during fasting, we studied metabolic fluxes in livers lacking hepatic cytosolic PEPCK by NMR using 2H and 13C tracers. After a 4-h fast, glucose production from glycogenolysis and conversion of glycerol to glucose remains normal, whereas gluconeogenesis from tricarboxylic acid (TCA) cycle intermediates was nearly absent. Upon an extended 24-h fast, livers that lack PEPCK exhibit both 2-fold lower glucose production and oxygen consumption, compared with the controls, with all glucose production being derived only from glycerol. The mitochondrial reduction-oxidation (red-ox) state, as indicated by the NADH/NAD+ ratio, is 5-fold higher, and hepatic TCA cycle intermediate concentrations are dramatically increased in the PEPCK null livers. Consistent with this, flux through the TCA cycle and pyruvate cycling pathways is 10- and 40-fold lower, respectively. Disruption of hepatic cataplerosis due to loss of PEPCK leads to the accumulation of TCA cycle intermediates and a nearly complete blockage of gluconeogenesis from amino acids and lactate (an energy demanding process) but intact gluconeogenesis from glycerol (which contributes to net NADH production). Inhibition of the TCA cycle and fatty acid oxidation due to increased TCA cycle intermediate concentrations and reduced mitochondrial red-ox state lead to the development of steatosis.     

Carbohydrate metabolism in fructose-fed and food-restricted running rats

In chronically catheterized rats hepatic glycogen was increased by fructose (approximately 10 g/kg) gavage (FF rats) or lowered by overnight food restriction (FR rats). [3-3H]- and [U-14C]glucose were infused before, during, and after treadmill running. During exercise the increase in glucose production (Ra) was always directly related to work intensity and faster than the increase in glucose disappearance, resulting in increased plasma glucose levels. At identical work-loads the increase in Ra and plasma glucose as well as liver glycogen breakdown were higher in FF and control (C) rats than in FR rats. Breakdown of muscle glycogen was less in FF than in C rats. Incorporation of [14C]glucose in glycogen at rest and mobilization of label during exercise partly explained that 14C estimates of carbohydrate metabolism disagreed with chemical measurements. In some muscles glycogen depletion was not accompanied by loss of 14C and 3H, indicating futile cycling of glucose. In FR rats a postexercise increase in liver glycogen was seen with 14C/3H similar to that of plasma glucose, indicating direct synthesis from glucose. In conclusion, in exercising rats the increase in glucose production is subjected to feedforward regulation and depends on the liver glycogen concentration. Endogenous glucose may be incorporated in glycogen in working muscle and may be used directly for liver glycogen synthesis rather than after conversion to trioses. Fructose ingestion may diminish muscular glycogen breakdown. The [14C]glucose infusion technique for determination of muscular glycogenolysis is of doubtful value in rats.     

Acute inhibition of hepatic glucose-6-phosphatase does not affect gluconeogenesis but directs gluconeogenic flux toward glycogen in fasted rats. A pharmacological study with the chlorogenic acid derivative S4048

Effects of acute inhibition of glucose-6-phosphatase activity by the chlorogenic acid derivative S4048 on hepatic carbohydrate fluxes were examined in isolated rat hepatocytes and in vivo in rats. Fluxes were calculated using tracer dilution techniques and mass isotopomer distribution analysis in plasma glucose and urinary paracetamol-glucuronide after infusion of [U-(13)C]glucose, [2-(13)C]glycerol, [1-(2)H]galactose, and paracetamol. In hepatocytes, glucose-6-phosphate (Glc-6-P) content, net glycogen synthesis, and lactate production from glucose and dihydroxyacetone increased strongly in the presence of S4048 (10 microm). In livers of S4048-treated rats (0.5 mg kg(-1)min(-)); 8 h) Glc-6-P content increased strongly (+440%), and massive glycogen accumulation (+1260%) was observed in periportal areas. Total glucose production was diminished by 50%. The gluconeogenic flux to Glc-6-P was unaffected (i.e. 33.3 +/- 2.0 versus 33.2 +/- 2.9 micromol kg(-1)min(-1)in control and S4048-treated rats, respectively). Newly synthesized Glc-6-P was redistributed from glucose production (62 +/- 1 versus 38 +/- 1%; p < 0.001) to glycogen synthesis (35 +/- 5% versus 65 +/- 5%; p < 0.005) by S4048. This was associated with a strong inhibition (-82%) of the flux through glucokinase and an increase (+83%) of the flux through glycogen synthase, while the flux through glycogen phosphorylase remained unaffected. In livers from S4048-treated rats, mRNA levels of genes encoding Glc-6-P hydrolase (approximately 9-fold), Glc-6-P translocase (approximately 4-fold), glycogen synthase (approximately 7-fold) and L-type pyruvate kinase (approximately 4-fold) were increased, whereas glucokinase expression was almost abolished. In accordance with unaltered gluconeogenic flux, expression of the gene encoding phosphoenolpyruvate carboxykinase was unaffected in the S4048-treated rats. Thus, acute inhibition of glucose-6-phosphatase activity by S4048 elicited 1) a repartitioning of newly synthesized Glc-6-P from glucose production into glycogen synthesis without affecting the gluconeogenic flux to Glc-6-P and 2) a cellular response aimed at maintaining cellular Glc-6-P homeostasis.     

Quantitation of gluconeogenesis by (2)H nuclear magnetic resonance analysis of plasma glucose following ingestion of (2)H(2)O

We present a simple (2)H NMR assay of the fractional contribution of gluconeogenesis to hepatic glucose output following ingestion of (2)H(2)O. The assay is based on the measurement of relative deuterium enrichment in hydrogens 2 and 3 of plasma glucose. Plasma glucose was enzymatically converted to gluconate, which displays fully resolved deuterium 2 and 3 resonances in its (2)H NMR spectrum at 14.1 T. The signal intensity of deuterium 3 relative to deuterium 2 in the gluconate derivative as quantitated by (2)H NMR was shown to provide a precise and accurate measurement of glucose enrichment in hydrogen 3 relative to hydrogen 2. This measurement was used to estimate the fractional contribution of gluconeogenesis to hepatic glucose output for two groups of rats; one group was fasted for 7 h and the other was fasted for 29 h. Rats were administered (2)H(2)O to enrich total body water to 5% over the last 4-5 h of each fasting period. For the 7-h fasted group, the hydrogen 3/hydrogen 2 enrichment ratio of plasma glucose was 0.32 +/- 0.09 (n = 7). This indicates that gluconeogenesis contributed 32 +/- 9% of total hepatic glucose output with glycogenolysis contributing the remainder. For the 29-h fasted group, the hydrogen 3/hydrogen 2 enrichment ratio of plasma glucose was 0.81 +/- 0.10 (n = 6), indicating that gluconeogenesis supplied the bulk of hepatic glucose output (81 +/- 10%).     

Role of liver nerves and adrenal medulla in glucose turnover of running rats

Sympathetic control of glucose turnover was studied in rats running 35 min at 21 m X min-1 on the level. The rats were surgically liver denervated, adrenodemedullated, or sham operated. Glucose turnover was measured by primed constant infusion of [3-3H]glucose. At rest, the three groups had identical turnover rates and concentrations of glucose in plasma. During running, glucose production always rose rapidly to steady levels. The increase was not influenced by liver denervation but was halved by adrenodemedullation. Similarly, hepatic glycogen depletion was identical in denervated and control rats but reduced after adrenodemedullation. Early in exercise, glucose uptake rose identically in all groups and, in adrenodemedullated rats, matched glucose production. Accordingly, plasma glucose concentration increased in liver-denervated and control rats but was constant in adrenodemedullated rats. Compensatory changes in hormone or substrate levels explaining the lack of effect of liver denervation were not found. In rats with intact adrenals, the plasma epinephrine concentration was increased after 2.5 min of running. It is concluded that, in rats carrying out exercise of moderate intensity and duration, hepatic glycogenolysis and glucose production are not influenced by the autonomic liver nerves but are enhanced by circulating epinephrine.     

Regulation of myocardial [(13)C]glucose metabolism in conscious rats

Administration of supplemental glucose and/or insulin is postulated to improve the outcome from myocardial ischemia by increasing the heart's relative utilization of glucose as an energy substrate. To examine the degree to which circulating glucose and insulin levels actually influence myocardial substrate preference in vivo, we infused conscious, chronically catheterized rats with D-[1-(13)C]glucose and compared steady-state (13)C enrichment of plasma glucose with that of myocardial glycolytic ([3-(13)C]alanine) and oxidative ([4-(13)C]glutamate) intermediary metabolites. In fasting rats, [3-(13)C]alanine-to-[1-(13)C]glucose and [4-(13)C]glutamate-to-[3-(13)C]alanine ratios averaged 0.16 +/- 0.12 and 0.14 +/- 0.03, respectively, indicating that circulating glucose contributed 32% of myocardial glycolytic flux, whereas subsequent flux through pyruvate dehydrogenase contributed 14% of total tricarboxylic acid (TCA) cycle activity. Raising plasma glucose to 11 mmol/l, or insulin to 500 pmol/l, increased these contributions equivalently. At supraphysiological (>6,500 pmol/l) insulin levels, the plasma glucose contribution to glycolysis increased further, and addition of hyperglycemia made it the sole glycolytic substrate, yet [4-(13)C]glutamate-to-[3-(13)C]alanine ratios remained /=40% of myocardial TCA cycle flux.     

Effects of glucose infusion in exercising rats

To determine whether feedforward control of liver glycogenolysis during exercise is subject to negative feedback by elevated blood glucose, glucose was infused into exercising rats at a rate that elevated blood glucose greater than 10 mM. Liver glycogen content decreased 22.4 mg/g in saline-infused rats compared with 13.6 mg/g in glucose-infused rats during the first 40 min of treadmill running (21 m/min, 15% grade). Liver adenosine 3',5'-cyclic monophosphate (cAMP) concentration was significantly lower in the glucose-infused rats during the exercise bout. The concentration of hepatic fructose 2,6-bisphosphate remained elevated throughout the exercise bout in glucose-infused rats but decreased markedly in saline-infused rats. Plasma insulin concentration was higher and plasma glucagon concentration lower in glucose-infused rats than in saline-infused rats during exercise. Early in exercise, liver glycogenolysis proceeds in the glucose-infused rats despite the fact that glucose and insulin concentrations are markedly elevated and liver cAMP is unchanged from resting values. These observations suggest the existence of a cAMP-independent feedforward system for activation of liver glycogenolysis that can override classical negative feedback mechanisms during exercise.     

Adenoviral vector-mediated glucagon-like peptide 1 gene therapy improves glucose homeostasis in Zucker diabetic fatty rats

BACKGROUND: Glucagon-like peptide-1 (GLP-1) is a gut-derived incretin hormone that plays an important role in glucose homeostasis. Its functions include glucose-stimulated insulin secretion, suppression of glucagon secretion, deceleration of gastric emptying, and reduction in appetite and food intake. Despite the numerous antidiabetic properties of GLP-1, its therapeutic potential is limited by its short biological half-life due to rapid enzymatic degradation by dipeptidyl peptidase IV. The present study aimed to demonstrate the therapeutic effects of constitutively expressed GLP-1 in an overt type 2 diabetic animal model using an adenoviral vector system. METHODS: A novel plasmid (pAAV-ILGLP-1) and recombinant adenoviral vector (Ad-ILGLP-1) were constructed with the cytomegalovirus promoter and insulin leader sequence followed by GLP-1(7-37) cDNA. RESULTS: The results of an enzyme-linked immunosorbent assay showed significantly elevated levels of GLP-1(7-37) secreted by human embryonic kidney cells transfected with the construct containing the leader sequence. A single intravenous administration of Ad-ILGLP-1 into 12-week-old Zucker diabetic fatty (ZDF) rats, which have overt type 2 diabetes mellitus (T2DM), achieved near normoglycemia for 3 weeks and improved utilization of blood glucose in glucose tolerance tests. Circulating plasma levels of GLP-1 increased in GLP-1-treated ZDF rats, but diminished 21 days after treatment. When compared with controls, Ad-ILGLP-1-treated ZDF rats had a lower homeostasis model assessment for insulin resistance score indicating amelioration in insulin resistance. Immunohistochemical staining showed that cells expressing GLP-1 were found in the livers of GLP-1-treated ZDF rats. CONCLUSIONS: These data suggest that GLP-1 gene therapy can improve glucose homeostasis in fully developed diabetic animal models and may be a promising treatment modality for T2DM in humans.     

Hepatic glycogen synthesis from duodenal glucose and alanine. An in situ 13C NMR study

An in situ and in vivo surface coil 13C NMR study was performed to study hepatic glycogen synthesis from [3-13C]alanine and [1-13C]glucose administered by intraduodenal infusion in 18-h fasted male Sprague-Dawley rats. Combined, equimolar amounts of alanine and glucose were given. Hepatic appearance and disappearance of substrate and concurrent glycogen synthesis was followed over 150 min, with 5-min time resolution. Active glycogen synthesis from glucose via the direct (glucose----glycogen) and indirect (glucose----lactate----glycogen) pathways and from alanine via gluconeogenesis was observed. The indirect pathway of glycogen synthesis from [1-13C]glucose accounted for 30% (+/- 6 S.E.) of total glycogen formed from labeled glucose. This estimate does not take into account dilution of label in the hepatic oxaloacetate pool and is, therefore, somewhat uncertain. Hepatic levels of [3-13C]alanine achieved were significantly lower than levels of [1-13C]glucose in the liver, and the period of active glycogen synthesis from [3-13C]alanine was longer than from glucose. However, the overall pseudo-first-order rate constant during the period of active glycogen synthesis from [3-13C]alanine (0.075 min-1 +/- 0.026 S.E.) was almost 3 times that from [1-13C]glucose via the direct pathway (0.025 min-1 +/- 0.005 S.E.). The most likely reason for the small rate constant governing direct glycogen formation from duodenally administered glucose compared to that from duodenally administered alanine is a low level of glucose phosphorylating capacity in the liver.     

Metabolism of glucose in hyper- and hypo-thyroid rats in vivo. Glucose-turnover values and futile-cycle activities obtained with 14C- and 3H-labelled glucose.

1. A trace amount of glucose labelled with 14C uniformly and with 3H at position 2, 3 or 6 was injected intravenously into starved rats to measure the turnover rate of blood glucose. 2. Reliable estimates were made based on the semilogarithmic plot of specific radioactivity of the glucose contained in whole blood samples taken from the tail vein. 3. Glucose turned over more rapidly in hyperthyroid and more slowly in hypothyroid than in euthyroid rats. The percentage contribution of glucose recycling (determined from the difference in replacement rates between [U-14C]glucose and [6-3H]glucose) to the glucose utilization increased on induction of hyperthyroidism. 4. Futile cycles between glucose and glucose 6-phosphate (determined from the difference between replacement rates of [2-3H]glucose and [6-3H]glucose) were activated and inactivated by induction of hyperthyroid and hypothyroid states respectively. 5. The hepatic content of glycogen was much lower in hyper- and hypo-thyroid than in euthyroid rats. The enhanced glucose production in hyperthyroid rats resulted from not only activationof hepatic gluconeogenesis but also diversion of the final product of gluconeogenesis from liver glycogen to blood glucose. In hypothyroidism, the inhibition of gluconeogensis led to suppression of both glucose production and glycogenesis in the liver.     

Glucose phosphorylation is not rate limiting for accumulation of glycogen from glucose in perfused livers from fasted rats

Incorporation of Glc and Fru into glycogen was measured in perfused livers from 24-h fasted rats using [6-3H]Glc and [U-14C]Fru. For the initial 20 min, livers were perfused with low Glc (2 mM) to deplete hepatic glycogen and were perfused for the following 30 min with various combinations of Glc and Fru. With constant Fru (2 mM), increasing perfusate Glc increased the relative contribution of Glc carbons to glycogen (7.2 +/- 0.4, 34.9 +/- 2.8, and 59.1 +/- 2.7% at 2, 10, and 20 mM Glc, respectively; n = 5 for each). During perfusion with substrate levels seen during refeeding (10 mM Glc, 1.8 mumol/g/min gluconeogenic flux from 2 mM Fru), Fru provided 54.7 +/- 2.7% of the carbons for glycogen, while Glc provided only 34.9 +/- 2.8%, consistent with in vivo estimations. However, the estimated rate of Glc phosphorylation was at least 1.10 +/- 0.11 mumol/g/min, which exceeded by at least 4-fold the glycogen accumulation rate (0.28 +/- 0.04 mumol of glucose/g/min). The total rate of glucose 6-phosphate supply via Glc phosphorylation and gluconeogenesis (2.9 mumol/g/min) exceeded reported in vivo rates of glycogen accumulation during refeeding. Thus, in perfused livers of 24-h fasted rats there is an apparent redundancy in glucose 6-phosphate supply. These results suggest that the rate-limiting step for hepatic glycogen accumulation during refeeding is located between glucose 6-phosphate and glycogen, rather than at the step of Glc phosphorylation or in the gluconeogenic pathway.     

Increased hepatic fructose 2,6-bisphosphate after an oral glucose load does not affect gluconeogenesis

The generally accepted metabolic concept that fructose 2,6-bisphosphate (Fru-2,6-P2) inhibits gluconeogenesis by directly inhibiting fructose 1,6-bisphosphatase is based entirely on in vitro observations. To establish whether gluconeogenesis is indeed inhibited by Fru-2,6-P2 in intact animals, a novel NMR method was developed using [U-13C]glucose and 2H2O as tracers. The method was used to estimate the sources of plasma glucose from gastric absorption of oral [U-13C]glucose, from gluconeogenesis, and from glycogen in 24-h fasted rats. Liver Fru-2,6-P2 increased approximately 10-fold shortly after the glucose load, reached a maximum at 60 min, and then dropped to base-line levels by 150 min. The gastric contribution to plasma glucose reached approximately 50% at 30 min after the glucose load and gradually decreased thereafter. Although the contribution of glycogen to plasma glucose was small, glucose formed from gluconeogenesis was substantial throughout the study period even when liver Fru-2,6-P2 was high. Liver glycogen repletion was also brisk throughout the study period, reaching approximately 30 micromol/g at 3 h. These data demonstrate that Fru-2,6-P2 does not inhibit gluconeogenesis significantly in vivo.     

Effects of hormone and glucose administration on hepatic glucose and glycogen metabolism in vivo. A 13C NMR study

Effects of peripheral venous injection of glucagon and insulin on [1-13C]glucose incorporation into hepatic glycogen of rats were studied by 13C NMR in vivo. Each animal was given a continuous somatostatin infusion and a 100-mg intravenous injection of [1-13C] glucose in NMR experiments or unlabeled glucose in parallel experiments for determination of serum glucose. Insulin administration caused serum glucose to fall below basal levels and accelerated the loss of hepatic [1-13C]glucose; these effects were counteracted by the addition of glucagon. Glucagon administration alone did not affect serum glucose or hepatic [1-13C] glucose but caused the loss of [1-13C]glucose from glycogen and inhibited [1-13C]glucose incorporation into glycogen. Insulin did not alter [1-13C]glucose incorporation into glycogen when given alone or in combination with glucagon. The data are consistent with a model in which liver glycogen synthesis increases linearly with hepatic glucose concentration above a threshold glucose concentration. Insulin did not alter the rate constant or the threshold for synthesis.     

Analysis of glucose metabolism in farmed European sea bass (Dicentrarchus labrax L.) using deuterated water

Glucose metabolism in free-swimming fasted and fed seabass was studied using deuterated water ((2)H(2)O). After transfer to seawater enriched with 4.9% (2)H(2)O for 6-h or for 72-h, positional and mole percent enrichment (MPE) of plasma glucose and water were quantified by (2)H NMR and ESI-MS/MS. Plasma water (2)H-enrichment reached that of seawater within 6h. In both fasted and fed fish, plasma glucose MPE increased asymptotically attaining ~55% of plasma water enrichment by 72 h. The distribution of (2)H-enrichment between the different glucose positions was relatively uniform. The gluconeogenic contribution to glucose that was synthesized during (2)H(2)O administration was estimated from the ratio of position 5 and 2 glucose enrichments. For both fed and fasted fish, gluconeogenesis accounted for 98±1% of the glucose that was produced during the 72-h (2)H(2)O administration period. For fasted fish, gluconeogenic contributions measured after 6h were identical to 72-h values (94±3%). For fed fish, the apparent gluconeogenic contribution at 6-h was significantly lower compared to 72-h (79±5% versus 98±1%, p<0.05). This may reflect a brief augmentation of gluconeogenic flux by glycogenolysis after feeding and/or selective enrichment of plasma glucose position 2 via futile glucose-glucose-6-phosphate cycling.     

Structure and metabolism of mammalian liver glycogen monitored by carbon-13 nuclear magnetic resonance

Natural-abundance 13C NMR signals from glycogen are observable in situ within the perfused livers of rats. The nuclear magnetic relaxation properties (T1, T2, eta + 1) of glycogen were measured for glycogen in situ and in vitro and were found to be identical. All of the carbon nuclei in glycogen contribute to the high-resolution NMR spectrum, in spite of glycogen's very large molecular weight. The metabolism of glycogen in situ in the perfused rat liver was followed by 13C NMR. Stimulation of the fed rat liver by physiological glucagon levels led to rapid glycogenolysis. Perfusion of the liver with [1-13C]glucose led to net glycolysis, with concomitant scrambling of the label from C1 to C6 due to triosephosphate isomerase activity.     

Quantitative analysis of the pathways of glycogen repletion in periportal and perivenous hepatocytes in vivo.

In order to examine the pathways of hepatic glycogen repletion in the periportal and perivenous zones of the liver, [1-13C]glucose (99% enriched) was infused intraduodenally into conscious, 24-h fasted rats for 3 h. The liver was then quickly perfused in situ, and the cytoplasmic contents of the periportal and perivenous hepatocytes were selectively sampled by modification of the dual-digitonin-pulse technique (Quistorff, B., and Grunnet, N. (1987) Biochem. J. 243, 87-95). The 13C isotopic enrichment at each carbon position of the glucosyl units of hepatic glycogen was determined by 13C NMR and that of the C-1 position by gas chromatography-mass spectroscopy. From comparison of hepatic glycogen repleted by direct incorporation of plasma glucose (glucose----glucose-6-P----glucose-1-P----UDP-glucose----glycogen) was calculated to be 29% in the periportal zone and 35% in the perivenous zone, assuming equal glycogen synthetic rates within the two zones. Thus, the majority of glycogen is derived by an indirect route (glucose--------3-carbon unit--------glucose --------UDP-glucose--------glycogen) in both the periportal zone and in the perivenous zone. In conclusion, in a 24-h fasted rat there does not appear to be a major difference between the periportal and perivenous hepatocytes in the percent of glycogen synthesized by the direct pathway following a glucose load.