Hepatic de novo synthesis of glucose 6-phosphate is not affected in peroxisome proliferator-activated receptor alpha-deficient mice but is preferentially directed toward hepatic glycogen stores after a short term fast

Apart from impaired beta-oxidation, Pparalpha-deficient (Pparalpha(-/-)) mice suffer from hypoglycemia during prolonged fasting, suggesting alterations in hepatic glucose metabolism. We compared hepatic glucose metabolism in vivo in wild type (WT) and Pparalpha(-/-) mice after a short term fast, applying novel isotopic methods. After a 9-h fast, mice were infused with [U-(13)C]glucose, [2-(13)C]glycerol, [1-(2)H]galactose, and paracetamol for 6 h, and blood and urine was collected in timed intervals. Plasma glucose concentrations remained constant and were not different between the groups. Hepatic glycogen content was 69 +/- 11 and 90 +/- 31 microM/g liver after 15 h of fasting in WT and Pparalpha(-/-) mice, respectively. The gluconeogenic flux toward glucose 6-phosphate was not different between the groups (i.e. 157 +/- 9 and 153 +/- 9 microM/kg/min in WT and Pparalpha(-/-) mice, respectively). The gluconeogenic flux toward plasma glucose, however, was decreased in PPARalpha(-/-) mice (i.e. 142 +/- 9 versus 124 +/- 13 microM/kg/min) (p < 0.05), accounting for the observed decrease (-15%) in hepatic glucose production in Pparalpha(-/-) mice. Expression of the gene encoding glucose-6-phosphate hydrolase (G6ph) was lower in the PPARalpha(-/-) mice compared with WT mice. In conclusion, Pparalpha(-/-) mice were able to maintain a normal total gluconeogenic flux to glucose 6-phosphate during moderate fasting, despite their inability to up-regulate beta-oxidation. However, this gluconeogenic flux was directed more toward glycogen, leading to a decreased hepatic glucose output. This was associated with a down-regulation of the expression of G6ph in PPARalpha-deficient mice.     

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.     

Blood glycerol is an important precursor for intramuscular triacylglycerol synthesis.

The utilization of blood glycerol and glucose as precursors for intramuscular triglyceride synthesis was examined in rats using an intravenous infusion of [2-(14)C]glycerol and [6-(3)H]glucose or [6-(14)C]glucose. In 24-h fasted rats, more glycerol than glucose was incorporated into intramuscular triglyceride glycerol in soleus (69 +/- 23 versus 4 +/- 1 nmol/micromol triglyceride/h, respectively, p = 0.02 glycerol versus glucose) and in gastrocnemius (25 +/- 5 versus 9 +/- 2 nmol/micromol triglyceride/h, respectively, p = 0.02). Blood glucose was utilized more than blood glycerol for triglyceride glycerol synthesis in quadriceps. In fed rats, the blood glycerol incorporation rates (4 +/- 2, 8 +/- 3, and 9 +/- 3 nmol/micromol triglyceride/h) were similar (p > 0.3) to those of glucose (5 +/- 2, 8 +/- 2, and 5 +/- 2 nmol/micromol triglyceride/h for quadriceps, gastrocnemius, and soleus muscle, respectively). Glucose incorporation into intramuscular triglycerides was less with [6-(3)H]glucose than with [6-(14)C]glucose, suggesting an indirect pathway for glucose carbon entry into muscle triglyceride. The isotopic equilibrium between plasma and intramuscular free glycerol ([U-(13)C]glycerol) was complete in quadriceps and gastrocnemius, but not soleus, within 2 h after beginning the tracer infusion. We conclude that blood glycerol is a direct and important precursor for muscle triglyceride synthesis in rats, confirming the presence of functionally important amounts of glycerol kinase in skeletal muscle.     

Alterations in carbohydrate metabolism and its regulation in PPARalpha null mouse hearts

Although a shift from fatty acids (FAs) to carbohydrates (CHOs) is considered beneficial for the diseased heart, it is unclear why subjects with FA beta-oxidation defects are prone to cardiac decompensation under stress conditions. The present study investigated potential alterations in the myocardial utilization of CHOs for energy production and anaplerosis in 12-wk-old peroxisome proliferator-activating receptor-alpha (PPARalpha) null mice (a model of FA beta-oxidation defects). Carbon-13 methodology was used to assess substrate flux through energy-yielding pathways in hearts perfused ex vivo at two workloads with a physiological substrate mixture mimicking the fed state, and real-time RT-quantitative polymerase chain reaction was used to document the expression of selected metabolic genes. When compared with that from control C57BL/6 mice, isolated working hearts from PPARalpha null mice displayed an impaired capacity to withstand a rise in preload (mimicking an increased venous return as it occurs during exercise) as reflected by a 20% decline in the aortic flow rate. At the metabolic level, beyond the expected shift from FA (5-fold down) to CHO (1.5-fold up; P < 0.001) at both preloads, PPARalpha null hearts also displayed 1) a significantly greater contribution of exogenous lactate and glucose and/or glycogen (2-fold up) to endogenous pyruvate formation, whereas that of exogenous pyruvate remained unchanged and 2) marginal alterations in citric acid cycle-related parameters. The lactate production rate was the only measured parameter that was affected differently by preloads in control and PPARalpha null mouse hearts, suggesting a restricted reserve for the latter hearts to enhance glycolysis when the energy demand is increased. Alterations in the expression of some glycolysis-related genes suggest potential mechanisms involved in this defective CHO metabolism. Collectively, our data highlight the importance of metabolic alterations in CHO metabolism associated with FA oxidation defects as a factor that may predispose the heart to decompensation under stress conditions even in the fed state.     

Glyceroneogenesis and the supply of glycerol-3-phosphate for glyceride-glycerol synthesis in liver slices of fasted and diabetic rats

The pathways of glycerol-3-phosphate (G3P) generation for glyceride synthesis were examined in precision-cut liver slices of fasted and diabetic rats. The incorporation of 5 mM [U-(14)C]glucose into glyceride-glycerol, used to evaluate G3P generation via glycolysis, was reduced by approximately 26-36% in liver slices of fasted and diabetic rats. The glycolytic flux was reduced by approximately 60% in both groups. The incorporation of 1.0 mM [2-(14)C]pyruvate into glyceride-glycerol (glyceroneogenesis) increased approximately 50% and approximately 36% in slices of fasted and diabetic rats, respectively, which also showed a two-fold increase in the activity phosphoenolpyruvate carboxykinase. The increased incorporation of 1.0 mM [2-(14)C]pyruvate into glyceride-glycerol by slices of fasted rats was not affected by the addition of 5 mM glucose to the incubation medium. The activity of glycerokinase and the incorporation of 1 mM [U-(14)C]glycerol into glyceride-glycerol, evaluators of G3P formation by direct glycerol phosphorylation, did not differ significantly from controls in slices of the two experimental groups. Rates of incorporation of 1 mM [2-(14)C]pyruvate and [U-(14)C]glycerol into glucose of incubation medium (gluconeogenesis) were approximately 140 and approximately 20% higher in fasted and diabetic slices than in control slices. It could be estimated that glyceroneogenesis by liver slices of fasted rats contributed with approximately 20% of G3P generated for glyceride-glycerol synthesis, the glycolytic pathway with approximately 5%, and direct phosphorylation of glycerol by glycerokinase with approximately 75%. Pyruvate contributed with 54% and glycerol with 46% of gluconeogenesis. The present data indicate that glyceroneogenesis has a significant participation in the generation of G3P needed for the increased glyceride-glycerol synthesis in liver during fasting and diabetes.     

Accelerated hepatic glycerol synthesis in rainbow smelt (Osmerus mordax) is fuelled directly by glucose and alanine: a 1H and 13C nuclear magnetic resonance study

At seawater temperatures below 1 degrees C, rainbow smelt (Osmerus mordax) accumulate plasma levels of glycerol up to 400 mM. Aspects of the synthesis of glycerol in liver and its regulation were previously investigated, but the pathways leading to glycerol synthesis remained unconfirmed. Here, we report nuclear magnetic resonance (NMR) studies which elucidate, in more detail, the fuel sources for rapid glycerol synthesis in rainbow smelt. Initial NMR analysis of liver homogenates from fish held at cold (-1 degrees C) temperatures and from fish transferred from 8 degrees C to -1 degrees C showed elevated glycerol, whereas those from fish held at 8 degrees C had far lower glycerol levels. These results confirm a temperature-responsive glycerol synthesis and show that NMR is a suitable approach to investigate the phenomenon. Further studies with fish held at low temperature and injected with labelled L-[2,3-(13)C(2)] alanine or D-[U-(13)C(6)]glucose revealed conversion of both alanine and glucose to glycerol. (13)C spectra showed satellites ((1)J(CC)=41.1 Hz) about the glycerol resonances indicating intact incorporation of a (13)C-(13)C unit in liver glycerol of fish injected with L-[2,3-(13)C(2)]alanine and a (13)C-(13)C-(13)C unit in liver glycerol of fish injected with D[U-(13)C(6)]glucose. Thus, glycerol can be efficiently produced directly from amino acid precursors by glyceroneogenesis, which is an abbreviated gluconeogenesis process leading to glycerol through dihydroxyacetone phosphate (DHAP). Glucose can also be metabolised to glycerol via an abbreviated form of glycolysis that similarly leads to glycerol through DHAP.     

Network flux analysis: impact of 13C-substrates on metabolism in Arabidopsis thaliana cell suspension cultures

The aim of this study was to test the assumption that (13)C-enrichment of respiratory substrate does not perturb metabolism. Cell suspension cultures of Arabidopsis thaliana were grown in MS medium containing unlabelled glucose (with (13)C at natural abundance), 100% [1-(13)C]glucose, 100% [U-(13)C(6)]glucose or 10% [U-(13)C(6)]glucose plus 90% unlabelled glucose. There was no significant difference in the metabolism of [U-(14)C]glucose between the cultures. Similarly, the pattern of (14)CO(2) release from specifically labelled [(14)C]-substrates was unaffected. Principal component analysis of (13)C-decoupled (1)H NMR metabolite fingerprints of cell extracts was unable to discriminate between the different culture conditions. It is concluded that (13)C-enrichment of the growth substrate has no effect on flux through the central pathways of carbon metabolism in higher plants. This conclusion supports the implicit assumption in metabolic flux analysis that steady-state (13)C-labelling does not perturb fluxes through the reactions of the metabolic network it seeks to quantify.     

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.     

Tricarboxylic acid cycle inhibition by Li+ in the human neuroblastoma SH-SY5Y cell line: a 13C NMR isotopomer analysis

Li+ effects on glucose metabolism and on the competitive metabolism of glucose and lactate were investigated in the human neuroblastoma SH-SY5Y cell line using 13C NMR spectroscopy. The metabolic model proposed for glucose and lactate metabolism in these cells, based on tcaCALC best fitting solutions, for both control and Li+ conditions, was consistent with: (i) a single pyruvate pool; (ii) anaplerotic flux from endogenous unlabelled substrates; (iii) no cycling between pyruvate and oxaloacetate. Li+ was shown to induce a 38 and 53% decrease, for 1 and 15 mM Li+, respectively, in the rate of glucose conversion into pyruvate, when [U-13C]glucose was present, while no effects on lactate production were observed. Pyruvate oxidation by the tricarboxylic acid cycle and citrate synthase flux were shown to be significantly reduced by 64 and 84% in the presence of 1 and 15 mM Li+, respectively, suggesting a direct inhibitory effect of Li+ on tricarboxylic acid cycle flux. This work also showed that when both glucose and lactate are present as energetic substrates, SH-SY5Y cells preferentially consumed exogenous lactate over glucose, as 62% of the acetyl-CoA was derived from [3-13C]lactate while only 26% was derived from [U-13C]glucose. Li+ did not significantly affect the relative utilisation of these two substrates by the cells or the residual contribution of unlabelled endogenous sources for the acetyl-CoA pool.     

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.     

Isotopomer analysis of myocardial substrate metabolism: a systems biology approach

The increasing accessibility of mass isotopomer data via GC-MS and NMR technology has necessitated the use of a systematic and reliable method to take advantage of such data for flux analysis. Here we applied a nonlinear, optimization-based method to study substrate metabolism in cardiomyocytes using (13)C data from perfused mouse hearts. The myocardial metabolic network used in this study accounts for 257 reactions and 240 metabolites, which are further compartmentalized into extracellular space, cytosol, and mitochondrial matrix. Analysis of the perfused mouse heart showed that the steady-state ATP production rate was 16.6 +/- 2.3 micromol/min . gww, with 30% of the ATP coming from glycolysis. Of the four substrates available in the perfusate (glucose, pyruvate, lactate, and oleate), exogenous glucose forms the majority of cytosolic pyruvate. Pyruvate decaboxylation is significantly higher than carboxylation, suggesting that anaplerosis is low in the perfused heart. Exchange fluxes were predicted to be high for reversible enzymes in the citric acid cycle (CAC), but low in the glycolytic pathway. Pseudoketogenesis amounted to approximately 50% of the net ketone body uptake. Sensitivity analysis showed that the estimated flux distributions were relatively insensitive to experimental errors. The application of isotopomer data drastically improved the estimation of reaction fluxes compared to results computed with respect to reaction stoichiometry alone. Further study of 12 commonly used (13)C glucose mixtures showed that the mixtures of 20% [U-(13)C(6)] glucose, 80% [3 (13)C] glucose and 20% [U-(13)C(6)] glucose, 80% [4 (13)C] were best for resolving fluxes in the current network.     

Stimulation of glycogen synthesis by proglycosyn (LY177507) by isolated hepatocytes of normal and streptozotocin diabetic rats

The phenacylimidazolium compound LY177507 was shown by Harris et al. (Harris, R. A., Yamanuchi, K., Roach, P. J., Yen, T. T., Dominiani, S. J., and Stephens, T. W. (1989) J. Biol. Chem. 264, 14674-14680) to stimulate glycogen synthesis greatly in isolated rat hepatocytes. We extended studies with this compound, designated proglycosyn (Yamaguchi, K., Stephens, T. W., Chikadar, K., Depaoli-Roach, A., And Harris, R. A. (1991) Diabetes 40, (Suppl. 1) 102 (abstr.] employing hepatocytes from normal and streptozotocin diabetic rats. Proglycosyn is more effective than amino acids in stimulating glycogen synthesis. In cells incubated with glucose, lactate, or dihydroxyacetone the effect of glutamine and proglycosyn was synergistic. In cells incubated with glucose plus lactate, or glucose plus dihydroxyacetone, the stimulation by the two agonists was additive. Proglycosyn diverted the gluconeogenic flux from glucose to glycogen. The maximal rates of glycogen deposition attained in the presence of glutamine and proglycosyn from cells incubated with glucose plus lactate, or glucose plus dihydroxyacetone, where about 80 and 110 mumols/h/g of liver, respectively. Proglycosyn depressed glycogenolysis in hepatocytes of fed rats and stimulated glycogen synthesis from lactate and dihydroxyacetone. The incorporation of [U-14C]glucose and [U-14C]lactate in these cells occurred in the presence of glycogen breakdown or exceeded net production, indicating the occurrence of recycling of glycogen in hepatocytes of fed rats. Hepatocytes from fasted streptozotocin diabetic rats contained high levels of glycogen. Glycogenolysis was markedly depressed by proglycosyn. Glycogen synthesis from lactate and dihydroxyacetone in these cells was stimulated by glutamine and proglycosyn in a fashion similar to that in cells from fasted control rats, and the rates of glycogen synthesis were similar in cells of control and diabetic rats. With glucose as sole substrate, glutamine did not stimulate glycogen synthesis. When both agonists were present, there was a marked synergism and substantial glycogen formation. Streptozotocin diabetic rats prior to the onset of cachexia have a normal capacity for glycogen synthesis.     

Dependence with nutritional status of the ethanol effects on fatty acid metabolism in rat hepatocytes

1. The effects of ethanol on fatty acid synthesis, esterification and oxidation were studied in hepatocytes isolated from fed and 24 hr fasted rats. 2. [3H]H2O was preferentially incorporated into the glycerol backbone of triglycerides and phospholipids. Addition of ethanol markedly increased the incorporation of this label in both classes of glycerolipids; the increase was higher in fasted rat hepatocytes, both in the glycerol backbone and acyl groups of glycerolipids. 3. Ethanol increased [U-14C]palmitate incorporation into triglycerides only in hepatocytes from fasted rats. 4. [14C]CO2 and total acid soluble product formation from [1-14C]palmitate resulted inhibited by ethanol both in the fed and the fasted state.     

Comparative utilization of glycerol and alanine as liver gluconeogenic substrates in the fed late pregnant rat

The appearance of plasma [14C]glucose in the inferior cava vein after a pulse of 0.2 mmol of [U-14C]L-alanine or [U-14C]glycerol/200 g body wt given through the portal vein was studied in fed 21 day pregnant rats and virgin controls under pentobarbital anesthesia. In both groups values were much higher when [U-14C]glycerol was the administered tracer than when [U-14C]L-alanine, and they were augmented in pregnant versus virgin animals at 1 min when receiving [U-14C]glycerol and at 2 min when receiving [U-14C]L-alanine. 20 min after the tracers rats receiving [U-14C]glycerol showed much higher liver [14C]glycogen and [14C]glyceride glycerol than those receiving [U-14C]L-alanine. Radioactivity present in liver as [14C]glyceride glycerol was greater in pregnant than in virgin rats receiving [U-14C]glycerol whereas radioactivity corresponding to [14C]fatty acids was lower in the former group receiving either tracer. At 20 min after maternal treatments fetuses showed lower plasma [14C]glycerol than [14C]alanine values but plasma [14C]glucose and liver [14C]glycogen values were much greater in fetuses from mothers receiving [U-14C]glycerol than [U-14C]L-amine. Besides showing the higher gluconeogenic efficiency in pregnant than in virgin rats, results indicate that at late gestation glycerol is used as a preferential substrate for both glucose and glyceride glycerol synthesis in liver.     

13C/31P NMR assessment of mitochondrial energy coupling in skeletal muscle of awake fed and fasted rats. Relationship with uncoupling protein 3 expression

To examine the relationship between mitochondrial energy coupling in skeletal muscle and change in uncoupling protein 3 (UCP3) expression during the transition from the fed to fasted state, we used a novel noninvasive (31)P/(13)C NMR spectroscopic approach to measure the degree of mitochondrial energy coupling in the hind limb muscles of awake rats before and after a 48-h fast. Compared with fed levels, UCP3 mRNA and protein levels in the gastrocnemius increased 1.7- (p < 0.01) and 2.9-fold (p < 0.001), respectively, following a 48-h fast. Tricarboxylic acid cycle flux measured using (13)C NMR as an index of mitochondrial substrate oxidation was 212 +/- 23 and 173 +/- 25 nmol/g/min (p not significant) in the fed and 48-h fasted groups, respectively. Unidirectional ATP synthesis flux measured using (31)P NMR was 79 +/- 15 and 57 +/- 9 nmol/g/s (p not significant) in the fed and 48-h fasted groups, respectively. Mitochondrial energy coupling as expressed by the ratio of ATP synthesis to tricarboxylic acid cycle flux was not different between the fed and fasted states. To test the hypothesis that UCP3 may be involved in the translocation of long chain free fatty acids (FFA) into the mitochondrial matrix under conditions of elevated FFA availability, [U-(13)C]palmitate/albumin was administered in a separate group of rats with (+) or without (-) etomoxir (an inhibitor of carnitine palmitoyltransferase I). The ratio of glutamate enrichment ((+) etomoxir/(-) etomoxir) in the hind limb muscles was the same between groups, indicating that UCP3 does not appear to function as a translocator for long chain FFA in skeletal muscle following a 48-h fast. In summary, these data demonstrate that despite a 2-3-fold increase in UCP3 mRNA and protein expression in skeletal muscle during the transition from the fed to fasted state, mitochondrial energy coupling does not change. Furthermore, UCP3 does not appear to have a major role in FFA translocation into the mitochondria. The physiological role of UCP3 following a 48-h fast in skeletal muscle remains to be elucidated.     

Glial Formation of Pyruvate and Lactate from TCA Cycle Intermediates: Implications for the Inactivation of Transmitter Amino Acids?

Abstract: Cerebral formation of lactate via the tricarboxylic acid (TCA) cycle was investigated through the labeling of lactate from [2-¹³C]acetate and [1-¹³C]glucose as shown by ¹³C NMR spectroscopy. In fasted mice that had received [2-¹³C]acetate intravenously, brain lactate C-2 and C-3 were labeled at 5, 15, and 30 min, reflecting formation of pyruvate and hence lactate from TCA cycle intermediates. In contrast, [1-¹³C]glucose strongly labeled lactate C-3, reflecting glycolysis, whereas lactate C-2 was weakly labeled only at 15 min. These data show that formation of pyruvate, and hence lactate, from TCA cycle intermediates took place predominantly in the acetate-metabolizing compartment, i.e., glia. The enrichment of total brain lactate from [2-¹³C]acetate reached ∼1% in both the C-2 and the C-3 position in fasted mice. It was calculated that this could account for 20% of the lactate formed in the glial compartment. In fasted mice, there was no significant difference between the labeling of lactate C-2 and C-3 from [2-¹³C]acetate, whereas in fed mice, lactate C-3 was more highly labeled than the C-2, reflecting adaptive metabolic changes in glia in response to the nutritional state of the animal. It is hypothesized that conversion of TCA cycle intermediates into pyruvate and lactate may be operative in the glial metabolism of extracellular glutamate and GABA in vivo. Given the vasodilating effect of lactate on cerebral vessels, which are ensheathed by astrocytic processes, conversion of glutamate and GABA into lactate could be one mechanism mediating increases in cerebral blood flow during nervous activity.     

Effect of macronutrients, age, and obesity on 6- and 24-h postprandial glucose metabolism in cats

Obesity and age are risk factors for feline diabetes. This study aimed to test the hypothesis that age, long-term obesity, and dietary composition would lead to peripheral and hepatorenal insulin resistance, indicated by higher endogenous glucose production (EGP) in the fasted and postprandial state, higher blood glucose and insulin, and higher leptin, free thyroxine, and lower adiponectin concentrations. Using triple tracer-(2)H(2)O, [U-(13)C(3)] propionate, and [3,4-(13)C(2)] glucose infusion, and indirect calorimetry-we investigated carbohydrate and fat metabolic pathways in overnight-fasted neutered cats (13 young lean, 12 old lean, and 12 old obese), each fed three different diets (high protein with and without polyunsaturated fatty acids, and high carbohydrate) in a crossover design. EGP was lowest in fasted and postprandial obese cats despite peripheral insulin resistance, indicated by hyperinsulinemia. Gluconeogenesis was the most important pathway for EGP in all groups, but glycogen contributed significantly. Insulin and leptin concentrations were higher in old than in young lean cats; adiponectin was lowest in obese cats but surprisingly highest in lean old cats. Diet had little effect on metabolic parameters. We conclude that hepatorenal insulin resistance does not develop in the fasted or postprandial state, even in long-term obese cats, allowing the maintenance of euglycemia through lowering EGP. Glycogen plays a major role in EGP, especially in lean fasted cats, and in the postprandial state. Aging may predispose to insulin resistance, which is a risk factor for diabetes in cats. Mechanisms underlying the high adiponectin of healthy old lean cats need to be further explored.     

Studies of glucose production in sheep using (6-3H)glucose and (U-14C)glucose.

Simultaneous primed-continuous intravenous infusions of [6-3H]glucose and [U-14C]glucose were performed on 13 fed, 4 fasted, and 4 dexamethasone-treated sheep. In 10 of the experiments on fed sheep, glucagon or insulin was infused intraportally for 2 h after control values were obtained. The 3H-labeled glucose gave glucose production values that were only 4.4 +/- 0.5, 5.4 +/- 1.0, and 5.8 +/- 0.8% higher than 14C-labeled glucose in the normal fed, fasted, and dexamethasone-treated sheep, respectively. Glucagon or insulin infusions did not significantly alter this recycling. It is condluced that a recycling of glucose carbon through metabolic intermediates is minimal in the sheep as compared with other species and also that it is not significantly altered by fasting or by hormones that affect glucose production.     

Studies on lipogenesis in vivo. Lipogenesis during extended periods of re-feeding after starvation

1. Lipogenesis was studied in mice re-fed for up to 21 days after starvation. At appropriate times [U-(14)]glucose was given by stomach tube and incorporation of (14)C into various lipid fractions measured. 2. In mice starved for 48hr. and then re-fed for 4 days with a diet containing 1% of corn oil, incorporation of (14)C from [U-(14)C]glucose into liver fatty acids and cholesterol was respectively threefold and eightfold higher than in controls fed ad libitum. The percentages by weight of fatty acids and cholesterol in the liver also increased and reached peaks after 7 days. Both the radioactivity and weights of the fractions returned to control values after 10-14 days' re-feeding. These changes could be diminished by re-feeding the mice with a diet containing 20% of corn oil. Incorporation of (14)C from [U-(14)C]glucose into extrahepatic fatty acids (excluding those of the epididymal fat pads) was not elevated during re-feeding with a diet containing either 1% or 20% of corn oil. However, incorporation of (14)C from [U-(14)C]glucose into the fatty acids of the epididymal fat pads was increased in mice re-fed with either diet, as compared with non-starved controls. 3. Lipogenesis was also studied in mice alternately fed and starved. Mice given a diet containing 1% of corn oil for 6hr./day for 4 weeks lost weight initially and never attained the weight or carcass fat content of controls fed ad libitum. Incorporation of (14)C from dietary [U-(14)C]-glucose into the fatty acids of the epididymal fat pads was elevated threefold in the mice allowed limited access to food, although the incorporation into the remainder of the extrahepatic fatty acids was not different from that found for controls. Mice given a diet containing 20% of corn oil for 6hr./day adapted to the limited feeding regimen quicker and in 4 weeks did attain the weight and carcass fat content of controls. Incorporation of (14)C from [U-(14)C]glucose into the fatty acids of the epididymal fat pads and the remainder of the extrahepatic fatty acids was respectively fivefold and threefold higher than in controls fed ad libitum. 4. The elevation in liver lipogenesis during re-feeding was greatest on a diet containing 1% of corn oil, whereas in extrahepatic tissues the increase in lipogenesis was greater when the mice were re-fed or were allowed limited access to a diet containing 20% of corn oil. These results suggest that the causes of the increased rate of incorporation of (14)C from [U-(14)C]glucose into fatty acids during re-feeding may be different in liver from that in extrahepatic tissues.     

Formation of glycerol from glucose in rat brain and cultured brain cells. Augmentation with kainate or ischemia

An increase in the concentration of glycerol in the ischemic brain is assumed to reflect degradation of phospholipids of plasma membranes. However, glycerol could, theoretically, be formed from glucose, which after glycolytic conversion to dihydroxyacetone phosphate, could be converted to glycerol-3-phosphate and hence to glycerol. We show here that (13)C-labeled glycerol accumulate in incubation media of cultured cerebellar granule cells and astrocytes incubated with [(13)C]glucose, 3 mmol/L, demonstrating the formation of glycerol from glucose. Co-incubation of cerebellar granule cells with kainate, 50 micromol/L, led to increased glucose metabolism and increased accumulation of [(13)C]glycerol. Accumulation of [(13)C]glycerol and its precursor, [(13)C]glycerol-3-phosphate, was evident in brain, but not in serum, of kainate-treated rats that received [U-(13)C]glucose, 5 micromol/g bodyweight, intravenously and survived for 5 min. Global ischemia induced by decapitation also caused accumulation of [(13)C]glycerol and [(13)C]glycerol-3-phosphate. These results show that glycerol can be formed from glucose in brain; they also demonstrate the existence of a cerebral glycerol-3-phosphatase activity. Ischemia-induced increases in brain glycerol may, in part, reflect an altered metabolism of glucose, in which glycerol formation, like lactate formation, acts as a redox sink.