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.     

Hormonal regulation of the tricarboxylic acid cycle in the isolated perfused rat liver

The effect of Ca2+-mobilizing hormones, vasopressin, angiotensin II and the alpha-adrenergic agonist phenylephrine, on the metabolic flux through the tricarboxylic acid cycle was investigated in isolated perfused rat livers. All three Ca2+-mobilizing agonists stimulated 14CO2 production and gluconeogenesis in livers of 24-h-fasted rats perfused with [2-14C]pyruvate. Prazosin blocked the phenylephrine-elicited stimulation of 14CO2 and glucose production from [2-14C]pyruvate whereas the alpha 2-adrenergic agonist, BHT-933, did not affect the rates of 14CO2 and glucose production from [2-14C]pyruvate indicating that the phenylephrine-mediated response involved alpha 1-adrenergic receptors. Phenylephrine, vasopressin and angiotensin II stimulated 14CO2 production from [2-14C]acetate in livers derived from fed rats but not in livers of 24-h-fasted rats. In livers of 24-h-fasted rats, perfused with [2-14C]acetate, exogenously added pyruvate was required for an increase in the rate of 14CO2 production during phenylephrine infusion. This last observation suggests increased pyruvate carboxylation as one of the mechanisms involved in stimulation of tricarboxylic acid cycle activity by the Ca2+-mobilizing agonists, vasopressin, angiotensin II and phenylephrine.     

13C and 31P NMR study of gluconeogenesis: utilization of 13C-labeled substrates by perfused liver from streptozotocin-diabetic and untreated rats

The metabolism of 13C-labeled substrates was followed by 13C and 31P NMR in perfused liver from the streptozotocin-treated rat model of insulin-dependent diabetes. Comparison was made with perfused liver from untreated littermates, fasted either 24 or 12 h. The major routes of pyruvate metabolism were followed by a 13C NMR approach that provided for the determination of the metabolic fate of several substances simultaneously. The rate of gluconeogenesis was 2-4-fold greater and beta-hydroxybutyrate production was 50% greater in liver from the chronically diabetic rats as compared with the control groups. Large differences in the distribution of 13C label in hepatic alanine were measured between diabetic and control groups. The biosyntheses of 13C-labeled glutathione and N-carbamoylaspartate were monitored in time-resolved 13C NMR spectra of perfused liver. Assignments for the resonances of glutathione and N-carbamoylaspartate were made with the aid of 13C NMR studies of perchloric acid extracts of the freeze-clamped livers. 13C NMR spectroscopy of the perfusates provided a convenient, rapid assay of the rate of oxidation of [2-13C]ethanol, the hepatic output of [2-13C]acetaldehyde, and the accumulation of [2-13C]acetate in the perfusate. By 31P NMR spectroscopy, carbamoyl phosphate was measured in all diabetic livers and an unusual P,P'-diesterified pyrophosphate was observed in one-fourth of the diabetic livers examined. Neither of these phosphorylated metabolites was detected in control liver. Both 13C and 31P NMR were useful in defining changes in hepatic metabolism in experimental diabetes.     

13C NMR study of effects of fasting and diabetes on the metabolism of pyruvate in the tricarboxylic acid cycle and the utilization of pyruvate and ethanol in lipogenesis in perfused rat liver

13C NMR has been used to study the competition of pyruvate dehydrogenase with pyruvate carboxylase for entry of pyruvate into the tricarboxylic acid (TCA) cycle in perfused liver from streptozotocin-diabetic and normal donor rats. The relative proportion of pyruvate entering the TCA cycle by these two routes was estimated from the 13C enrichments at the individual carbons of glutamate when [3-13C]alanine was the only exogenous substrate present. In this way, the proportion of pyruvate entering by the pyruvate dehydrogenase route relative to the pyruvate carboxylase route was determined to be 1:1.2 +/- 0.1 in liver from fed controls, 1:7.7 +/- 2 in liver from 24-fasted controls, and 1:2.6 +/- 0.3 in diabetic liver. Pursuant to this observation that conversion of pyruvate to acetyl coenzyme A (acetyl-CoA) was greatest in perfused liver from fed controls, the incorporation of 13C label into fatty acids was monitored in this liver preparation. Livers were perfused under steady-state conditions with labeled substrates that are converted to either [2-13C]acetyl-CoA or [1-13C]acetyl-CoA, which in the de novo synthesis pathway label alternate carbons in fatty acids. With the exception of the repeating methylene carbons, fatty acyl carbons labeled by [1-13C]acetyl-CoA (from [2-13C]pyruvate) gave rise to resonances distinguishable on the basis of chemical shift from those observed when label was introduced by [3-13C]alanine plus [2-13C]ethanol, which are converted to [2-13C]acetyl-CoA. Thus, measurement of 13C enrichment at several specific sites in the fatty acyl chains in time-resolved spectra of perfused liver offers a novel way of monitoring the kinetics of the biosynthesis of fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of hepatic energy metabolism by epidermal growth factor

Employing the non-recirculating perfused rat liver preparation, we have investigated the regulation of hepatic gluconeogenesis, and metabolic fluxes through the tricarboxylic acid cycle and 2-oxoglutarate dehydrogenase reaction by epidermal growth factor (EGF) which mimics the actions of both insulin and Ca(2+)-mobilizing hormones (e.g. vasopressin). As monitored by the rate of 14CO2 production from [2-14C]pyruvate (0.5 mM), EGF (10 nM) transiently stimulated the activity of the tricarboxylic acid cycle. EGF also transiently stimulated hepatic gluconeogenesis from pyruvate. The transient stimulation of tricarboxylic acid cycle activity and gluconeogenesis were accompanied by an increase in perfusate Ca2+ content indicating that EGF also altered hepatic Ca2+ fluxes. EGF-elicited stimulation of gluconeogenesis was, at least in part, the result of a transient (50%) inhibition of pyruvate kinase activity. Likewise, EGF-mediated stimulation of tricarboxylic acid cycle activity can, in part, be attributed to EGF-elicited stimulation of metabolic flux through the mitochondrial, Ca(2+)-sensitive, 2-oxoglutarate dehydrogenase reaction. The regulation of hepatic metabolism by EGF appears to be the manifestation of alteration in cellular Ca2+ content since in experiments performed under conditions known to abolish the ability of EGF to alter cytosolic free-Ca2+ concentrations, i.e. in livers of pertussis-toxin-treated rats, EGF did not alter either perfusate Ca2+ content or any of the metabolic parameters monitored. Additionally, experiments involving pulsatile infusion of either EGF or phenylephrine into livers demonstrated that, unlike the alpha 1-adrenergic receptor, homologous desensitization of the EGF receptor occurs. Such a homologous desensitization of the EGF receptor can explain the transient nature of EGF-elicited stimulation of various metabolic processes. Since protein kinase C activation by EGF can lead to receptor desensitization, experiments were performed with phorbol esters which either activate or do not alter protein kinase C activity. While the inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate did not modulate the hepatic actions of EGF, activation of protein kinase C by 4 beta-phorbol 12-myristate 13-acetate (70 nM) abolished the ability of EGF to stimulate gluconeogenesis, tricarboxylic acid cycle activity and metabolic flux through the 2-oxoglutarate dehydrogenase complex.     

Brain pyruvate recycling and peripheral metabolism: an NMR analysis ex vivo of acetate and glucose metabolism in the rat

The occurrence of pyruvate recycling in the rat brain was studied in either pentobarbital anesthetized animals or awake animals receiving a light analgesic dose of morphine, which were infused with either [1-13C]glucose + acetate or glucose + [2-13C]acetate for various periods of time. Metabolite enrichments in the brain, blood and the liver were determined from NMR analyses of tissue extracts. They indicated that: (i) Pyruvate recycling was revealed in the brain of both the anesthetized and awake animals, as well as from lactate and alanine enrichments as from glutamate isotopomer composition, but only after infusion of glucose + [2-13C]acetate. (ii) Brain glucose was labelled from [2-13C]acetate at the same level in anaesthetized and awake rats (approximately 4%). Comparing its enrichment with that of blood and liver glucose indicated that brain glucose labelling resulted from hepatic gluconeogenesis. (iii) Analysing glucose 13C-13C coupling in the brain, blood and the liver confirmed that brain glucose could be labelled in the liver through the activities of both pyruvate recycling and gluconeogenesis. (iv) The rate of appearance and the amount of brain glutamate C4-C5 coupling, a marker of pyruvate recycling when starting from [2-13C]acetate, were lower than those of brain glucose labelling from hepatic metabolism. (v) The evaluation of the contributions of glucose and acetate to glutamate metabolism revealed that more than 60% of brain glutamate was synthesized from glucose whereas only 7% was from acetate and that glutamate C4-C5 coupling was mainly due to the metabolism of glucose labelled through hepatic gluconeogenesis. All these results indicate that, under the present conditions, the pyruvate recycling observed through the labelling of brain metabolites mainly originates from peripheral metabolism.     

Regulation of gluconeogenesis from pyruvate and lactate in the isolated perfused rat liver

The effects of glucagon and the alpha-adrenergic agonist, phenylephrine, on the rate of 14CO2 production and gluconeogenesis from [1-14C]lactate and [1-14C]pyruvate were investigated in isolated perfused livers of 24-h-fasted rats. Both glucagon and phenylephrine stimulated the rate of 14CO2 production from [1-14C]lactate but not from [1-14C]pyruvate. Neither glucagon nor phenylephrine affected the activation state of the pyruvate dehydrogenase complex in perfused livers derived from 24-h-fasted rats. 3-Mercaptopicolinate, an inhibitor of the phosphoenolpyruvate carboxykinase reaction, inhibited the rates of 14CO2 production and glucose production from [1-14C]lactate by 50% and 100%, respectively. Furthermore, 3-mercaptopicolinate blocked the glucagon- and phenylephrine-stimulated 14CO2 production from [1-14C]lactate. Additionally, measurements of the specific radioactivity of glucose synthesized from [1-14C]lactate, [1-14C]pyruvate and [2-14C]pyruvate indicated that the 14C-labeled carboxyl groups of oxaloacetate synthesized from 1-14C-labeled precursors were completely randomized and pyruvate----oxaloacetate----pyruvate substrate cycle activity was minimal. The present study also demonstrates that glucagon and phenylephrine stimulation of the rate of 14CO2 production from [1-14C]lactate is a result of increased metabolic flux through the phosphoenolpyruvate carboxykinase reaction, and phenylephrine-stimulated gluconeogenesis from pyruvate is regulated at step(s) between phosphoenolpyruvate and glucose.     

The effects of ethanol concentration on glycero-3-phosphate accumulation in the perfused rat liver. A reassessment of ethanol-induced inhibition of glycolysis using 31P-NMR spectroscopy and HPLC.

The dose-dependent effect of ethanol on the hepatic metabolism of the perfused rat liver has been investigated by (a) 31P-NMR spectroscopy for the follow-up of intracellular phosphorylated metabolites and (b) HPLC for compounds released in the effluents. Perfusion of livers from fed rats with ethanol induced an increase in the level of sn-glycerol 3-phosphate and net accumulations of 3.30 +/- 0.33 and 0.69 +/- 0.15 mumol x g-1 wet liver were reached after 20 min, for 70 mM and 0.5 mM ethanol, respectively. sn-Glycerol-3-phosphate accumulation was fully detected by 31P NMR as indicated by comparing quantitations based on NMR and biochemical assays. Ethanol administration up to a concentration of 10 mM induced a dose-dependent decrease in the release of lactate + pyruvate by the liver. Lactate release decreased from 1129 +/- 39 to 674 +/- 84 nmol x min-1 x g-1, while pyruvate decreased from 230 +/- 9 to 6.2 +/- 0.4 nmol x min-1 x g-1, after 20 min of perfusion with 10 mM ethanol. Nevertheless, the flux through 6-phosphofructo-1-kinase, as measured by both the accumulation of sn-glycerol 3-phosphate and release of lactate + pyruvate, was not affected in the early phase of ethanol oxidation. Finally, data obtained from oxygen consumption, the release of acetate and the accumulation of sn-glycerol 3-phosphate do not support the involvement of the microsomal ethanol-oxidizing system in the catalysis of ethanol oxidation, even at high doses of alcohol.     

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.     

Differing mechanisms of hepatic glucose overproduction in triiodothyronine-treated rats vs. Zucker diabetic fatty rats by NMR analysis of plasma glucose

The metabolic mechanism of hepatic glucose overproduction was investigated in 3,3'-5-triiodo-l-thyronine (T3)-treated rats and Zucker diabetic fatty (ZDF) rats (fa/fa) after a 24-h fast. 2H2O and [U-13C3]propionate were administered intraperitoneally, and [3,4-13C2]glucose was administered as a primed infusion for 90 min under ketamine-xylazine anesthesia. 13C NMR analysis of monoacetone glucose derived from plasma glucose indicated that hepatic glucose production was twofold higher in both T3-treated rats and ZDF rats compared with controls, yet the sources of glucose overproduction differed significantly in the two models by 2H NMR analysis. In T3-treated rats, the hepatic glycogen content and hence the contribution of glycogenolysis to glucose production was essentially zero; in this case, excess glucose production was due to a dramatic increase in gluconeogenesis from TCA cycle intermediates. 13C NMR analysis also revealed increased phosphoenolpyruvate carboxykinase flux (4x), increased pyruvate cycling flux (4x), and increased TCA flux (5x) in T3-treated animals. ZDF rats had substantial glycogen stores after a 24-h fast, and consequently nearly 50% of plasma glucose originated from glycogenolysis; other fluxes related to the TCA cycle were not different from controls. The differing mechanisms of excess glucose production in these models were easily distinguished by integrated 2H and 13C NMR analysis of plasma glucose.     

Diets enriched in sucrose or fat increase gluconeogenesis and G-6-Pase but not basal glucose production in rats

High-fat (HFD) and high-sucrose diets (HSD) reduce insulin suppression of glucose production in vivo, increase the capacity for gluconeogenesis in vitro, and increase glucose-6-phosphatase (G-6-Pase) activity in whole cell homogenates. The present study examined the effects of HSD and HFD on in vivo gluconeogenesis, the catalytic and glucose-6-phosphate translocase subunits of G-6-Pase, glucokinase (GK) translocation, and glucose cycling. Rats were fed a high-starch control diet (STD; 68% cornstarch), HSD (68% sucrose), or HFD (45% fat) for 7-13 days. The ratio of 3H in C6:C2 of glucose after 3H2O injection into 6- to 8-h-fasted rats was significantly increased in HSD (0.68 +/- 0.07) and HFD (0.71 +/- 0.08) vs. STD (0.40 +/- 0.10). G-6-Pase activity was significantly higher in HSD and HFD vs. STD in both intact and disrupted liver microsomes. HSD and HFD significantly increased the amount of the p36 catalytic subunit protein, whereas the p46 glucose-6-phosphate translocase protein was increased in HSD only. Despite increased nonglycerol gluconeogenesis and increased G-6-Pase, basal glucose and insulin levels as well as glucose production were not significantly different among groups. Hepatocyte cell suspensions were used to ascertain whether diet-induced adaptations in glucose phosphorylation and GK might serve to compensate for upregulation of G-6-Pase. Tracer-estimated glucose phosphorylation and glucose cycling (glucose <--> glucose 6-phosphate) were significantly higher in cells isolated from HSD only. After incubation with either 5 or 20 mM glucose and no insulin, GK activity (nmol. mg protein(-1). min(-1)) in digitonin-treated eluates (translocated GK) was significantly higher in HSD (32 +/- 4 and 146 +/- 6) vs. HFD (4 +/- 1 and 83 +/- 10) and STD (9 +/- 2 and 87 +/- 9). Thus short-term, chronic exposure to HSD and HFD increase in vivo gluconeogenesis and the G-6-Pase catalytic subunit. Exposure to HSD diet also leads to adaptations in glucose phosphorylation and GK translocation.     

Simultaneous 13C and 31P NMR studies of perfused rat liver. Effects of insulin and glucagon and a 13C NMR assay of free Mg2+

Metabolism of [2-13C]pyruvate, [1,2-13C]ethanol, and NH4+ in the presence and absence of 7 nM insulin has been followed at 35 degrees C by alternate scan 13C and 31P NMR at 90.5 and 145.8 MHz, respectively, in isolated perfused liver from 16-h fasted rats. With this technique, 31P and 13C NMR spectra are recorded simultaneously so that both phosphate metabolites and 13C-labeled metabolites could be followed, noninvasively, in perfused liver to give a comprehensive view of the response to a variety of stimuli. 13C-labeled glycogen increased synchronously, at a rate of 17 mumol of glucose units/g of liver/h, with the synthesis of 13C-labeled glucose, which also proceeded at a rate of 17 mumol/g of liver/h; glycogenesis was essentially a gluconeogenic process under these conditions and was not affected by the presence of insulin. From the position of the 13C-labeled citrate peak observed in liver, the measurement of Kd for the citrate-Mg complex under our conditions, and the expression relating these quantities to the concentration of free Mg2+, the intracellular level of free Mg2+ is estimated to be 0.46 +/- 0.05 mM in perfused rat liver. After subsequent administration of glucagon, a rapid decrease in glycogen and citrate was seen by 13C NMR and a 44% increase in glycero-3-phosphocholine was seen by 31P NMR; increase in glycero-3-phosphocholine is consistent with stimulation of liver phospholipase activity by glucagon. The co-administration of two different 13C-labeled substrates introduced multiplet structure arising from spin-spin interaction between labeled adjacent carbons into the peaks of several key metabolites. 13C enrichments at specific carbons of citrate, glutamate, glutamine, beta-hydroxybutyrate, and glucose and the distribution of intensity within the multiplets of specific carbons were measured in spectra of perfusates and extracts of the freeze-clamped livers. Within the context of a first order model for fluxes into the Krebs cycle and into glucose, analytical expressions were written that describe the intensity distributions within the several multiplets. In this way, a set of simultaneous equations was generated and solved in general form; when the measured intensity ratios are substituted into these expressions, relative fluxes under the conditions of the experiment can be estimated. Because a redundancy of information is available, checks on self-consistency are built into the estimated fluxes.     

13C NMR study of hepatic pyruvate carboxylase activity in tumor rats

Alanine and lactate, as major gluconeogenic substrates, must be converted into oxaloacetate by way of pyruvate carboxylase before their entry into gluconeogenesis. Although it is well known that hepatic gluconeogenesis from these substrates is increased in tumor hosts, the involvement of pyruvate carboxylase has not been demonstrated. In the present study, we examined pyruvate carboxylase activity in the perfused livers of tumor rats using 13C NMR spectroscopy with [3-13C]-alanine as the gluconeogenic substrate. A substantial increase in hepatic [3-13C]-aspartate production was found in the tumor rats. Since aspartate accumulation directly reflects fluxes of alanine through pyruvate carboxylase, the observed increase in hepatic production of [3-13C]-aspartate in tumor rats indicates that pyruvate carboxylase activity is significantly enhanced.     

Futile hydrogen cycling in liver cells from triiodothyronine treated rats

Aminooxyacetate does not inhibit gluconeogenesis from sorbitol or glycerol, and ethanol does not inhibit gluconeogenesis from L-lactate, in liver cells prepared from triiodothyronine (T3) treated rats. These results are in accord with the previously documented marked increase in α-glycerol phosphate shuttle activity induced by thyroid hormones. Aminooxyacetate inhibits gluconeogenesis from L-lactate in hepatocytes from T3 treated rats by only about 30% (vs 90% in hepatocytes from normal rats). Also pyruvate kinase flux during gluconeogenesis from L-lactate is markedly increased in liver cells from fasted, T3 treated, rats.     

Antagonizing effects of phorbol 12-myristate 13-acetate on hormonally stimulated gluconeogenesis in isolated rat hepatocytes involve activity changes of pyruvate kinase

The tumor-promoting phorbol ester phorbol 12-myristate 13-acetate partially neutralized the stimulatory effects of epinephrine (alpha 1-adrenergic actions), glucagon, and dibutyryl-cAMP on gluconeogenesis in isolated hepatocytes of fasted rats, when lactate or dihydroxyacetone was used as the substrate. By constructing metabolic crossover plots and by comparing rates of lactate production from dihydroxyacetone with K0.5 values of extracted pyruvate kinase for phosphoenolpyruvate, we obtained evidence that phorbol ester actions on hormonally stimulated gluconeogenesis were accompanied by proportionate increases in activity of pyruvate kinase. Although purified pyruvate kinase from rat liver was a substrate for protein kinase C in vitro, phosphorylation was not accompanied by modulation of kinetic parameters. Furthermore, incubation of pyruvate kinase extracted from hormone-treated hepatocytes with protein kinase C revealed no activation of the prephosphorylated enzyme. This and the absence of effects of the phorbol ester on basal rates of gluconeogenesis and lactate production suggest that effects of protein kinase C on pyruvate kinase activity in hepatocytes may result from impairment of steps at the level of hormone-induced signal transduction.     

An in vivoC NMR study on the effect of dietary sugar on pyruvate cycling during glucogenesis from (2-C)pyruvate in Manduca sexta L.

Pyruvate cycling from (2-¹³C)pyruvate was detected in vivo in intact 5th instar Manduca sexta larvae by application of NMR spectroscopy. Cycling was evident from the enrichment of C3 in alanine following transamination of recycled pyruvate in larvae maintained on casein-based diets with or without sucrose. This metabolism is assumed to principally occur in the fat body. Analysis of ¹³C enriched metabolites released into the hemolymph indicated that isotopic dilution of recycled pyruvate was sufficiently great that further metabolism of the recycled metabolite did not occur to any significant extent under these dietary conditions. The C3/C2 ¹³C-enrichment ratio of alanine, therefore, accurately reflected the relative degree of pyruvate cycling and indicated that the rate of cycling was approximately three-fold lower in larvae maintained on diets lacking sucrose. Moreover, based on the distribution of ¹³C in trehalose, these larvae displayed significantly greater rates of gluconeogenesis. Enrichment of C1, C2, C5 and C6 were principally due to carboxylation of the isotopically substituted substrate catalyzed by pyruvate carboxylase and, therefore, reflected net carbohydrate synthesis. Trehalose C3 and C4 enrichments were principally due to pyruvate dehydrogenase-catalyzed decarboxylation and reflected incorporation of label following metabolism through the TCA cycle. Pentose cycling following glucogenesis significantly affected the ¹³C distribution in trehalose in insects on both diets, and the relative intensity of trehalose C6 was, therefore, used for comparing the rates of gluconeogenesis and pyruvate cycling. Based on the ¹³C enrichment of trehalose C6 relative to C3 of alanine the mean rate of pyruvate cycling relative to the rate of gluconeogenesis was approximately 60% in larvae on the diet lacking sucrose, while the rate of pyruvate cycling in larvae maintained on the diet supplemented with sucrose was greater than the gluconeogenic flux. The results were consistent with the conclusion that pyruvate kinase likely plays an important role in regulating gluconeogenesis in M. sexta larvae.     

Metabolomic and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle. I. Interrelation between gluconeogenesis and cataplerosis; formation of methoxamates from aminooxyacetate and ketoacids

We conducted a study coupling metabolomics and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle. Rat livers were perfused with lactate or pyruvate +/- aminooxyacetate or mercaptopicolinate in the presence of 40% enriched NaH(13)CO(3). Other livers were perfused with dimethyl [1,4-(13)C(2)]succinate +/- mercaptopicolinate. In this first of two companion articles, we show that a substantial fraction of gluconeogenic carbon leaves the liver as citric acid cycle intermediates, mostly alpha-ketoglutarate. The efflux of gluconeogenic carbon ranges from 10 to 200% of the rate of liver gluconeogenesis. This cataplerotic efflux of gluconeogenic carbon may contribute to renal gluconeogenesis in vivo. Multiple crossover analyses of concentrations of gluconeogenic intermediates and redox measurements expand previous reports on the regulation of gluconeogenesis and the effects of inhibitors. We also demonstrate the formation of adducts from the condensation, in the liver, of (i) aminooxyacetate with pyruvate, alpha-ketoglutarate, and oxaloacetate and (ii) mercaptopicolinate and pyruvate. These adducts may exert metabolic effects unrelated to their effect on gluconeogenesis.     

The interaction between the cytosolic pyridine nucleotide redox potential and gluconeogenesis from lactate/pyruvate in isolated rat hepatocytes. Implications for investigations of hormone action

By using very low concentrations of cells to minimize alterations in substrate concentrations, we demonstrated that the lactate/pyruvate ratio of the incubation medium, which determines the cytosolic NADH/NAD+ ratio, affects gluconeogenic flux in suspensions of isolated hepatocytes from fasted rats. At a fixed extracellular pyruvate concentration of 1 mM and with the lactate/pyruvate ratio varied from 0.6 to 10 and to 50, glucose production rates increased from 2.5 to 5.5 and then decreased to 1.8 nmol/mg of cell protein/min. This finding paralleled the observation of Sugano et al. (Sugano, T., Shiota, M., Tanaka, T., Miyamae, Y., Shimada, M., and Oshino, N. (1980) J. Biochem. (Tokyo) 87, 153-166) who noted a similar biphasic response in the perfused liver system when lactate was held constant and pyruvate varied. The biphasic relationship can be explained by the influence of the NADH/NAD+ ratio on the near-equilibrium reactions catalyzed by glyceraldehyde-3-phosphate dehydrogenase and malate dehydrogenase in the hepatocyte cytosol. By shifting the equilibrium of the glyceraldehyde-3-phosphate dehydrogenase reaction, a rise in the NADH/NAD+ ratio decreases the concentration of 3-phosphoglycerate which, because of the linkage of 3-phosphoglycerate to phosphoenolpyruvate through two near-equilibrium reactions, reduces the concentration of phosphoenolpyruvate and therefore causes a decline in flux through pyruvate kinase. This decrease in pyruvate kinase flux results in an enhanced gluconeogenic flux. At higher NADH/NAD+ ratios, however, the oxalacetate concentration drops to such an extent that the consequent decreased flux through phosphoenolpyruvate carboxykinase exceeds the decline in flux through pyruvate kinase, producing a decrease in gluconeogenic flux. The lactate/pyruvate ratio was found to influence the actions of three hormones thought to stimulate gluconeogenesis by different mechanisms. Except for an inhibition by glucagon seen at the lowest lactate/pyruvate ratio tested, the stimulations by this hormone were relatively insensitive to lactate/pyruvate ratios, while angiotensin II produced greater stimulations of gluconeogenesis as the lactate/pyruvate ratio was increased. Dexamethasone, added in vitro, stimulated gluconeogenesis significantly only at very low and very high lactate/pyruvate ratios.     

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.     

Application of 13C and 31P NMR to the study of hepatic metabolism

Alternate scan 13C and 31P NMR has been used to follow the metabolism of 13C-labeled substrates, in the presence and absence of insulin, in isolated perfused liver from fasted rats. Because both 31P and 13C NMR spectra are recorded almost simultaneously with this method, both phosphate metabolites and 13C-labeled metabolites are measured, noninvasively and repetitively, to give an immediate, broad survey of the hepatic response to a variety of stimuli. During the metabolism of [2-13C]pyruvate, [1,2-13C]ethanol, and NH4+, 13C-labeled glycogen increases synchronously with, and at the same rate as, the synthesis of 13C-labeled glucose; thus, glycogenesis was essentially a gluconeogenic process under our conditions and was unaltered by the presence of insulin. From the position of the 13C-labeled citrate peak observed in liver, the measurement of KD for the citrate-magnesium complex under our conditions, and the expression relating these quantities to the concentration of free Mg2+, the intracellular level of free Mg2+ is estimated to be 0.46 +/- 0.05 mM. Later administration of glucagon led to a rapid decrease in glycogen and citrate and a 44% increase in glycero-3-phosphocholine (GPC); increase in GPC is consistent with stimulation of liver phospholipase activity by glucagon. Simultaneous administration of two different 13C-labeled substrates, or one doubly labeled substrate, introduced multiplet structure arising from spin-spin interaction between labeled adjacent carbons into the peaks of several key metabolites. The 13C NMR intensity distributions within the several multiplets are used, within the context of a first-order model for fluxes into the Krebs cycle, to estimate relative fluxes under the conditions of the experiment.