Determinants in the pathways followed by the carbons of acetone in their conversion to glucose

[2-14C]Acetone was infused into rats that were fed or fasted. Each was infused with either a trace quantity of acetone or a large quantity that resulted in a blood concentration of acetone of at least 4 mM. The distribution of 14C in the carbons of glucose from each rat was determined. Two of the rats were given acetone in their drinking water and one was diabetic. Whether a rat was chronically exposed to acetone, fed or fasted, normal or diabetic, if given the trace dose, over 80% of the 14C in the glucose it formed was in carbons 1, 2, 5, and 6 of the glucose. If a rat was given the large dose, about 50% was in carbons 3 and 4. Thus, the major determinant of the pathways followed by acetone when it is metabolized is its concentration and not the prior dietary state of the animal or its previous exposure to acetone. Incorporation into carbons 1, 2, 5, and 6 occurs in the conversion of the carbons of [2-14C]lactate into glucose, whereas incorporation into carbons 3 and 4 occurs in the conversion of the carbons of [1-14C]acetate into glucose. Therefore, at high acetone concentration, the pathway that has been proposed for acetone's metabolism via acetate predominates, and via acetate there can be no net synthesis of glucose from acetone. When rats were given cyanamide and then the large dose of acetone, 74% of the 14C in the glucose they formed was in carbons 3 and 4 of the glucoses. Thus, the relative contribution of the pathway to lactate, or its metabolic equivalent, that has been proposed appears to be lessened by the administration of an aldehyde dehydrogenase inhibitor.     

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)     

Quantitative estimation of the pathways followed in the conversion to glycogen of glucose administered to the fasted rat

When [6-3H,6-14C]glucose was given in glucose loads to fasted rats, the average 3H/14C ratios in the glycogens deposited in their livers, relative to that in the glucoses administered, were 0.85 and 0.88. When [3-3H,3-14C]lactate was given in trace quantity along with unlabeled glucose loads, the average 3H/14C ratio in the glycogens deposited was 0.08. This indicates that a major fraction of the carbons of the glucose loads was converted to liver glycogen without first being converted to lactate. When [3-3H,6-14C]glucose was given in glucose loads, the 3H/14C ratios in the glycogens deposited averaged 0.44. This indicates that a significant amount of H bound to carbon 3, but not carbon 6, of glucose is removed within liver in the conversion of the carbons of the glucose to glycogen. This can occur in the pentose cycle and by cycling of glucose-6-P via triose phosphates: glucose----glucose-6-P----triose phosphates----glucose-6-P----glycogen. The contributions of these pathways were estimated by giving glucose loads labeled with [1-14C]glucose, [2-14C]glucose, [5-14C]glucose, and [6-14C]glucose and degrading the glucoses obtained by hydrolyzing the glycogens that deposited. Only a few per cent of the glucose carbons deposited in glycogen were deposited in liver via glucose-6-P conversion to triose phosphates. Between 4 and 9% of the glucose utilized by the liver was utilized in the pentose cycle. While these are relatively small percentages, since three NADP3H molecules are formed from each molecule of [3-3H]glucose-6-P utilized in the cycle, a major portion of the difference between the ratios obtained with [3-3H]glucose and with [6-3H]glucose is attributable to metabolism in the pentose cycle. Because 3H of [3-3H]glucose is extensively removed during the conversion of the glucose to glycogen within liver the extent of incorporation of the 3H into liver glycogen is not the measure of glucose's metabolism in other tissues before its carbons are deposited in liver glycogen. The distributions of 14C from the 14C-labeled glucoses into the carbons of the liver glycogens mean that at a minimum about 30% of the carbons of the glucose deposited in the glycogen were first converted to lactate or its metabolic equivalent.     

Contribution of omega-oxidation to fatty acid oxidation by liver of rat and monkey.

Contributions of omega-oxidation to overall fatty acid oxidation in slices from livers of ketotic alloxan diabetic rats and of fasted monkeys are estimated. Estimates are made from a comparison of the distribution of 14C in glucose formed by the slices from omega-14C-labeled compared to 2-14C-labeled fatty acids of even numbers of carbon atoms and from [1-14C]acetate compared to [2-14C]acetate. These estimates are based on the fact that 1) the dicarboxylic acid formed via omega-oxidation of a omega-14C-labeled fatty acid will yield [1-14C]acetate and [1-14C]succinate on subsequent beta-oxidation, if beta-oxidation is assumed to proceed to completion; 2) only [2-14C]acetate will be formed if the fatty acid is metabolized solely via beta-oxidation; and 3) 14C from [1-14C]acetate and [1-14C]succinate is incorporated into carbons 3 and 4 of glucose and 14C from [2-14C]acetate is incorporated into all six carbons of glucose. From the distributions found, the contribution of omega-oxidation to the initial oxidation of palmitate by liver slices is estimated to between 8% and 11%, and the oxidation of laurate between 17% and 21%. Distributions of 14C in glucose formed from 14C-labeled palmitate infused into fasted and diabetic rats do not permit quantitative estimation of the contribution of omega-oxidation to fatty acid oxidation in vivo. However, the distributions found also indicate that, of the fatty acid metabolized by the whole animal in the environment of glucose formation, at most, only a minor portion is initially oxidized via omega-oxidation. As such, omega-oxidation cannot contribute more than a small extent to the formation of glucose.     

Proline and hepatic lipogenesis

The effects of proline on lipogenesis in isolated rat hepatocytes were determined and compared with those of lactate, an established lipogenic precursor. Proline or lactate plus pyruvate increased lipogenesis (measured with 3H2O) in hepatocytes from fed rats depleted of glycogen in vitro and in hepatocytes from starved rats. Lactate plus pyruvate but not proline increased lipogenesis in hepatocytes from starved rats. ( - )-Hydroxycitrate, an inhibitor of ATP-citrate lyase, partially inhibited incorporation into saponifiable fatty acid of 3H from 3H2O and 14C from [U-14C]lactate with hepatocytes from fed rats. Incorporation of 14C from [U-14C]proline was completely inhibited. Similar complete inhibition of incorporation of 14C from [U-14C]proline by ( - )-hydroxycitrate was observed with glycogen-depleted hepatocytes or hepatocytes from starved rats. Inhibition of phosphoenolpyruvate carboxykinase by 3-mercaptopicolinate did not inhibit the incorporation into saponifiable fatty acid of 3H from 3H2O or 14C from [U-14C]proline or [U-14C]lactate. Both 3-mercaptopicolinate and ( - )-hydroxycitrate increased lipogenesis (measured with 3H2O) in the absence or presence of lactate or proline with hepatocytes from starved rats. The results are discussed with reference to the roles of phosphoenolpyruvate carboxykinase, mitochondrial citrate efflux, ATP-citrate lyase and acetyl-CoA carboxylase in proline- or lactate-stimulated lipogenesis.     

Acute control of fatty acid synthesis by cyclic AMP in the chick liver cell: possible site of inhibition of citrate formation.

Glucagon and N,(6)O(2)-dibutyryl cyclic adenosine 3',5'-cyclic monophosphate (Bt(2)cAMP) inhibit fatty acid synthesis from acetate by more than 90% and prevent citrate formation in chick hepatocytes metabolizing glucose. With substrates that enter glycolysis at or below triose-phosphates, e.g., fructose, lactate, or pyruvate, Bt(2)cAMP has no effect on the citrate level and its inhibitory effect on fatty acid synthesis is substantially reversed. Because acetyl-CoA carboxylase requires a tricarboxylic acid activator for activity, it is proposed that regulation of fatty acid synthesis by Bt(2)cAMP is due, in part, to changes in the citrate level. Reduced citrate formation appears to result from a cAMP-induced inhibition of glycolysis. Bt(2)cAMP inhibits (14)CO(2) production from [1-(14)C]-, [6-(14)C]-, and [U-(14)C]glucose and has little effect on (14)CO(2) formation from [1-(14)C]- or [2-(14)C]pyruvate or from [1-(14)C]fructose. [(14)C]Lactate formation from glucose is depressed 50% by Bt(2)cAMP. In the presence of an inhibitor of mitochondrial pyruvate transport lactate accumulation is enhanced, but continues to be lowered 50% by Bt(2)cAMP. The activity of phosphofructokinase is greatly decreased in Bt(2)cAMP-treated cells while the activities of pyruvate kinase and acetyl-CoA carboxylase are unaffected. It appears that decreased glycolytic flux and decreased citrate formation result from depressed phosphofructokinase activity. Fatty acid synthesis from [(14)C]acetate is partially inhibited by Bt(2)cAMP in the presence of fructose, lactate, and pyruvate despite a high citrate level. Incorporation of [(14)C]fructose, [(14)C]pyruvate, or [(14)C]lactate into fatty acids is similarly depressed by Bt(2)cAMP. Synthesis of cholesterol from [(14)C]acetate or [2-(14)C]pyruvate is unaffected by Bt(2)cAMP. These results implicate a second site of inhibition of fatty acid synthesis by Bt(2)cAMP that involves the utilization, but not the production, of cytoplasmic acetyl-CoA.-Clarke, S. D., P. A. Watkins, and M. D. Lane. Acute control of fatty acid synthesis by cyclic AMP in the chick liver cell: possible site of inhibition of citrate formation.     

Intracellular pyruvate flux in the methane-producing archaeon Methanococcus maripaludis

During growth of the methanogenic archaeon Methanococcus maripaludis on alanine as the sole nitrogen source under H(2)/CO(2), alanine was incorporated into amino acids derived from pyruvate including leucine, isoleucine, and valine. Thus, growth with alanine was an efficient means of labeling intracellular pools of pyruvate in this lithotroph. Cells were grown with 18% [U-(13)C]alanine, and the distribution of the isotope in the branched-chain amino acids was determined by (13)C-NMR. Carbons derived from pyruvate contained 14.5% (13)C, indicating that most of the cellular pyruvate was obtained from alanine. In contrast, carbons derived from acetyl-CoA contained only 3-5% (13)C, indicating that only small amounts of acetyl-CoA were formed from pyruvate. Thus, autotrophic acetyl-CoA biosynthesis continued even in the presence of an organic carbon source. Moreover, the labeling of acetyl-CoA was lower than would be predicted if pyruvate was a C-1 donor for acetyl-CoA biosynthesis. Carbon derived from the C-1 of acetyl-CoA contained less (13)C than carbon derived from the C-2 of acetyl-CoA, and this difference was attributed to the acetyl-CoA:CO(2) exchange activity of acetyl-CoA synthase. No enrichment was detected for the C-1 of valine, which was derived from the C-1 of pyruvate. This result was attributed to the pyruvate:CO(2) exchange activity of pyruvate oxidoreductase and may have important implications for isotope tracer studies utilizing pyruvate. Lastly, these results demonstrate that the breakdown of pyruvate by methanococci is very limited even under conditions where it is the sole nitrogen and major carbon source.     

Carbon flux through citric acid cycle pathways in perfused heart by 13C NMR spectroscopy

Mathematical models of the TCA cycle derived previously for 14C tracer studies have been extended to 13C NMR to measure the 13C fractional enrichment of [2-13C]acetyl-CoA entering the cycle and the relative activities of the oxidative versus anaplerotic pathways. The analysis is based upon the steady-state enrichment of 13C into the glutamate carbons. Hearts perfused with [2-13C]acetate show low but significant activity of the anaplerotic pathways. Activation of two different anaplerotic pathways is demonstrated by addition of unlabeled propionate or pyruvate to hearts perfused with [2-13C]acetate. In each case, the amount of [2-13C]acetate being oxidized and the relative carbon flux through anaplerotic versus oxidative pathways are evaluated.     

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.     

13C n.m.r. studies of gluconeogenesis in rat liver suspensions and perfused mouse livers

Our early 31P n.m.r. studies of compartmentation in suspensions of rat liver cells have been extended by following fructose-1-phosphate peaks, known to be in the cytosol, which gave the same pH as the Pi peak previously assigned to the cytosol. Gluconeogenesis have been followed from [13C]glycerol labelled at C1,3 or at C2 and from labelled [3-13C]alanine. With the glycerol substrate it was possible to follow the label into alpha-glycerophosphate and to determine its distribution in the glucose formed. To a first approximation (i.e. 90%) the glucose level could be followed from its original glycerol position, e.g. [1,3-13C]glycerol to strongly labelled positions 1, 3, 4 and 6 of glucose. Slightly more than 10% of the label was scrambled (i.e. 10% movement of C2 to C1 and ca. 10% of C1 was lost, the remainder being unchanged). These are consistent with a flux through the pentose shunt, dominated by the transketolase pathway. With [3-13C]alanine, about 14 resonances are assigned to different carbons of the intermediates beta-hydroxybutyrate, acetoacetate, lactate, pyruvate, glutamate, glutamine, asparate, as well as C2-alanine, while another 7 resonances are observed from the different anomeric carbons of glucose. The effects of thyroid hormone treatment of the rats upon numerous in vivo rates are clearly observed and will be illustrated.     

A sensitive radioisotopic assay of pyruvate dehydrogenase complex in human muscle tissue.

A radioactive assay for the determination of pyruvate dehydrogenase complex activity in muscle tissue has been developed. The assay measures the rate of acetyl-CoA formation from pyruvate in a reaction mixture containing NAD+ and CoASH. The acetyl-CoA is determined as [14C]citrate after condensation with [14C]-oxaloacetate by citrate synthase. The method is specific and sensitive to the picomole range of acetyl-CoA formed. In eleven normal subjects, the active form of pyruvate dehydrogenase (PDCa) in resting human skeletal muscle samples obtained using the needle biopsy technique was 0.44 +/- 0.16 (SD) mumol acetyl-CoA.min-1.g-1 wet wt. Total pyruvate dehydrogenase complex (PDCt) activity was determined after activation by pretreating the muscle homogenate with Ca2+, Mg2+, dichloroacetate, glucose, and hexokinase. The mean value for PDCt was 1.69 +/- 0.32 mumol acetyl-CoA.min-1.g-1 wet wt, n = 11. The precision of the method was determined by analyzing 4-5 samples of the same muscle piece. The coefficient of variation for PDCa was 8% and for PDCt 5%.     

Effect of insulin on the metabolic distribution of carbons 1, 2, and 3 of pyruvate

It has long been known that the carbons of pyruvate are converted to CO2 at different points in the metabolic process. This report deals with the observation that insulin affects the oxidation of carbons 2 and 3 primarily and has little effect on the oxidation of the carboxyl carbon. Oxidation of different carbons of pyruvate and their incorporation into various metabolic components was studied in isolated rat hepatocytes. Insulin stimulated the 14CO2 production from [2-14C]- and [3-14C]pyruvate and from [U-14C]alanine. However, it had little or no effect on the activity of the pyruvate dehydrogenase complex as measured by the evolution of 14CO2 from [1-14C]pyruvate or [1-14C] alanine. Insulin also stimulated the incorporation of carbons 2 and 3 of pyruvate into protein but had no effect on the incorporation of carbon 1. Incorporation of [1-14C]- and [U-14C]alanine into protein was differentially enhanced by insulin in a manner similar to that of the pyruvate carbons. The fact that insulin stimulates the incorporation of [1-14C]alanine into protein but not [1-14C]pyruvate suggests the possibility of a compartmentation of pyruvate metabolism in the isolated hepatocytes. These studies show that the stimulation of [2-14C]- and [3-14C]pyruvate incorporation into protein involves the stimulatory effect of insulin on the activity of the Krebs cycle which is evident from the fact that insulin did not stimulate the pyruvate carbons to enter protein via alanine but the incorporation via glutamate was increased by about 40%.     

Carbon flux through tricarboxylic acid cycle in rat renal tubules

Our aim was to delineate the effect(s) of chronic metabolic acidosis on renal TCA-cycle metabolism. Renal tubules isolated from control and chronically acidotic rats were incubated at pH 7.4 with either 2 mM [2,3-13C]pyruvate or [2-13C]acetate. GC-MS and/or 13C-NMR were utilized to monitor the flux of 13C through pyruvate dehydrogenase, pyruvate carboxylase and the TCA-cycle. With either, precursor acidosis was associated with significantly decreased formation of 13C-labelled citrate, malate, aspartate and alanine and increased formation of glucose, lactate and acetyl-CoA as compared with the control. The results indicate that adaptation of renal metabolism to chronic metabolic acidosis is associated with diminished flux through citrate synthetase and concomitantly increased flux through pyruvate carboxylase. The data suggest that depletion of TCA-cycle intermediates and enhanced ammoniagenesis in the kidney of chronically acidotic rats may be regulated at the site of mitochondrial citrate-condensing enzyme.     

Differential modulation of glucose,lactate, and pyruvate oxidation by insulin and dichloroacetate in the rat heart

Despite the fact that lactate and pyruvate are potential substrates for energy production in vivo, our understanding of the control and regulation of carbohydrate metabolism is based principally on studies where glucose is the only available carbohydrate. Therefore, the purpose of this study was to determine the contributions of lactate, pyruvate, and glucose to energy production in the isolated, perfused rat heart over a range of insulin concentrations and after activation of pyruvate dehydrogenase with dichloroacetate (DCA). Hearts were perfused with physiological concentrations of [1-13C]glucose, [U-13C]lactate, [2-13C]pyruvate, and unlabeled palmitate for 45 min. Hearts were freeze clamped, and 13C NMR glutamate isotopomer analysis was performed on tissue extracts. Glucose, lactate, and pyruvate all contributed significantly to myocardial energy production; however, in the absence of insulin, glucose contributed only 25-30% of total pyruvate oxidation. Even under conditions where carbohydrates represented >95% of substrate entering the tricarboxylic acid (TCA) cycle, we found that glucose contributed at most 50-60% of total carbohydrate oxidation. Despite being present at only 0.1 mM, pyruvate contributed between approximately 10% and 30% of total acetyl-CoA entry into the TCA cycle. We also found that insulin and DCA not only increased glucose oxidation but also exogenous pyruvate oxidation; however, lactate oxidation was not increased. The differential effects of insulin and DCA on pyruvate and lactate oxidation provide further evidence for compartmentation of cardiac carbohydrate metabolism. These results may have important implications for understanding the mechanisms underlying the beneficial effects of increasing cardiac carbohydrate metabolism.     

The effects of alpha-adrenergic stimulation on the regulation of the pyruvate dehydrogenase complex in the perfused rat liver

The regulation of the pyruvate dehydrogenase multienzyme complex was investigated during alpha-adrenergic stimulation with phenylephrine in the isolated perfused rat liver. The metabolic flux through the pyruvate dehydrogenase reaction was monitored by measuring the production of 14CO2 from infused [1-14C] pyruvate. In livers from fed animals perfused with a low concentration of pyruvate (0.05 mM), phenylephrine infusion significantly inhibited the rate of pyruvate decarboxylation without affecting the amount of pyruvate dehydrogenase in its active form. Also, phenylephrine caused no significant effect on tissue NADH/NAD+ and acetyl-CoA/CoASH ratios or on the kinetics of pyruvate decarboxylation in 14CO2 washout experiments. Phenylephrine inhibition of [1-14C]pyruvate decarboxylation was, however, closely associated with a decrease in the specific radioactivity of perfusate lactate, suggesting that the pyruvate decarboxylation response simply reflected dilution of the labeled pyruvate pool due to phenylephrine-stimulated glycogenolysis. This suggestion was confirmed in additional experiments which showed that the alpha-adrenergic-mediated inhibitory effect on pyruvate decarboxylation was reduced in livers perfused with a high concentration of pyruvate (1 mM) and was absent in livers from starved rats. Thus, alpha-adrenergic agonists do not exert short term regulatory effects on pyruvate dehydrogenase in the liver. Furthermore, the results suggest either that the rat liver pyruvate dehydrogenase complex is insensitive to changes in mitochondrial calcium or that changes in intramitochondrial calcium levels as a result of alpha-adrenergic stimulation are considerably less than suggested by others.     

Examination of primary metabolic pathways in a murine hybridoma with carbon-13 nuclear magnetic resonance spectroscopy

Primary metabolism of a murine hybridoma was probed with (13)C nuclear magnetic resonance (NMR) spectroscopy. Cells cultured in a hollow fiber bioreactor were serially infused with [1-(13)C] glucose, [2-(13)C] glucose, and [3-(13)C] glutamine. In vivo spectroscopy of the culture was used in conjunction with off-line spectroscopy of the medium to determine the intracellular concentration of several metabolic intermediates and to determine fluxes for primary metabolic pathways. Intracellular concentrations of pyruvate and alanine were very high relative to levels observed in normal quiescent mammalian cells. Estimates made from labeling patterns in lactate indicate that 76% of pyruvate is derived directly from glycolysis; some is also derived from the malate shunt, the pyruvate/melate shuttle associated with lipid synthesis and the pentose phosphate pathway. The rate of formation of pyruvate from the pentose phosphate pathway was estimated to be 4% of that from glycolysis; This value is a lower limit and the actual value may be higher. Incorporation of pyruvate into the tricarboxylic acid (TCA) cycle appears to occur through only pyruvate dehydrogenase; no pyruvate carboxylase activity was detected. The malate shunt rate was approximately equal to the rate of glutamine uptake. The rate of incorporation of glucosederived acetyl-CoA into lipids was 4% of the glucose uptake rate. The TCA cycle rate between isocitrate and alpha-ketoglutarate was 110% of the glutamine uptake rate. (c) 1994 John Wiley & Sons, Inc.     

Insulin activation of lipogenesis in isolated mammary acini from lactating rats fed on a high-fat diet. Evidence that acetyl-CoA carboxylase is a site of action.

Feeding lactating rats on high-fat cheese crackers in addition to laboratory chow increased the dietary intake of fat from 2 to 20% of the total weight of food eaten and decreased mammary-gland lipogenesis in vivo by approx. 50%. This lipogenic inhibition was also observed in isolated mammary acini, where it was accompanied by decreased glucose uptake. These inhibitions were completely reversed by incubation with insulin. Insulin had no effect on the rate of glucose transport into acini, nor on pyruvate dehydrogenase activity as estimated by the accumulation of pyruvate and lactate, suggesting that these are not the sites of lipogenic inhibition. Insulin stimulated the incorporation of [1-14C]acetate into lipid in acini from high-fat-fed rats. In the presence of alpha-cyanohydroxycinnamate, a potent inhibitor of mitochondrial pyruvate transport, and with glucose as the sole substrate, neither [1-14C]glucose incorporation into lipid nor glucose uptake were stimulated by insulin. Insulin did stimulate the incorporation of [1-14C]acetate into lipid in the presence of alpha-cyanohydroxycinnamate, and this was accompanied by an increase in glucose uptake by the acini. This indicated that increased glucose uptake was secondary to the stimulation of lipogenesis by insulin, which therefore must occur via activation of a step in the pathway distal to mitochondrial pyruvate transport. Insulin stimulated acetyl-CoA carboxylase activity measured in crude extracts of acini from high-fat-fed rats, restoring it to values close to those of chow-fed controls. The effects of insulin on acetyl-CoA carboxylase activity and lipogenesis were not antagonized by adrenaline or dibutyryl cyclic AMP.     

Gluconeogenesis from acetone in starved rats.

To non-anaesthetized rats starved for 3 days, [U-14C]acetone, NaH14CO3, L-[U-14C]lactate, [2-14C]acetate or D-[U-14C]- plus D-[3-3H]-glucose was injected intravenously. From the change in the plasma concentration of labelled acetone versus time after the injection, the metabolic clearance rate of acetone was calculated as 2.25 ml/min per kg body wt., and its rate of turnover as 0.74 mumol/min per kg. The extent and time course of the labelling of plasma glucose, lactate, urea and acetoacetate were followed and compared with those observed after the injection of labelled lactate, acetate and NaHCO3. The labelling of plasma lactate was rapid and extensive. Some 1.37% of the 14C atoms of circulating glucose originated from plasma acetone, compared with 44% originating from lactate. By deconvolution of the Unit Impulse Response Function of glucose, it was shown that the flux of C atoms from acetone to glucose reached a peak at about 100 min after injection of labelled acetone. In comparable experiments the transfer from lactate reached a peak at 14 min after the injection of labelled lactate. It was concluded that acetone is converted into lactate to a degree sufficient to account for the labelling of plasma glucose and is thus a true, albeit minor, substrate of glucose synthesis in starved rats.     

Plasmodium berghei: gluconeogenesis in the infected mouse liver studied by 13C nuclear magnetic resonance

Using 13C nuclear magnetic resonance, we have compared the gluconeogenic activity of perfused livers isolated from normal starved mice and mice highly parasitized with Plasmodium berghei, using [2-13C]pyruvate as substrate. In both types of livers, 13C labeling of glucose carbons occurred in positions 1, 2, 5, and 6. The equal proportions of [1,6-13C]- and [2,5-13C]glucose in livers from malarial and normal mice suggests that pyruvate enters the gluconeogenic pathway directly and, to an equal extent, via the tricarboxylic acid cycle. The normalized signal heights indicated that at a given time after the addition of [2-13C]pyruvate the degree of 13C labeling in glucose carbons was reduced in livers from malarial animals, when compared to livers from normal animals. During the course of the perfusion experiment, the [2-13C]lactate resonance signal was always more intense from livers of malarial animals than from normal animals. A reduced activity of hepatic gluconeogenesis in malarial animals was further confirmed by a separate set of perfusion experiments which showed a 56% reduction of the measured rate of glucose production in livers from malarial animals, with respect to that of normal animals. A lowered NAD/NADH ratio in livers from malarial animals would explain the increased proportion of lactate observed in the spectra and be related to a decreased gluconeogenic rate. A more reduced oxidoreduction level in the hepatocytes of a malarial animal would result from a defect in the oxidative phosphorylation activity of mitochondria.     

Acute effects of ethanol on the perfused rat liver. Studies on lipid and carbohydrate metabolism, substrate cycling and perfusate amino acids

1. Livers from fed rats were perfused in situ with whole rat blood containing glucose labelled uniformly with (14)C and specifically with (3)H at positions 2, 3 or 6. 2. When ethanol was infused at a concentration of 24mumol/ml of blood the rate of utilization was 2.8mumol/min per g of liver. 3. Ethanol infusion raised perfusate glucose concentrations and caused a 2.5-fold increase in hepatic glucose output. 4. Final blood lactate concentrations were decreased in ethanol-infused livers, but the mean uptake of lactate from erythrocyte glycolysis was unaffected. 5. Production of ketone bodies (3-hydroxybutyrate+3-oxobutyrate) and the ratio [3-hydroxybutyrate]/[3-oxobutyrate] were raised by ethanol. 6. Formation of (3)H(2)O from specifically (3)H-labelled glucoses increased in the order [6-(3)H]<[3-(3)H]<[2-(3)H]. Production of (3)H(2)O from [2-(3)H]glucose was significantly greater than that from [3-(3)H]glucose in both control and ethanol-infused livers. Ethanol significantly decreased (3)H(2)O formation from all [(3)H]glucoses. 7. Liver glycogen content was unaffected by ethanol infusion. 8. Production of very-low-density lipoprotein triacylglycerols was inhibited by ethanol and there was a small increase in liver triacylglycerols. Very-low-density-lipoprotein secretion was negatively correlated with the ratio [3-hydroxybutyrate]/[3-oxobutyrate]. Perfusate fatty acid concentrations and molar composition were unaffected by perfusion with ethanol. 9. Ethanol decreased the incorporation of [U-(14)C]glucose into fatty acids and cholesterol. 10. The concentration of total plasma amino acids was unchanged by ethanol, but the concentrations of alanine and glycine were decreased and ([glutamate]+[glutamine]) was raised. 11. It is proposed that the observed effects of ethanol on carbohydrate metabolism are due to an increased conversion of lactate into glucose, possibly by inhibition of pyruvate dehydrogenase. The increase in gluconeogenesis is accompanied by diminished substrate cycling at glucose-glucose 6-phosphate and at fructose 6-phosphate-fructose 1,6-bisphosphate.