Inhibition of lipogenesis by vasopressin and angiotensin II in glycogen-depleted hepatocytes

Vasopressin and angiotensin II inhibited lipogenesis (measured with 3H2O) in hepatocytes from fed rats. Inhibition was also observed with hepatocytes from fed rats which had been depleted of glycogen in vitro and incubated with lactate + pyruvate (5 mM + 0.5 mM) as substrates. The inhibitory actions of the hormones are therefore independent of hormone-mediated changes in glycogenolytic or glycolytic flux from glycogen, and thus the site(s) of hormone action must be subsequent to the formation of lactate. (-)Hydroxycitrate, a specific inhibitor of ATP-citrate lyase, decreased lipogenesis in hepatocytes from fed rats incubated with lactate + pyruvate by approx. 51% but had little effect on lipogenesis in glycogen-depleted hepatocytes similarly incubated. There was parallel inhibition of incorporation of 14C from [U-14C]lactate into fatty acid and lipogenesis as measured with 3H2O in each case. Thus depletion of glycogen, or conceivably the process of glycogen-depletion (incubation with dibutyryl cyclic AMP) causes a change in the rate-determining step(s) for lipogenesis from lactate. Vasopressin and angiotensin II also decreased lipogenesis and incorporation of 14C into fatty acids in glycogen-depleted hepatocytes provided with [U-14C]proline as opposed to [U-14C]-lactate. However, proline-stimulated lipogenesis was inhibited by (-)hydroxycitrate, and proline-stimulated lipogenesis and incorporation of 14C from [U-14C]-proline were not decreased in parallel by this inhibitor (inhibition of 52% and 85% respectively). It is inferred that lactate and proline stimulate lipogenesis by different mechanisms and incorporation of 14C from [U-14C]proline and [U-14C]lactate into fatty acid occurs via different routes.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The effects of specific lipogenic substrates and metabolic inhibitors on de Novo fatty acid synthesis in isolated hepatocytes from chow-fed female rats

The effects of glucose (10 mm), glycerol (3 mm), and lactate/pyruvate (10 mm) on the incorporation of ³H from ³H₂O into fatty acids were studied in isolated hepatocytes prepared from chow-fed female rats. Lactate/pyruvate markedly increased lipogenic rates, while glucose and glycerol did not significantly affect rates of lipogenesis. In cells incubated with lactate/pyruvate plus glycerol, the increase in ³H incorporation was greater than observed with lactate/pyruvate alone. In hepatocytes isolated from 24-h starved rats, lactate/pyruvate again increased de novo fatty acid synthesis to a greater extent than either glucose or glycerol. Glycerol significantly increased lipogenesis compared to the endogenous rates and when incubated with lactate/pyruvate produced an increase above lactate/pyruvate alone. (?)-Hydroxycitrate, a potent inhibitor of ATP-citrate lyase (EC 4.1.3.8), and agaric acid, an inhibitor of tricarboxylate anion translocation, were studied in hepatocytes to determine their effects on lipogenesis by measuring ³H₂O, [1-¹⁴C]acetate, and [2-¹⁴C]lactate incorporation into fatty acids. ³H incorporation into fatty acids was markedly inhibited by both inhibitors with agaric acid (60 μm) producing the greater inhibition. (?)-Hydroxycitrate (2 mm) increased acetate incorporation into fatty acids from [1-¹⁴C]acetate and agaric acid produced a strong inhibitory effect. Combined effects of (?)-hydroxycitrate and agaric acid on lipogenesis from [1-¹⁴C]acetate showed an inhibitory response to a lesser extent than with agaric acid alone. With substrate concentrations of acetate present, there was no significant increase in rates of lipogenesis from [1-¹⁴C]acetate and the increase previously observed with (?)-hydroxycitrate alone was minimized. Agaric acid significantly inhibited fatty acid synthesis from acetate in the presence of exogenous substrate, but the effect was decreased in comparison to rates with only endogenous substrate present. With [2-¹⁴C]lactate as the lipogenic precursor, agaric acid and (?)-hydroxycitrate strongly inhibited fatty acid synthesis. However, agaric acid despite its lower concentration (60 μm vs 2 mm) was twice as effective as (?)-hydroxycitrate. A similar pattern was observed when substrate concentrations of lactate/pyruvate (10 mm) were added to the incubations. When (?)-hydroxycitrate and agaric acid were simultaneously incubated in the presence of endogenous substrate, there was an additive effect of the inhibitors on decreasing fatty acid synthesis. Results are discussed in relation to the origin of substrate for hepatic lipogenesis and whether specific metabolites increase lipogenic rates.     

Characteristics of glycogen synthesis from [1-14C]glucose and its labeled precursors in the piglet liver

The in vivo experiments have established that the rapid decrease in the glycogen content in the liver of piglets during the first 24 hours after birth is associated with the reduction of the degree of label inclusion from [1-14C]glucose into polysaccharide. The level of label inclusion from [1-14C]pyruvate and [1-14C]lactate into the liver glycogen in new-born piglets is higher than from [1-14C]alanine and [1-14C]glutamic acid. During the days immediately after birth the extension of the pool of glucogenic substrates occurs at the expense of alanine and other amino acids during catabolism of which pyruvate is formed. The degree of label inclusion from the investigated substrates into the liver glycogen of piglets of early age decreases in the series: [1-14C]glucose greater than [1-14C]lactate greater than [1-14C]pyruvate greater than [1-14C]alanine. Glutamic acid in the liver of piglets of early age is not a glucogenic substrate.     

Temperature dependence of lipogenesis in isolated hepatocytes from rainbow trout (Salmo gairdneri)

Temperature dependence of lipogenesis in trout liver cells was investigated in the presence of 5 mM lactate using either [14C]lactate or [3H]water. A ratio of 3H/14C-incorporation greater than one is found, irrespective of temperature. Acclimation of fish to 4, 10 or 16 degrees C affects neither the height of lipid synthesis nor its temperature sensitivity. The distribution of [14C]lactate between the main lipid classes and the capacities for cholesterol- and triacylglycerol-synthesis are correlated to the glycogen stores of the hepatocytes. A comparison of fatty acid synthesis and cholesterogenesis in livers of normal fed rat and of trout suggests a capability for lipogenesis in trout somewhat similar to that in mammals.     

Stimulation by alpha-adrenergic agonists of Ca2+ fluxes, mitochondrial oxidation and gluconeogenesis in perfused rat liver.

Glucose output from perfused livers of 48 h-starved rats was stimulated by phenylephrine (2 microM) when lactate, pyruvate, alanine, glycerol, sorbitol, dihydroxyacetone or fructose were used as gluconeogenic precursors. Phenylephrine-induced increases in glucose output were immediately preceded by a transient efflux of Ca2+ and a sustained increase in oxygen uptake. Phenylephrine decreased the perfusate [lactate]/[pyruvate] ratio when sorbitol or glycerol was present, but increased the ratio when alanine, dihydroxyacetone or fructose was present. Phenylephrine induced a rapid increase in the perfusate [beta-hydroxybutyrate]/[acetoacetate] ratio and increased total ketone-body output by 40-50% with all substrates. The oxidation of [1-14C]octanoate or 2-oxo[1-14C]glutarate to 14CO2 was increased by up to 200% by phenylephrine. All responses to phenylephrine infusion were diminished after depletion of the hepatic alpha-agonist-sensitive pool of Ca2+ and returned toward maximal responses after Ca2+ re-addition. Phenylephrine-induced increases in glucose output from lactate, sorbitol and glycerol were inhibited by the transaminase inhibitor amino-oxyacetate by 95%, 75% and 66% respectively. Data presented suggest that the mobilization of an intracellular pool of Ca2+ is involved in the activation of gluconeogenesis by alpha-adrenergic agonists in perfused rat liver. alpha-Adrenergic activation of gluconeogenesis is apparently accompanied by increases in fatty acid oxidation and tricarboxylic acid-cycle flux. An enhanced transfer of reducing equivalents from the cytoplasmic to the mitochondrial compartment may also be involved in the stimulation of glucose output from the relatively reduced substrates glycerol and sorbitol and may arise principally from an increased flux through the malate-aspartate shuttle.     

Synthesis of fatty acids in the perfused mouse liver

1. Fatty acid synthesis de novo was measured in the perfused liver of fed mice. 2. The total rate, measured by the incorporation into fatty acid of (3)H from (3)H(2)O (1-7mumol of fatty acid/h per g of fresh liver), resembled the rate found in the liver of intact mice. 3. Perfusions with l-[U-(14)C]lactic acid and [U-(14)C]glucose showed that circulating glucose at concentrations less than about 17mm was not a major carbon source for newly synthesized fatty acid, whereas lactate (10mm) markedly stimulated fatty acid synthesis, and contributed extensive carbon to lipogenesis. 4. The identification of 50% of the carbon converted into newly synthesized fatty acid lends further credibility to the use of (3)H(2)O to measure hepatic fatty acid synthesis. 5. The total rate of fatty acid synthesis, and the contribution of glucose carbon to lipogenesis, were directly proportional to the initial hepatic glycogen concentration. 6. The proportion of total newly synthesized lipid that was released into the perfusion medium was 12-16%. 7. The major products of lipogenesis were saturated fatty acids in triglyceride and phospholipid. 8. The rate of cholesterol synthesis, also measured with (3)H(2)O, expressed as acetyl residues consumed, was about one-fourth of the basal rate of fatty acid synthesis. 9. These results are discussed in terms of the carbon sources of hepatic newly synthesized fatty acids, and the effect of glucose, glycogen and lactate in stimulating lipogenesis, independently of their role as precursors.     

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.     

Cultured trout liver cells: Utilization of substrates and response to hormones

Summary The characterization of a recently established system for the short-term culture of rainbow trout (Oncorhynchus mykiss) liver cells in chemically defined medium has been extended to studies on the metabolic competence of the cells and the characterization of their response to hormones. Three areas of metabolism have been addressed: a) the utilization of the exogenously added substrates fructose, lactate, glucose, dihydroxyacetone, and glycerol for glucose and lactate formation; b) the effects of the pancreatic hormones insulin and glucagon on cellular glucose formation, lactate formation, and fatty acid synthesis; and c) the effects of insulin and dexamethasone on the estradiol-dependent production of vitellogenin. Incubation of trout liver cells with fructose, lactate, glucose, dihydroxyacetone, or glycerol resulted in enhanced rates of cellular glucose and lactate production. Substrate-induced effects usually were more clearly expressed after extended (20 h) than after acute (5 h) culture periods. Addition of the hormones insulin or glucagon caused dose-dependent alterations in the flux of substrates to glucose and lactate. Rates of de novo synthesis of fatty acids from [¹⁴C]acetate were stimulated by insulin and inhibited by glucagon during acute and extended incubation periods. Treatment of liver cells isolated from male trout for 72 h with estradiol induced vitellogenin production and secretion into the medium. However, the addition of insulin or dexamethasone drastically reduced this estrogen-induced vitellogenesis. These results indicate that trout liver cells cultured in defined medium maintain central metabolic pathways, including glycolysis, gluconeogenesis, lipogenesis, and vitellogenesis as well as their responsiveness to various hormones, for at least 72 h. This cell culture system should provide an excellent model to further characterize metabolic processes in fish liver.     

Inhibition of lipogenesis by halothane in isolated rat liver cells.

1. Halothane at clinically effective concentrations [2.5 and 4% (v/v) of the gas phase of the incubation flask] was found to inhibit significantly lipogenesis from endogenous substrates, e.g., glycogen, or from added lactate plus pyruvate. This was accompanied by a decrease in the ratio of the free [NAD+]/[NADH] of the mitochondrion and the cytoplasm, as shown by the [3-hydroxybutyrate]/[acetoacetate] ratio and the [lactate]/[pyruvate] ratio. 2. Acetoacetate or pyruvate decreased the inhibitory effect of halothane and restored lipogenesis to control rates. They were reduced rapidly by 3-hydroxybutyrate dehydrogenase or lactate dehydrogenase respectively, with the concomitant oxidation of NADH and the generation of NAD+. 3. These results suggest that the mechanism by which halothane inhibits lipogenesis from glycogen or lactate is by inhibition of the oxidation of NADH; this results in inhibition of flux of carbon through pyruvate dehydrogenase and a shortage of acetyl-CoA for fatty acid synthesis. Thus when NADH acceptors are added in the presence of halothane, the concentration of mitochondrial NAD+ is raised so that the flux of carbon through pyruvate dehydrogenase increases and lipogenesis is restored.     

Short-term insulin infused through the portal vein enhances liver gluconeogenesis and glycogenesis from [3-14C]pyruvate in the starved rat

Insulin infusion through the portal vein immediately after a pulse of [3-14C]pyruvate in 24 hr starved rats enhanced the appearance of [14C]glucose at 2, 5 and 10 min and glucose specific activity at 1, 2 and 20 min in blood collected from the cava vein at the level of the suprahepatic veins. Insulin infusion for 5 min decreased liver pyruvate concentration and enhanced both liver and plasma lactate/pyruvate ratio, and it decreased the plasma concentration of all amino acids. When insulin was infused together with glucose, [14C]glucose levels and glucose specific activity decreased in blood but there was a marked increase in liver [14C]glycogen, glycogen specific activity and glycogen concentration, and an increase in liver lactate/pyruvate ratio. The effect of insulin plus glucose infusion on plasma amino acids concentration was smaller than that found with insulin alone. It is proposed that insulin effect enhancing liver gluconeogenesis is secondary to its effect either enhancing liver glycolysis which modifies the liver's cytoplasmic oxidoreduction state to its more reduced form, increasing liver amino acids consumption or both. In the presence of glucose, products of gluconeogenesis enhanced by insulin are diverted into glycogen synthesis rather than circulating glucose. This together with results of the preceding paper (Soley et al., 1985), indicates that glucose enhances liver glycogen synthesis from C3 units in the starved rat, the process being further enhanced in the presence of insulin.     

Lipogenesis in rat brown adipocytes. Effects of insulin and noradrenaline, contributions from glucose and lactate as precursors and comparisons with white adipocytes.

1. Brown adipocytes were isolated from the interscapular depot of male rats maintained at approx. 21 degrees C. In some experiments parallel studies were made with white adipocytes from the epididymal depot. 2. Insulin increased and noradrenaline decreased [U-14C]glucose incorporation into fatty acids by brown adipocytes. Brown adipocytes differed from white adipocytes in that exogenous fatty acid (palmitate) substantially decreased fatty acid synthesis from glucose. Both noradrenaline and insulin increased lactate + pyruvate formation by brown adipocytes. Brown adipocytes converted a greater proportion of metabolized glucose into lactate + pyruvate and a smaller proportion into fatty acids than did white adipocytes. 3. In brown adipocytes, when fatty acid synthesis from [U-14C]glucose was decreased by noradrenaline or palmitate, incorporation of 3H2O into fatty acids was also decreased to an extent which would not support proposals for extensive recycling into fatty acid synthesis of acetyl-CoA derived from fatty acid oxidation. 4. In the absence of glucose, [U-14C]lactate was a poor substrate for lipogenesis in brown adipocytes, but its use was facilitated by glucose. When brown adipocytes were incubated with 1 mM-lactate + 5 mM-glucose, lactate-derived carbon generally provided at least 50% of the precursor for fatty acid synthesis. 5. Both insulin and noradrenaline increased [U-14C]glucose conversion into CO2 by brown adipocytes (incubated in the presence of lactate) and, in combination, stimulation of glucose oxidation by these two agents showed synergism. Rates of 14CO2 formation from glucose by brown adipocytes were relatively small compared with maximum rates of oxygen consumption by these cells, suggesting that glucose is unlikely to be a major substrate for thermogenesis. 6. Brown adipocytes from 6-week-old rats had considerably lower maximum rates of fatty acid synthesis, relative to cell DNA content, than white adipocytes. By contrast, rates of fatty acid synthesis from 3H2O in vivo were similar in the interscapular and epididymal fat depots. Expressed relative to activities of fatty acid synthase or ATP citrate lyase, however, brown adipocytes synthesized fatty acids as effectively as did white adipocytes. It is suggested that the cells most active in fatty acid synthesis in the brown adipose tissue are not recovered fully in the adipocyte fraction during cell isolation. Differences in rates of fatty acid synthesis between brown and white adipocytes were less apparent at 10 weeks of age.     

Control of glycolysis and lipogenesis in the liver by glucagon at the phosphofructokinase-fructose 1,6-diphosphatase site

We have examined the effects of glucagon on lipogenesis from fasted-refed rats incubated under two conditions, either without added substrate or with 10 mml-lactate. Net glycolysis (from glycogen) occurs in the absence of glucagon. This glycolysis is inhibited by glucagon under conditions of no added lactate, and reversed by glucagon to a net gluconeogenesis in the presence of 10 mm lactate. Glucagon markedly inhibits fatty acid synthesis (estimated by incorporation of tritium from THO) in hepatocytes incubated without added substrate; but, in the presence of 10 mml-lactate, the inhibition of fatty acid synthesis is only about 10%. The inhibition of lipogenesis from endogenous glycogen is primarily caused by inhibition of glycolysis. Glucagon markedly lowers the $\text{C}\text{-4,5,6}\text{C}\text{-1,2,3}$ ratio in glucose produced from [1-¹⁴C]galactose, indicating a strong inhibition of phosphofructokinase flux. The $\text{C}\text{-1,2,3}\text{C}\text{-4,5,6}$ ratio in glucose from [1-¹⁴C]glycerol is only slightly less than 1, indicating an active fructose diphosphatase flux even under conditions of active net glycolysis. Glucagon increases this ratio only slightly, suggesting that an acute increase of fructose diphosphatase activity by glucagon may occur, but is of much less importance than the decrease of phosphofructokinase.     

Effects of inhibition of protein synthesis by cycloheximide on lipogenesis in mammary gland and liver of lactating rats.

1. Administration of cycloheximide (an inhibitor of protein synthesis) to lactating rats raised the concentrations of amino acids, and in particular, the branched-chain amino acids (valine, leucine and isoleucine) in blood, liver and mammary gland. 2. Inhibition of protein synthesis increased the incorporation in vivo of L-[U-14C]leucine into lipids of mammary gland and liver. 3. Cycloheximide treatment caused no immediate change in the overall rate of lipogenesis in vivo (measured with 3H2O) in mammary gland but increased the rate in liver 3-fold; this latter effect also occurred in livers of virgin rats. 4. The increased rate of hepatic lipogenesis was not accompanied by significant changes in the plasma insulin concentration or the activity of acetyl-CoA carboxylase. 5. Although cycloheximide decreased the entry of total triacylglycerol into the circulation it did not alter the rate of secretion of newly synthesized saponifiable lipid. 6. Cycloheximide slightly stimulated lipogenesis from endogenous substrates in isolated hepatocytes, but this effect was abolished when lactate was the exogenous substrate. 7. Administration of cycloheximide to virgin rats decreased liver glycogen and increased the hepatic content of glucose 6-phosphate, pyruvate and lactate. 8. It is concluded that (a) there is no short-term link between the rate of protein synthesis and lipogenesis in the lactating mammary gland and (b) the increased rate of hepatic lipogenesis in cycloheximide-treated rats is mainly due to stimulation of glycogenolysis, glycolytic flux and consequent increased availability of pyruvate.     

Interrelationship and control of glucose metabolism and lipogenesis in isolated fat-cells. Effect of the amount of glucose uptake on the rates of the pentose phosphate cycle and of fatty acid synthesis

In order to study the quantitative relationship between fatty acid synthesis and pentose phosphate-cycle activity under different hormonal and dietary conditions affecting the extent of glucose uptake, cells isolated from rat epididymal adipose tissue were incubated in bicarbonate buffer containing [U-(14)C]-, [1-(14)C]- or [6-(14)C]-glucose. From the amount of glucose taken up, the production of lactate and pyruvate, and the incorporation of (14)C from differently labelled [(14)C]glucose into CO(2), fatty acids and glyceride glycerol, the rates of glucose metabolism via different pathways and the extent of lipogenesis under various experimental conditions were determined. The contribution of the pentose phosphate-cycle to glucose metabolism under normal conditions was calculated to be 8%. Starvation and re-feeding, and the presence of insulin, caused an enhancement of glucose uptake, pentose phosphate-cycle activity and fatty acid synthesis. Plots of both pentose phosphate-cycle activity and fatty acid synthesis versus glucose uptake revealed that the extent of glucose uptake, over a wide range, determines the rates of fatty acid synthesis and glucose metabolism via the pentose phosphate cycle. A balance of formation and production of nicotinamide nucleotides in the cytoplasm was established. The total amount of cytoplasmic NADH and NADPH formed was only in slight excess over the hydrogen equivalents required for the synthesis of fatty acids, glyceride glycerol and lactate. Except in cells from starved animals, the pentose phosphate cycle was found to provide only about 60% of the NADPH required for fatty acid synthesis. The results are discussed with respect to an overall control of the different metabolic and biosynthetic reactions in the fat-cells by the amount of glucose transported into the cell.     

Lactate production in the perfused rat liver

1. In aerobic conditions the isolated perfused liver from well-fed rats rapidly formed lactate from endogenous glycogen until the lactate concentration in the perfusion medium reached about 2mm (i.e. the concentration of lactate in blood in vivo) and then production ceased. Pyruvate was formed in proportion to the lactate, the [lactate]/[pyruvate] ratio remaining between 8 and 15. 2. The addition of 5mm- or 10mm-glucose did not affect lactate production, but 20mm- and 40mm-glucose greatly increased lactate production. This effect of high glucose concentration can be accounted for by the activity of glucokinase. 3. The perfused liver released glucose into the medium until the concentration was about 6mm. When 5mm- or 10mm-glucose was added to the medium much less glucose was released. 4. At high glucose concentrations (40mm) more glucose was taken up than lactate and pyruvate were produced; the excess of glucose was probably converted into glycogen. 5. In anaerobic conditions, livers of well-fed rats produced lactate at relatively high rates (2.5mumol/min per g wet wt.). Glucose was also rapidly released, at an initial rate of 3.2mumol/min per g wet wt. Both lactate and glucose production ceased when the liver glycogen was depleted. 6. Addition of 20mm-glucose increased the rate of anaerobic production of lactate. 7. d-Fructose also increased anaerobic production of lactate. In the presence of 20mm-fructose some glucose was formed anaerobically from fructose. 8. In the perfused liver from starved rats the rate of lactate formation was very low and the increase after addition of glucose and fructose was slight. 9. The glycolytic capacity of the liver from well-fed rats is equivalent to its capacity for fatty acid synthesis and it is pointed out that hepatic glycolysis (producing acetyl-CoA in aerobic conditions) is not primarily an energy-providing process but part of the mechanism converting carbohydrate into fat.     

Early effects of phytohaemagglutinin on glucose metabolism of normal human lymphocytes

1. Phytohaemagglutinin induced early changes in the catabolism of glucose by normal human lymphocytes suspended in a bicarbonate buffer. During 4hr. incubation glucose utilization was almost doubled. 2. The rates of several reactions in the metabolism of glucose were estimated. Total pyruvate formation, lactate production and fatty acid synthesis were stimulated to the same degree as was glucose utilization. The pentose cycle and the glycogen synthesis were also stimulated but less than was glucose utilization. The pentose cycle was found to account for 1.4% and 0.9% of the total glucose utilization without and with phytohaemagglutinin respectively. In these cells rates of triose phosphate iso-merization were at least six to seven times the rate of glucose phosphorylation. On an average 55-60% of the total carbon dioxide evolved was derived from decarboxylation of pyruvate, 25-30% from the tricarboxylic acid cycle and about 15% from the pentose cycle. Observed ratios of (14)C specific yields in glycogen from [1-(14)C]- and [6-(14)C]-glucose could possibly be explained by assuming the existence of two separate glucose 6-phosphate pools. 3. During 4hr. incubation in bicarbonate buffer (14)C from [U-(14)C]serine was incorporated into perchloric acid-insoluble material. This incorporation was stimulated by phytohaemagglutinin but was almost completely inhibited by puromycin. Puromycin also abolished phytohaemagglutinin-induced stimulation of glycolysis.     

Effects of 2,4-dihydroxybutyrate on lipogenesis in rat hepatocytes.

Hepatocytes were prepared from rats fasted 2 days and refed a high carbohydrate diet for 1 day. These cells contained very high levels of glycogen (about half the defatted dry weight) and carried out high rates of lipogenesis (up to 800 micron at tritium incorporation from 3HOH/g (defatted dry weight)/h), even in the absence of added substrates. Pentose cycle flux was estimated by a method involving the use of [1-14C]galactose (Rognstad, R. (1976) Int. J. Biochem. 7, 221-228). In hepatocytes from normal fasted refed rats, the amount of NADPH produced by the pentose cycle was sufficient for about one-half to three-fourths of that required for fatty acid synthesis. 2,4-Dihydroxybutyrate, a malic enzyme inhibitor (Schimerlik, M.I. & Cleland, W.W. (1977) Biochemistry 16, 565-570) markedly depressed the randomization of 14C in lactate from [6-14C]hexoses, indicating an inhibition of the pyruvate cycle. 2,4-Dihydroxybutyrate (10 mM) had only a slight inhibitory effect on overall lipogenesis, but increased the rate of the pentose cycle by 40 to 90%.     

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.     

The effect of 2,4-dinitrophenol on adipose-tissue metabolism

1. The effect of dinitrophenol on the metabolism of glucose labelled with (14)C and tritium by epididymal fat-pad segments from fed rats was studied. Dinitrophenol at concentrations of 0.1-0.3mm: (a) had little effect on glucose utilization; (b) depressed synthesis of fatty acids and greatly increased that of lactate; (c) increased the T/(14)C ratio in fatty acids synthesized from [U-(14)C,3-T]glucose and decreased that in fatty acids synthesized from [U-(14)C,4-T]glucose; (d) abolished randomization of (14)C from [6-(14)C]glucose in lactate. 2. Dinitrophenol stimulated oxidation of pyruvate and greatly inhibited the oxidation of lactate. It inhibited lipogenesis from pyruvate and lactate. 3. From the isotope data it was calculated that: (a) dinitrophenol stimulates oxidation via the tricarboxylic acid cycle three- to six-fold; (b) dinitrophenol depresses markedly the operation of the pentose cycle; (c) in the presence of dinitrophenol, NADPH formed in the pentose cycle provides all the hydrogen equivalents for fatty acid reduction, whereas, in its absence, NADPH provides 50-70% of the hydrogen equivalents; (d) in the presence of dinitrophenol, there is an excess of ATP produced in the cytoplasm, which flows into the mitochondria. A reverse flow operates in the absence of dinitrophenol. 4. A balance of formation and utilization of reduced nicotinamide nucleotides in the cytoplasm was established. With dinitrophenol there is some excess of NADH. There are indications that this excess may be transferred into mitochondria in the form of malate. 5. Our results are interpreted to indicate the absence from adipose tissue of the alpha-glycerophosphate shuttle for transferring reducing equivalents from the cytoplasm to mitochondria. 6. The effects of dinitrophenol are accounted for in terms of decreased ATP concentrations in the cells, leading to marked decrease in pyruvate carboxylation in the mitochondria and depression of fatty acid synthesis in the cytoplasm.