Control of hepatic gluconeogenesis: role of fatty acid oxidation.

Octanoate has been found to activate the gluconeogenic pathway in perfused isolated rat liver. Whether a net increase in the production of glucose is observed is a function of the relative concentrations of the glucose precursor and the fatty acid. The kinetics of octanoate interaction with the gluconeogenic pathway are influenced by the rate changes induced by decreases in pyruvate concentration as a result of the increased NAD redox potential produced by the oxidation of fatty acid. Taking this into account, two distinct effects of octanoate were identified. The first is an increase in the Vmax even at the lowest (25 microM) concentration of the fatty acid tested. The second is a progressive decrease in [pyruvate]0.5 as a function of octanoate concentration. The latter occurs at low (less than 0.1 mM), presumably physiological, pyruvate concentrations, when its mitochondrial transport is limiting, indicating that this process must have been activated. The former is observable even at high (greater than 0.5 mM), supraphysiological, concentrations of pyruvate, when its mitochondrial transport is not limiting, indicating that a distal step, presumably pyruvate carboxylation, is activated. The action of octanoate in increasing gluconeogenesis has been found not to be related to a decreased flux through pyruvate dehydrogenase, neither to changes in the NAD redox potential nor to its ability to increase energy production. Actually, the oxygen uptake induced by octanoate was largely accounted for by the production of ketone body and the latter process was found to be independent of variations in energy demand.

Synergistic inhibition of hepatic ketogenesis in the presence of insulin and a cAMP antagonist.

The separate or combined effects of insulin and the cAMP antagonist, the Rp-diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp-cAMPS), were examined on fatty acid-stimulated ketogenesis in hepatocytes from normal fasted rats. Addition of 0.4 mM oleic acid or 0.4 mM octanoic acid resulted in a linear increase in ketone production measured over 60 min. When oleic acid was the substrate, incubation with 1 to 30 microns Rp-cAMPS alone or 0.1 to 10 nM insulin alone caused a variable decrease in the production of ketones which did not exceed an average value of 30% in any one experiment. The simultaneous addition of Rp-cAMPS and insulin resulted in a greater than additive inhibition which reached average values between 47-60% when the theoretical combined inhibitory effect of the insulin alone plus the Rp-cAMPS alone was less than 18%. No significant effects of either insulin or Rp-cAMPS, alone or in combination, were seen when octanoic acid was the substrate. These data imply that Rp-cAMPS can potentiate insulin inhibition of hepatic ketogenesis through inhibition of a cAMP-mediated process.

Induction of the SOS function sfiA in E. coli by systems which generate triplet ketones.

Generation of triplet ketones, either chemically through thermal decomposition of 3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxetane and 3-[N-(pyridino)carbamoyl]methyl-3,4,4-trimethyl-1,2-dioxetane++ + or enzymatically via the aerobic oxidation of isobutyraldehyde trimethylsilyl enol ether catalyzed by horse-radish peroxidase, triggers the SOS function sfiA in E. coli. Although the observed effects are relatively weak and the triplet ketone scavenger tryptophan was ineffective in this system, our results provide evidence for the involvement of triplet ketones in this type of DNA damage. Possible mechanisms are discussed.

Medium-chain triglyceride feeding over the long term: the metabolic fate of [14C]octanoate and [14C]oleate in isolated rat hepatocytes.

Feeding medium-chain triglycerides (MCT) is reported to result in less weight gain as fat than feeding long-chain triglycerides (LCT). In this experiment, fatty acid metabolism was studied in hepatocytes isolated from rats fed high fat diets containing MCT (70.9% of total energy), LCT (72.3% total energy) or a low fat (LF) (10.8% total energy). In the MCT group, hepatocyte utilization of [1-14C]oleate or [1-14C]octanoate, unlabeled ketone body (KB) production and lipogenesis (LG) were greater than in the other groups. Esterified products (EST) from both fatty acids were greater in the MCT groups, and 5-tetradecycloxy-2-furoic acid, an effective inhibitor of LG, did not abolish this. Lactate/pyruvate increased EST from octanoate and total LG but had little effect on octanoate oxidation. These data suggest that the capacity of medium-chain fatty acid esterification is adaptable to MCT content of the diet and may help regulate their metabolic fate. Despite high rates of KB production in the MCT group, the NADH-NAD ratio was not elevated. Rapid lipogenesis may contribute to the utilization of the excess energy equivalents formed. High LG and EST in the presence of a glycerol precursor may limit the usefulness of MCT in dietetic products for weight control. The animal fed an MCT diet is unusual in that the antithetical pathways of fatty acid catabolism and anabolism are being carried out simultaneously and at rapid rates. The lesser food efficiency of MCT may be related to the energy losses necessarily associated with cycling between these processes.(ABSTRACT TRUNCATED AT 250 WORDS)

Fungal volatiles associated with moldy grain in ventilated and non-ventilated bin-stored wheat.

The fungal odor compounds 3-methyl-1-butanol, 1-octen-3-ol and 3-octanone were monitored in nine experimental bins in Winnipeg, Manitoba containing a hard red spring wheat during the autumn, winter and summer seasons of 1984-85. Quality changes were associated with seed-borne microflora and moisture content in both ventilated and non-ventilated bins containing wheat of 15.6 and 18.2% initial moisture content. All three odor compounds occurred in considerably greater amounts in bulk wheat in non-ventilated than in ventilated bins, particularly in those with wheat having 18.2% moisture content. The presence of these compounds usually coincided with infection of the seeds by the fungi Alternaria alternata (Fr.) Keissler, Aspergillus repens DeBarry, A. versicolor (Vuill.) Tiraboschi, Penicillium crustosum Thom, P. oxalicum Currie and Thom, P. aurantiogriesum Dierckx, and P. citrinum Thom. High production of all three odor compounds in damp wheat stored in non-ventilated bins was associated with heavy fungal infection of the seeds and reduction in seed germinability. High initial moisture content of the harvested grain accelerated the production of all three fungal volatiles in non-ventilated bins.

Fetal rat hyperinsulinism and hyperglucagonism: effects on hepatic ketogenesis, lipogenesis, and gluconeogenesis.

To study the direct effects of hyperglucagonism and hyperinsulinism (both with glucose excess) on fetal intermediary metabolism, rat liver explants from 19-day-gestated fetuses were maintained in culture for 48 h. The liver cubes were exposed to 0, 250, or 500 mU/ml porcine insulin or 5 micrograms/ml glucagon. In addition, lipogenesis from 3H2O was cumulated throughout the 48 h. Chronic hyperinsulinism in the fetal rat doubled hepatic lipogenesis and curtailed hepatic gluconeogenesis and ketogenesis by 80% and 50%, respectively. Chronic hyperglucagonism was without effect; however, the fetal liver did yet respond to 1 mM (Bu)2cAMP.

In vitro hepatic gluconeogenesis and ketogenesis as affected by prolonged ketonemia-glucosuria and fasting in steers.

Both 1,3-butanediol, which causes ketonemia, and phlorizin, which causes glucosuria, were given to four steers for 28 days to determine effects of prolonged ketonemia and glucosuria on in vitro hepatic gluconeogenesis and ketogenesis. Treatments were: control ration; control with butanediol plus phlorizin; and fasting for 9 days. Liver slices, obtained by biopsy, were incubated with carbon-14 substrates. Substrate converted to glucose [mumol/(h X g liver)] during control, butanediol plus phlorizin, and fasting averaged 2.34, 7.21, and 12.00 for propionate; .99, 3.80, and 12.26 for lactate; .30, .76, and 2.20 for alanine; and 2.06, 5.37, and 5.78 for glycerol. Omission of calcium++ eliminated increases of gluconeogenesis caused by butanediol plus phlorizin and by fasting. Ketone bodies, octanoate, and bovine serum albumin did not affect glucose production markedly. Stearate inhibited gluconeogenesis during all periods except fasting. Production of beta-hydroxybutyrate [mumol/(h X g liver)] during control, butanediol plus phlorizin, and fasting averaged 2.07, 4.27, and 3.25 from butyrate and .06, .27, and .02 from palmitate. Results demonstrate that the gluconeogenic capacity of bovine liver is responsive to physiological and nutritional status.

Hepatic gluconeogenic and ketogenic interrelationships in the lactating cow.

Interrelationships between propionate, palmitate, and butyrate metabolism were investigated in vitro with [1-carbon-14] carboxyl substrates. Production of labeled glucose, ketone bodies, and carbon dioxide was used to estimate rates of bovine hepatic gluconeogenesis and ketogenesis. Incubations were with liver slices from eight lactating Holstein cows fed either a control or high concentrate-low fiber diet. Liver samples were acquired by trochar biopsy at 30, 60, 90, and 180 days postpartum. Ketone production from both palmitate and butyrate was highest in liver slices obtained at 30 days. Glucose production from labeled propionate was also highest in early lactation. The higher rates of gluconeogenesis and ketogenesis in early lactation were associated with higher hepatic carnitine palmitoyltransferase (EC 2.3.1.21) activity. Feeding the high concentrate enhanced gluconeogenesis from propionate and decreased ketogenesis from palmitate. Propionate addition (10 mM) to incubation media also decreased the total amount of palmitate oxidized [( carbon-14] dioxide plus [carbon-14] ketones). Diet had no effect on hepatic butyrate metabolism. Results indicated that ketogenesis is regulated via rate of long chain fatty acid transport into the mitochondria. Stage of lactation has a greater influence on long and short chain fatty acid metabolism than does diet composition.

Role of basal glucagon levels in the regulation of splanchnic glucose output and ketogenesis in insulin-deficient humans.

The aim of the present study was to investigate the influence of hepatic glycogen depletion and increased lipolysis on the response of splanchnic glucose output and ketogenesis to combined glucagon and insulin deficiency in normal man. Healthy subjects were studied after a 60-h fast and compared with a control group studied after an overnight fast. Net splanchnic exchange of glucose, gluconeogenic precursors, free fatty acids (FFA) and ketone acids were measured in the basal state and during intravenous infusion of somatostatin (9 micrograms/min) for 90-140 min (overnight fasted subjects) or for 5 h (60-h fasted subjects). During the infusion of somatostatin, euglycemia was maintained by a variable intravenous infusion of glucose. Prior to somatostatin infusion, after an overnight (12-14 h) fast, splanchnic uptake of glucose precursors (alanine, lactate, pyruvate, glycerol) could account for 26% of splanchnic glucose output (SGO) indicating primarily glycogenolysis. Somatostatin infusion resulted in a 50% reduction in both insulin and glucagon concentrations and a transient decline in SGO which returned to baseline values by 86 +/- 11 min at which point the glucose infusion was no longer necessary to maintain euglycemia. Arterial concentrations of FFA and beta-OH-butyrate and splanchnic beta-OH-butyrate production rose 2.5-fold, 6-fold and 7.5-fold, respectively, in response to somatostatin infusion. In the 60-h fasted state, basal SGO (0.29 +/- 0.03 mmol/min) was 60% lower than after an overnight fast and basal splanchnic uptake of glucose precursors could account for 85% of SGO, indicating primarily gluconeogenesis. Somatostatin administration suppressed the arterial glucagon and insulin concentrations to values comparable to those observed during the infusion in the overnight fasted state. SGO fell promptly in response to the somatostatin infusion and in contrast to the overnight fasted state, remained inhibited by 50-100% for 5 h. Infusion of glucose was consequently necessary to maintain euglycemia throughout the 5-h infusion of somatostatin. Splanchnic uptake of gluconeogenic precursors was unchanged during somatostatin despite the sustained suppression of SGO. Basal arterial concentration and splanchnic exchange of beta-OH-butyrate were respectively 22-fold and 6- to 7-fold elevated and basal FFA concentration was 70% increased as compared to the corresponding values in the overnight fasted state.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of fatty acids on energy metabolism. 2. Kinetics of changes in metabolic rates and changes in subcellular adenine nucleotide contents and pH gradients following addition of octanoate and oleate in perfused rat liver.

The uncoupling-like effect of fatty acids [ Scholz , R., Schwabe , U., and Soboll , S. (1984) Eur. J. Biochem. 141, 223-230] was further substantiated in experiments with perfused rat livers by two ways: firstly the kinetics of changes in metabolic rates (oxygen consumption, ketogenesis, fatty acid oxidation) were analysed; secondly subcellular contents of adenine nucleotides and pH gradients across the mitochondrial membrane were determined following fractionation of freeze-fixed and dried tissues in non-aqueous solvents. The following results were obtained. The relaxation kinetics of the increase in oxygen consumption following fatty acid infusion revealed two components, a rapid one with a half-time around 10 s and a slow one with a half-time of more than 100 s. The rapid component was similar to the kinetics of fatty acid oxidation (ketogenesis and 14CO2 production from labelled fatty acids) whereas the half-time of the slow component was in the range of half-times observed with the increase in oxygen consumption following addition of carbonylcyanide p-trifluoromethoxyphenylhydrazone. In the presence of fatty acids, the cytosolic ATP concentrations and ATP/ADP ratios decreased, whereas the corresponding parameters for the mitochondrial space were either increased (oleate) or decreased (octanoate). The effects of oleate were dependent on the albumin concentrations in the perfusate. The normally large difference between cytosolic and mitochondrial ATP/ADP ratios became smaller. Similar observations were obtained with uncoupling agents. The pH gradient across the mitochondrial membrane as calculated from the subcellular distribution of 5,5 dimethyl[2-14C]oxazolidine-2,4-dione was inversed following the addition of both carbonylcyanide p-trifluoromethoxyphenylhydrazone and fatty acids, i.e. the mitochondrial matrix became more acidic than the cytosol. The pH gradient was not affected when oleate was added in the presence of high albumin concentrations. The data support the hypothesis that the increase in hepatic oxygen consumption due to octanoate or oleate is, in part, caused by a mechanism similar to uncoupling of oxidative phosphorylation. This mechanism seems not to be an artifact of isolated systems; it may be of physiological importance for processes in which reducing equivalents are removed independently of the ATP demand of the hepatocyte.

Influence of fatty acids on energy metabolism. 1. Stimulation of oxygen consumption, ketogenesis and CO2 production following addition of octanoate and oleate in perfused rat liver.

Changes in metabolic rates (oxygen consumption, ketogenesis, 14CO2 production from labelled fatty acids, glycolysis) following the addition of octanoate or oleate were studied in isolated livers from fed and starved rats perfused with Krebs-Henseleit bicarbonate buffer in a non-recirculating system. The following results were obtained. The infusion of fatty acids caused a large increase in the rate of oxygen consumption. The effect was greater with octanoate than with oleate and was half-maximal with fatty acid concentrations (free plus albumin bound) around 0.1 mM. The effects of oleate were only partially suppressed when the perfusate contained albumin concentrations near the physiological range. When fatty acids were oxidized at high rates, the glycolytic rate was diminished by 50%. The increase in oxygen consumption could not be explained fully by the increased ATP demand for fatty acid metabolism or by a compensation for the diminished extramitochondrial ATP generation. In the presence of phenylalkyl oxirane carboxylic acid, an inhibitor of the transport of long-chain acyl-CoA derivates into the mitochondria, ketogenesis and 14CO2 production from labelled oleate were strongly inhibited, whereas the increase in oxygen consumption was only slightly affected. In the presence of antimycin A, the increase in oxygen consumption due to fatty acids was totally abolished. Following pretreatment of rats with ciprofibrate (induction of enzymes for peroxisomal beta-oxidation of long-chain fatty acids), ketogenesis (but not 14CO2 production) from oleate was enhanced threefold. The increase in oxygen consumption, however, was not affected. In conclusion, the increase in hepatic oxygen consumption due to addition of fatty acids reflects a mitochondrial process; it is, in part, independent of the ATP demand of the cell. An uncoupling-like effect of fatty acids on the respiratory chain and its possible physiological significance in ketogenesis are discussed.

The ketogenic effect of glucose in rumen epithelium of ovine (Ovis aries) and bovine (Bos taurus) origin.

In experiments with rumen epithelium incubated in vitro the ratio of 3-hydroxybutyrate: acetoacetate produced was similar to the ratio reported for portal blood, and the ratio ketogenesis: oxidized to CO2 of butyrate was also close to values reported in vivo. Ovine and bovine epithelium incubated with butyrate differed significantly by the values of about 12-17 and 4-7 obtained for the ratio of 3-hydroxybutyrate: acetoacetate. Increasing levels of butyrate in the incubation medium resulted in a decreasing proportion of butyrate oxidized to CO2 and an increasing proportion of ketogenesis. The addition of glucose to butyrate in the incubation medium significantly increased the rate of ketogenesis from butyrate by ovine and bovine tissues. The addition of glucose to butyrate in the incubation medium significantly decreased the rate of butyrate oxidation to CO2 by ovine and bovine tissues. The ketogenic effect of glucose was also apparent in perfused rumen epithelium with butyrate at the mucosal side and glucose at the serosal side.

Possible interrelationship between gluconeogenesis and ketogenesis in the liver.

Effects of various ketogenic substrates on gluconeogenesis from lactate were examined. D,L-3-Hydroxybutyrate (5 mM) stimulated gluconeogenesis by 41%, the effect being the same as that of 5 mM acetate (49%). No stimulating effect of acetoacetate was observed; conversely, acetoacetate (up to 40 mM) partially or completely abolished the observed stimulating effects of acetate, oleate, and 3-hydroxybutyrate. The results suggest that, in intact liver cells, pyruvate is transported into mitochondria in exchange for acetoacetate and that an interrelationship between gluconeogenesis and ketogenesis at the level of mitochondrial pyruvate carrier may exist in the liver.

Oleate metabolism in isolated hepatocytes from lean and obese Zucker rats. Influence of a high fat diet and in vitro response to glucagon.

The uptake and metabolism of [1-14C]oleate (0.3 mmol/L) were studied in isolated hepatocytes from lean and obese Zucker rats fed either a control (low-fat) diet or a high-fat diet. With the control diet, [1-14C]oleate uptake was increased by 70% in the obese rats, and fat-feeding decreased this uptake to values comparable to that of their lean littermates. Interestingly, the hepatocyte mean surface area was increased in the obese mutants by 21% with the control diet and by 30% with the high-fat diet. The possible reasons for the differences in oleate uptake are discussed. With the control diet, cells from the obese rats showed a four-fold rise in [1-14C]oleate esterification, while ketogenesis (beta-hydroxybutyrate + acetoacetate production) as well as the radioactive acid-soluble products were greatly depressed. Production of CO2 was very low and similar in both groups of animals. Adaptation to the high-fat diet in the obese rats resulted in a reversal between esterification and oxidation of oleate: the latter became the major metabolic pathway as in the lean rats. The ketogenic capacity was greatly if not completely restored. In the lean animals, glucagon stimulated ketogenesis both in the presence or absence of oleate and decreased [1-14C]oleate esterification. In the obese rats, the hormone exerted a significant ketogenic effect only if oleate was present and did not influence its esterification. The data demonstrate the following abnormalities in the hepatocytes of obese Zucker rats: (1) an enlargement of cell size, (2) an increased oleate uptake, (3) a virtual absence of a ketogenic response to exogenous oleate, and (4) a markedly increased esterification of the latter. The metabolic defects, but not the cell size, appear to be largely corrected by an adaptation to a high-fat diet. The hepatic response to glucagon was decreased in the obese rats at the level of endogenous ketogenesis.

Comparison of the effects of 2-chloropropionate and dichloroacetate on ketogenesis and lipogenesis in isolated rat hepatocytes.

The respective effects of 2-chloropropionate and dichloroacetate on the pyruvate metabolic crossroads, lipogenesis and ketogenesis, were compared in hepatocytes isolated from fed rats. 2-Chloropropionate acts as an exclusive pyruvate dehydrogenase activator: it increases ketogenesis, lipogenesis, Krebs cycle intermediates and mitochondrial NADH/NAD+ ratio. The effects of dichloroacetate depend on experimental conditions and the intensity of its catabolization into oxalate: the resultant action of dichloroacetate on tested parameters combines the effects of pyruvate dehydrogenase activation on the one hand, and pyruvate carboxylase inhibition by oxalate on the other. A mixture of 2-chloropropionate plus oxalate mimics the effects of dichloroacetate. In hepatocytes from fed rats, endogenous lipogenesis is correlated with the mitochondrial NADH/NAD+ ratio, irrespective of the effector added.

Metabolic fate of non-esterified fatty acids in isolated hepatocytes from newborn and young pigs. Evidence for a limited capacity for oxidation and increased capacity for esterification.

In hepatocytes isolated from 48 h-old starved of suckling newborn pigs or from 15-day-old starved piglets, the rate of ketogenesis from oleate or from octanoate is very low. This is not due to an inappropriate fatty acid uptake by the isolated liver cells, but results from a limited capacity for fatty acid oxidation. Some 80-95% of oleate taken up is converted into esterified fats, whatever the age or the nutritional conditions. Three lines of indirect evidences suggest that fatty acid oxidation is not controlled primarily by malonyl-CoA concentration in newborn pig liver. Firstly, the addition of glucagon does not increase fatty acid oxidation or ketogenesis. Secondly, the rate of lipogenesis is very low in isolated hepatocytes from newborn pigs. Thirdly, the rates of oxidation and ketogenesis from octanoate are also decreased in isolated hepatocytes from newborn and young piglets. The huge rate of esterification of fatty acids in the liver of the newborn pigs probably represents a species-specific difference in intrahepatic fatty acid metabolism.