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Effects of dietary proteins on lipogenic enzymes in rat liver.

When fasted rats were fed fat-free diets containing various sources of protein for 3 d, the activities of liver glucose-6-phosphate dehydrogenase, malic enzyme, acetyl-CoA carboxylase and fatty acid synthetase were markedly lower in rats fed soybean protein or gluten than in those fed casein or fish protein. Since malic enzyme mRNA activity was not low in the soybean protein-fed animals, the translation of malic enzyme appears to be suppressed by dietary soybean protein. The incorporation of tritiated water into liver fatty acids was significantly lower in animals fed soybean protein than in those fed casein. The triglyceride levels in plasma and especially in liver were also lower in the groups fed soybean and gluten than in the groups fed casein and fish. In addition, when dietary soybean protein was replaced with amino acids to simulate casein or soybean protein, the effects on the levels of lipogenic enzymes were still found but were not as great. Thus, some effects can be ascribed to the protein itself and some to the amino acid composition of the diet.

Acetyl-CoA Carboxylase↗

The effect of starvation and starvation followed by feeding on enzyme activity and the metabolism of [U-14C]glucose in liver from growing chicks.

1. The conversion of [U-(14)C]glucose into carbon dioxide, cholesterol and fatty acids in liver slices and the activities of ;malic' enzyme, citrate-cleavage enzyme, NADP-linked isocitrate dehydrogenase and hexose monophosphate-shunt dehydrogenases in the soluble fraction of homogenates of liver were measured in chicks that were starved or starved then fed. 2. In newly hatched chicks the incorporation of [U-(14)C]glucose and the activity of ;malic' enzyme did not increase unless the birds were fed. The response to feeding of [U-(14)C]glucose incorporation into fatty acids increased as the starved chicks grew older. 3. Citrate-cleavage enzyme activity increased slowly even when the newly hatched chicks were unfed. On feeding, citrate-cleavage enzyme activity increased at a much faster rate. 4. In normally fed 20-day-old chicks starvation decreased the incorporation of [U-(14)C]glucose into all three end products and depressed the activities of ;malic' enzyme and citrate-cleavage enzyme. Re-feeding increased all of these processes to normal or higher-than-normal levels. 5. In both newly hatched and 20-day-old chicks starvation increased the activity of isocitrate dehydrogenase and feeding or re-feeding decreased it. 6. Very little change in hexose monophosphate-shunt dehydrogenase activity was observed during the dietary manipulations. 7. The results indicate that increased substrate delivery to the liver is the principal stimulus to the increased rate of glucose metabolism observed in newly hatched chicks. The results also suggest that changes in the activities of ;malic' enzyme and citrate-cleavage enzyme are secondary to an increased flow of metabolites through the glucose-to-fatty acid pathway and that the dehydrogenases of the hexose monophosphate shunt play a minor role in NADPH production for fatty acid synthesis.

Age Factors↗

Tumor-associated metabolic alterations in patients with gastric and esophageal cancer.

BACKGROUND/AIMS: This study was conducted to elucidate tumor-induced alterations in pace maker enzymes and possible effects of pre-operative nutritional support on those enzymes in gastrointestinal (GI) cancer patients. METHODOLOGY: Three pacemaker enzymes; phosphoenolpyruvate carboxykinase (PEPck) for gluconeogenesis, malic enzyme for de novo fatty acid synthesis, and lipoprotein lipase (LPL) for triglyceride clearance in liver and adipose tissues were examined in 22 patients with gastrointestinal cancers, both with or without pre-operative total parenteral nutritional support. Five patients with normal nutrition and liver function but undergoing cholecystectomy were used as controls. RESULTS: Activity of hepatic malic enzyme was significantly decreased (p < 0.05) and activity of hepatic PEPck was slightly elevated in patients with advanced stage cancer. LPL activity in the abdominal subcutaneous tissue was increased by advanced tumor bearing (p < 0.05). Pre-operative total parenteral nutrition for 1 week resulted in significant stimulation of LPL activity in adipose tissue from patients with advanced cancer compared to that from controls (6.53 +/- 4.99 U/g tissue and 1.32 +/- 0.18 U/g tissue, respectively). CONCLUSIONS: Effects of tumor bearing on key regulatory enzymes were evident only at the advanced stages. Pre-operative hyperalimentation may increase fat storage at the peripheral tissue by stimulating tissue LPL activity even at the advanced stage disease.

Adult↗

Influence of age and exercise training on lipid metabolism in Fischer-344 rats.

The influence of training on fatty acid and glyceride synthesis by liver and adipose tissue homogenates of young and old Fischer-344 rats was examined. Four groups of rats (10 animals/group) were studied: young untrained, young trained, old untrained, and old trained. Training of each group was for 10 wk at 75% maximal O2 uptake. Young rats were killed at 6 mo of age and old rats were killed at 27 mo of age. Fatty acid synthesis was assessed by measuring the activities of acetyl-CoA carboxylase, fatty acid synthase, ATP citrate-lyase, "malic" enzyme, and glucose-6-phosphate dehydrogenase. Glyceride synthesis was evaluated by determining the rate of incorporation of [14C]glycerol 3-phosphate into lipids. In addition, lipoprotein lipase activity was measured in acetone-ether powders of adipose tissue from the four groups of rats. In liver, training had no effect on fatty acid or glyceride synthesis in either group. However, aging caused a significant decrease in the activities of four of the lipogenic enzymes but had no effect on glyceride synthesis. Training caused an increase in fatty acid synthase and glyceride synthesis in adipose tissue, and aging decreased lipoprotein lipase activity. It was concluded that training enhances the synthetic capacity of lipids by adipose tissue but that aging had a more profound effect in that the activities of the enzymes involved in these processes were lower in the old rats. Furthermore, the decreased activity of lipoprotein lipase in the older rats may explain the higher plasma triglyceride levels that were observed in these animals.

ATP Citrate (pro-S)-Lyase↗

The effect of prolonged hyperglycemia on metabolic alterations in the subtotally pancreatectomized rat.

A delayed onset of diabetes is characteristic of subtotally pancreatectomized patients in whom persistent hyperglycemia per se is documented to lead to the development of insulin resistance. This study was conducted to elucidate the metabolic alterations seen during transition of the acute to chronic phase after subtotal pancreatectomy (SP). Eight male Sprague-Dawley rats were studied 2 weeks after surgery in the acute phase, and the other eight at 4 weeks in the chronic phase. Phosphoenolpyruvate carboxykinase (PEPck) for gluconeogenesis and the malic enzyme for de novo fatty acid synthesis in the liver showed a reciprocal change, the former activity being increased, while the latter was suppressed. Both alterations were more pronounced in the chronic phase. In the acute phase, lipoprotein lipase (LPL) for triglyceride clearance decreased in the adipose tissue, while that in the cardiac and skeletal muscle became significantly elevated. The latter elevations were decreased in the chronic phase. Sustained hyperglycemia in the SP rats not only increased the changes in PEPck and malic enzyme activities but reversed the tissue-specific muscle LPL elevations. These changes might help to explain the wasting condition seen in surgically induced diabetic patients.

Adipose Tissue↗

Time-dependent effects of insulin on lipid synthesis in cultured fetal rat hepatocytes: a comparison between lipogenesis and glycogenesis.

The lipogenic effect of insulin was studied in 18-day-old fetal rat hepatocytes after 2 to 3 days of culture in the presence of glucocorticoids when an acute stimulatory effect of insulin on glycogenesis was present. The rate of [1-14C]-acetate incorporation into lipids measured for 4 hours was much higher than with [U-14C]-glucose (30 v 3.8 nmol/h/mg protein). The stimulatory effect of insulin on lipid labeling remained weak (1.2-fold) and contrasted with its striking stimulatory effect on [U-14C]-glucose incorporation into glycogen (fourfold). When lipid labeling was assessed in longer experiments, increasing acetate concentrations in the medium stimulated the incorporation rate of [1-14C]-acetate into lipids (3.5-fold from 1 to 5 mmol/L after 36 hours) and decreased that of [U-14C]-glucose (by twofold). The stimulatory effect of insulin on the rate of lipid labeling developed with both precursors from 12 to 36 hours after insulin exposure (by approximately twofold) independently of acetate concentration and was not glucocorticoid-dependent, contrary to the glycogenic response. Addition of a glucose, load simultaneously with insulin increased the stimulation of lipogenesis when measured with [U-14C]-glucose (twofold to 3.7-fold). Besides contributing to an accumulation of larger and numerous lipid droplets in the cells, insulin increased fatty acid synthase activity by 26%, whereas malic enzyme was not affected. Thus, insulin-dependent lipogenesis in cultured fetal hepatocytes appears to be mostly regulated by a long-term mechanism, contrary to the glycogenic effect of insulin.

Acetates↗

Sesamol as an inhibitor of growth and lipid metabolism in Mucor circinelloides via its action on malic enzyme.

Sesamol, a nonoil component of sesame seed oil, inhibited growth, fatty acid synthesis, and desaturation by Mucor circinelloides in vivo. Although sesamol also inhibited the growth of other fungi and yeasts, its effect on the lipid metabolism of M. circinelloides was exceptional. An enzymological study demonstrated that sesamol affected lipid synthesis primarily by the inhibition of malic enzyme activity, thereby limiting the NADPH supply for fatty acid synthesis and desaturation. Sesamol itself had no inhibitory effect on malic enzyme activity in vitro. A metabolite of sesamol is therefore probably responsible for the in vivo effects of sesamol on lipid metabolism.

Antioxidants↗

Structural identity of the subunits of pigeon liver malic enzyme.

Pigeon liver malic enzyme was found to have arginine, alanine, and tyrosine as the only N-terminal, N-1, and N-2 amino acids, respectively. Hydrolysis of the reduced and carboxymethylated malic enzyme by carboxypeptidase A yielded quantitative evidence for the following C-terminal sequence: -Leu-(Phe-Ala)-Ile-Leu-COOH. Fifty-five trypsin-digested peptides were separated by HPLC, in accordance with the arginine and lysine contents of each subunit. This more direct structural evidence strongly supports the conclusion that pigeon liver malic enzyme is composed of four chemically identical subunits.

Amino Acid Sequence↗

Mapping the active site topography of the NAD-malic enzyme via alanine-scanning site-directed mutagenesis.

The NAD-malic enzyme cDNA has been subcloned into the pQE expression vector, expressed with a six-His tag, and purified. The His-tagged enzyme is purified by a combination of Ni-NTA and orange A agarose column chromatography with a yield of 45% and an estimated purity of >90%. The tag and linker have no effect on the kinetic parameters of the enzyme compared to the wild-type enzyme. Alanine-scanning site-directed mutagenesis has been carried out on all of the conserved neutral acid residues of the NAD-malic enzyme from Ascaris suum. Data obtained confirm the predicted role of D178 and D295 in metal ion binding, the likely role of D294, D361, and E440 in the NAD binding site, and the role of E58 and D272 in malate binding. Decreases in V/E(t) by 10(4)-fold and in V/K(malate)E(t) by 10(7)-fold, when D295 is changed to alanine, suggest that it is a likely candidate for the general base that accepts a proton from the malate hydroxyl in the oxidation step.

Alanine↗

Weight cycling-induced alteration in fatty acid metabolism.

Epidemiological studies have suggested that repeated weight cycling over time may increase the risk of coronary heart disease. The mechanism involved remains poorly understood, but the change in lipid metabolism during weight cycling has been offered as a possible explanation. The present study investigated the effect of weight cycling on the size and fatty acid composition of rat fat pads as well as serum cholesterol, triglyceride, glucose, insulin, and glucagon in rats. Two consecutive weight cycles were induced by 40% energy restriction followed by ad libitum refeeding of either a moderate-fat (MF; 22% energy) or a high-fat (HF; 45% energy) diet. The lipogenic enzymes, including fatty acid synthase, acetyl-CoA carboxylase, malic enzyme, pyruvate kinase, and lipoprotein lipase in the weight-cycled (WC) rats fed only the HF diet, yielded an overshoot of activities at the end of two weight cycles. These changes were accompanied by an 80% increase in the size of the adipocyte and a 40-50% increase in the size of perirenal and epididymal fat tissues in HF-WC rats. Regardless of whether the rats were fed the HF or MF diet, all WC rats showed a gradual reduction in linoleic and alpha-linolenic acid and an increase in palmitic, palmitoleic, and stearic acid in total body lipid. It is concluded that weight cycling in rats may promote body fatness if an HF diet is consumed and can significantly alter whole body fatty acid balance irrespective of whether they consumed an MF or HF diet. Most importantly, the weight cycling led to an overshoot or fluctuation of serum cholesterol, triglyceride, glucose, insulin, and glucagon. If weight cycling is associated with an increased risk of cardiovascular disease, then, part of the mechanism may involve the changes in these risk factors.

Acetyl-CoA Carboxylase↗

Effects of corticosterone infusion on the lipogenic activity and ultrastructure of the liver of laying hens.

The effects of infusion of corticosterone (30 micrograms/h) for periods of 14 and 28 days on the ultrastructure and activity levels of lipogenic enzymes (fatty acid synthetase, glucose-6-phosphate dehydrogenase, malic enzyme) of the liver of laying hens, were studied. The liver and the abdominal fat pad in the corticosterone treated birds weighed significantly more than those of the control birds while the ovary and oviduct were substantially regressed in the former. Ultrastructure of the hepatocytes of the treated birds showed heavy deposition of lipid. The respective levels of total activities of the three enzymes studied were also higher in the liver of the treated birds, even though the specific enzyme activities were higher only in the case of fatty acid synthetase.

Animals↗

Selective inhibition of bacterial enzymes by free fatty acids.

Octanoic acid inhibits, in vitro, the bacterial enzymes glucose-6-phosphate dehydrogenase, phosphofructokinase, pyruvate kinase, fumarase, lactate dehydrogenase, and the malic enzyme of Arthrobacter crystallopoietes. The free fatty acid appears to act as an inhibitor of lipogenesis, although it does not affect the rate of gluconeogenesis. To demonstrate that this inhibition may be of physiological significance in vivo, those enzymes not involved in lipogenesis, such as fructose-1, 6-diphosphatase, phosphoglucomutase, phosphohexoisomerase, aconitase, nicotinamide adenine dinucleotide phosphate (NADP) isocitrate dehydrogenase, NADP glutamate dehydrogenase, malate dehydrogenase, and isocitrate lyase, were assayed and found not to be inhibited by the free fatty acid.

Arthrobacter↗

Localization of genes coding for biochemical traits on the second chromosome of Drosophila imeretensis Sokolov (D. littoralis Meigen).

We have investigated 13 alleles of four genes coding for acid phosphatase, alpha- and beta-esterases, and malic enzyme. The genes were localized and their positions regarding the centromere are as follows: Acph-1--centromere--Me--cu--dt--alpha-Est--[Inversion 2t]--beta-Est. The occurrence of crossing over in Drosophila imeretensis males, as well as the tetrameric structure of malic enzyme, was confirmed.

Acid Phosphatase↗

A pre-genetic study of the isoforms of malic enzyme associated with lipid accumulation in Mucor circinelloides.

The oleaginous fungus Mucor circinelloides possesses at least six isoforms of malic enzyme (EC 1.1.1.40), a key lipogenic enzyme in filamentous fungi. These isoforms were detected using a specific stain for activity after native PAGE of cell extracts. Only one isoform (isoform IV) was associated with lipid accumulation, appearing only after N-exhaustion from the medium (which is a pre-requisite for lipid accumulation) in glucose-growing cells. Isoforms I, II, V and VI were involved in anaerobic growth and only appeared under O(2)-limited conditions. Isoform III appeared to be constitutive and was formed under conditions of active (balanced) growth and is therefore thought to play a crucial role in basic metabolism. Growth on acetate increased the amount of cell lipid (from 25-27% in glucose-grown cells to 37-38% in acetate-grown cells) accumulated by M. circinelloides and this was associated with the appearance of isoform IV of malic enzyme prior to N-exhaustion in these cultures. Amino acid sequence analysis of isoforms III and IV suggests that these two malic enzymes may be encoded by a single gene and that isoform IV is formed from isoform III by post-translational modification initiated by either N-limitation (when glucose was the carbon source) or growth on acetate as the sole carbon source.

Acetates↗

Duodenal rapeseed oil infusion in early and midlactation cows. 5. Milk fatty acids and adipose tissue lipogenic activities.

Lipogenic activities of perirenal adipose tissue were investigated in early (wk 3) and midlactation (wk 19 to 26) cows that received a duodenal rapeseed oil infusion (1.0 to 1.1 kg/d). In midlactation, oil infusion resulted in a decreased rate of fatty acid synthesis from acetate and a decreased rate of the activities of fatty acid synthetase and glucose-6-phosphate dehydrogenase, whereas lipoprotein lipase activity tended to increase. The rate of glucose incorporation into glyceride-glycerol and the activities of glycerol-3-phosphate dehydrogenase and malic enzyme were not significantly affected. Fatty acid C14:0 content of perirenal adipose tissue was decreased, and fatty acid C18:2 and C18:3 contents were increased in oil-infused cows. In early lactation, rates of acetate incorporation into fatty acids and activities of fatty acid synthetase and lipoprotein lipase were very low. Activities of glucose-6-phosphate dehydrogenase and glycerol-3-phosphate dehydrogenase were lower in the early than in the midlactation trial. Oil infusion did not change the measured parameters. In both trials, percentages and yields of milk fatty acids C18:1, C18:2, and C18:3 were increased, whereas those of C14:0 and C16:0 were decreased by oil. Calculated transfer rates of absorbed fatty acid C18:2 from oil to milk fat were 16 to 26%. Results suggested that oil fatty acids affected adipose and mammary de novo lipogenesis in a direct way without affecting fatty acid esterification in adipose tissue or total fat secretion in mammary tissue.

Acetates↗

Conceptual Error in Determination of NAD-Malic Enzyme in Extracts Containing NAD-Malic Dehydrogenase.

The typical procedure for determining NAD(+)-malic enzyme (EC 1.1.1.39) is to calculate the enzyme rate to be DeltaA(340)/Delta time after the endogenous NAD(+)-malic dehydrogenase (EC 1.1.1.37) catalyzed reaction has reached equilibrium. This ignores the equilibrium shift of oxaloacetic acid and NADH during the course of the NAD(+)-malic enzyme reaction and causes an error that varies depending on the reagent [malate], [NAD(+)], pH and final [NADH]. For a DeltaA(340) of 0.02, the error is about 80% and for a DeltaA(340) of 0.30, 20%. We develop this argument, give supportive data and present a simple method to circumvent the error.

Journal Article↗

Effects of dietary phytosterols on liver lipids and lipid metabolism enzymes.

The effects of dietary phytosterols on lipid metabolism have been assessed through determination of liver lipids (sterols and fatty acids) and lipid metabolism enzymes (acetyl-CoA carboxylase, malic enzyme, glucose-6-phosphate dehydrogenase) in rats fed 12 or 24 mg cholesterol a day and 0-96 mg phytosterols. The results indicate that, provided the dietary phytosterol to cholesterol ratio is at least 1 and in the presence of a dietary cholesterol excess, phytosterols do exert a regulatory role through decreases of both acetyl-CoA carboxylase and malic enzyme activities. A ratio of 2 enhances this effect. At the same time, liver fatty acids and cholesterol contents significantly decrease.

Acetyl-CoA Carboxylase↗