Vitamin B6 metabolism in Morris hepatomas

The enzymes involved in the metabolism of vitamin B6 were measured in Morris hepatomas and livers of female Buffalo rats fed pyridoxine-sufficient and deficient diets. Pyridoxal phosphate levels in plasmas hepatomas, and livers were also determined. Nontumor-bearing animals were maintained as controls. Regardless of the B6 nutritional status, the concentration of pyridoxal phosphate was lower in the hepatomas than in the livers of the host animals. The apoenzyme levels of ornithine decarboxylase, a pyridoxal phosphate-dependent enzyme, were higher in the hepatomas from animals fed the B6-deficient diet. Liver pyridoxine kinase activity was higher in B6-sufficient animals. In contrast, tumor pyridoxine kinase activity was influenced by B6 intake and was significantly lower than that in host liver. Liver pyridoxine phosphate oxidase activity was not significantly affected by B6 intake or by the presence of tumor. In contrast, hepatomas had little or no pyridoxine phosphate oxidase activity. Pyridoxine phosphate phosphatase activity was elevated in tumors relative to livers. These data indicate that the metabolism of vitamin B6 is markedly different in the hepatomas than in host or control livers and suggest that the tumor is apparently incapable of the complete synthesis of co-enzymatically active pyridoxal phosphate from inactive precursor forms such as pyridoxine.     

Stearyl coenzyme A desaturase activity in mouse liver microsomes of varying lipid composition

Liver microsomes derived from mice fed fat-free diets contained tenfold more stearyl coenzyme A desaturase activity than microsomes derived from mice fed a safflower oil supplemented diet. Removal of the native lipid from each of the microsomal desaturase proteins was performed and the native lipid was replaced by a mixture of phosphatidylcholine and oleic acid. Desaturase activity could only be restored to the level present in the original microsomes in both instances and it was concluded that, although the lipid composition of the two kinds of microsomes was markedly different, this was not directly responsible for the difference in enzyme activity. Microsomal electron transport chain intermediates remained unchanged during the feeding time tested, however, the rate of oxidation of cytochrome b₅could be stimulated sevenfold by stearyl coenzyme A only in microsomes that contained a high desaturase activity. This demonstrates that a blockage in the electron transport chain exists in microsomes from mice fed safflower oil supplemented diets and that this correlates with a markedly reduced desaturase activity in these microsomes.     

Selective modulation of nucleotide levels in rat liver and hepatomas by high-orotate or arginine-deficient diets and by carbamoylating agents

Transplanted Morris hepatomas in Buffalo-strain rats were found to be resistant to the changes in ribonucleotide levels in rat liver caused by a high-orotate diet or an arginine-deficient diet. The increase in UTP levels and decrease in ATP levels seen in the livers of rats on a 1%-orotate diet were less marked in the livers of BUB- and DBA-strain mice on this diet. Although the changes were less than in rat liver, there was a 2-3-fold increase in UTP concentration in the livers of mice on the high-orotate diet. However, there was a similar response in nucleotide levels in the two species when the animals were maintained on an arginine-deficient diet, and there was a greater than 10-fold increase in the UTP level in the livers of both rats and mice. These diets had much less effect on the levels of deoxyribonucleotides than of ribonucleotides. In contrast to the insensitivity of hepatomas to dietary modulation of nucleotide levels, treatment of hepatoma-bearing rats with carbamoylating agents (sodium cyanate and 2-chloroethyl isocyanate) caused decreases in the levels of nucleotides in the tumors which were generally greater than in host livers. For example, 2-chloroethyl isocyanate depressed ATP levels in the Morris hepatomas 5123C and 20 under conditions in which there was no significant effect on host liver ATP. The data revealed selective modulation of nucleotide levels in normal and neoplastic liver which may be achieved by either dietary modification or drug treatment.     

Effect of essential fatty acid deficiency on release of triglycerides by the perfused rat liver

Studies are reported on release of triglycerides during perfusion of livers of male Sprague-Dawley rats fed a fat-free diet or diets containing hydrogenated coconut oil or corn oil. Perfusions were carried out with Krebs-Ringer bicarbonate buffer containing albumin with and without infusion of oleate or linoleate. Infusion with sodium oleate or linoleate caused an accumulation of triglycerides in the livers of the corn oil-fed animals and stimulated the release of triglycerides into the perfusing medium. In similar experiments with essential fatty acid-deficient animals, which were fed fat-free diets or diets containing hydrogenated coconut oil, there was no increase in secretion of triglycerides into the perfusate, and the amount of triglyceride which accumulated in the liver was greater than in the livers of the control (corn oil-fed) animals. Tracer experiments with oleate-1-(14)C or linoleate-1-(14)C also showed that with livers of essential fatty acid-deficient animals, secretion of triglyceride into the perfusate was not stimulated by infusion of fatty acids into the perfusing medium. It is concluded that impairment of the secretion of triglycerides is a factor in the accumulation of fat in the livers of essential fatty acid-deficient animals.     

The regulation of hepatic stearoyl-coenzyme A desaturase in obese-hyperglycaemic (ob/ob) mice by food intake and the fatty acid composition of the diet.

1. The effects of food intake and the fatty acid composition of the diet on the hepatic stearoyl-CoA desaturase activity of obese-hyperglycaemic (ob/ob) mice were investigated. 2. Obese mice fed on a commercial mouse diet, ad libitum, had 6.5-fold more activity per liver cell than had lean mice. 3. On a diet containing 14% corn oil the activity was 65% less in obese mice and 62% less in lean mice compared with animals fed on the commercial diet. 4. Feeding with 14% saturated fat in the diet doubled the activity in lean mice compared with those on the commercial diet, but had no effect on the activity in obese mice. 5. Obese mice fed on the corn-oil diet contained a higher proportion of linoleic acid in the liver lipids than did lean mice fed on the commercial diet, but the acyl-CoA desaturase activity was 125% higher than in the lean mice. 6. Limiting the food intake of obese mice by pair-feeding with lean mice decreased their acyl-CoA desaturase activity when the animals were fed on the saturated-fat diet, but the activity remained 75% higher than in lean mice, whereas in obese mice pair-fed on the corn-oil diet the activity was the same as in lean mice. 7. During starvation the acyl-CoA desaturase activity in livers from obese mice decreased more slowly and proportionately less than in livers from lean mice. 8. It is concluded that increased substrate supply as a result of hyperphagia and not low concentration of linoleic acid is the main factor causing high acyl-CoA desaturase activity in obese mice.     

Early effect of myo-inositol deficiency on phosphatidylinositol metabolism in rat liver

Young rats (100 g) were fed either a myo-inositol-deficient or supplemented (control) diet for up to 14 days following a 12 h fast. At various times during this period animals were killed, livers were removed, and a microsomal fraction was prepared and assayed for CDPdiacylglycerol inositol transferase activity and for phosphatidylinositol-inositol exchange activity. Within 2 days after beginning the regimen, rats consuming the deficient diet had a 40% lower activity of the transferase than rats consuming the control diet. This difference was maintained throughout the feeding period and developed simultaneously with the accumulation of triacylglycerol in the deficient livers. In contrast, the specific activity of the exchange enzyme was unchanged by feeding the deficient diet.     

STEARYL COENZYME A DESATURASE ACTIVITIES IN RAT BRAIN MICROSOMES

—A quantitative assay for the rat brain microsomal stearyl CoA desaturase is described. The method was used to determine the developmental pattern and regional distribution of the desaturase in rat brain. Even though the specific activity of the enzyme decreased to one-third from 8 to 60 days of age, the total activity per brain remained the same. The regional distribution of the activity in adult brain was of the order midbrain > medulla oblongata > cerebral hemispheres > cerebellum, with midbrain activity being about twice as that of the cerebellum.     

COLD RESISTANCE OF THE BRAIN DURING HIBERNATION: THE ROLE OF STEARYL CoA DESATURASE IN BRAIN AND LIVER AS THE SOURCE FOR MONOENES

Brain and liver stearyl CoA desaturase activity and its associated microsomal electron transport chain was investigated in both the warm-adapted and hibernating hamster. It was shown that the activity of this enzyme in brain was essentially the same in both the warm-adapted and hibernating hamster. In liver an 8-fold increase in desaturase activity was observed for the hibernator without corresponding increases in the activity of the microsomal electron transport chain. It is concluded that the increase of monoenes in brain that contributes to the lipid adaptation probably results from peripheral production of these fatty acids.     

Fate of Mycobacterium tuberculosis inside rate peritoneal macrophages in vitro

Streptozotocin-induced diabetic rats were maintained on 0.5% curcumin containing diet for 8 weeks. Blood cholesterol was lowered significantly by dietary curcumin in these diabetic animals. Cholesterol decrease was exclusively from LDL-VLDL fraction. Significant decrease in blood triglyceride and phospholipids was also brought about by dietary curcumin in diabetic rats. In a parallel study, wherein diabetic animals were maintained on a high cholesterol diet, the extents of hypercholesterolemia and phospholipidemia were still higher compared to those maintained on control diet. Curcumin exhibited lowering of cholesterol and phospholipid in these animals also. Liver cholesterol, triglyceride and phospholipid contents were elevated under diabetic conditions. Dietary curcumin showed a distinct tendency to counter these changes in lipid fractions of liver. This effect of curcumin was also seen in diabetic animals maintained on high cholesterol diet. Dietary curcumin also showed significant countering of renal cholesterol and triglycerides elevated in diabetic rats.In order to understand the mechanism of hypocholesterolemic action of dietary curcumin, activities of hepatic cholesterol-7a-hydroxylase and HMG CoA reductase were measured. Hepatic cholesterol-7a-hydroxylase activity was markedly higher in curcumin fed diabetic animals suggesting a higher rate of cholesterol catabolism. (Mol Cell Biochem 166: 169-175, 1997)     

Effects of fat content in the diet on hepatic peroxisomes of the rat

Effects of fat content in the diet on rat liver peroxisomes was examined. In the livers of rats fed for one week on the high-fat diet containing 30% fat, the cyanide-insensitive palmitoyl-CoA oxidation was accelerated to eight times that of control and the enzymic activities of catalase, carnitine acetyltransferase and carnitine palmitoyltransferase were elevated by the factors of 1.3, 5 and 2, respectively. In contrast, the activities of D-amino acid oxidase in addition to the three enzymes mentioned above were all lowered by 20% when the animals were maintained on a fat-free diet for the same period of time. It appears that the high-fat diet-induced increase in the activity of carnitine palmitoyltransferase is a result of the raised activity of this enzyme in mitochondria only while the apparent high activity reflects stimulation of carnitine acetyltransferase in all the subcellular fractions. Another notable effect of the high-fat diet was a remarkable increase in the quantity of a peroxisome-associated polypeptide which was separable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It is noteworthy that this effect of the high-fat diet resemble that of clofibrate. If the diet was deprived of fat, however, this polypeptide species, with an estimated molecular weight of 80 000, decreased to a level slightly lower than normal. On the basis of the electron micrographic criteria, the high-fat diet provoked a marked proliferation of hepatic peroxisomes.     

Characterization of fatty acid desaturase activity in rat lung microsomes.

Preparations of rat lung microsomes containing 0.030-0.050 nmole of cytochromes P-450 and b5 per mg microsomal protein have been observed to contain significant levels of fatty acid desaturase activity. Both stearoyl CoA and palmitoyl CoA are desaturated to their monounsaturated analogues, oleic acid and palmitoleic acid, respectively. Activity (per mg microsomal protein) of the lung preparations varied according to the diet of the animals prior to killing in the order: fat free diet greater than normal rat chow greater than starvation. All preparations exhibited approximately 50% inhibition when incubated in the presence of 0.10 mM CN-. Maximal activity was obtained with the 0.50 mM NADH less activity with equal amounts of NADPH, and there was no synergistic interaction of NADH and NADPH together. The rate of desaturation was linear with protein concentrations between 0.15-1.5 mg microsomal protein/incubation at incubation times up to 8 min. A pH optimum range of 7.0-7.4 was observed. For all variables of fatty acid desaturase activity which were examined, the rate of desaturation of stearoyl CoA was approximately twice that for palmitoyl CoA. These results indicate that the same fatty acid desaturation system which is functional in the liver is also present in significant amounts in mammalian lungs.     

Assay for the terminal enzyme of the stearoyl coenzyme A desaturase system using chick embryo liver microsomes.

The NADH-dependent stearoyl CoA desaturase of hepatic microsomes (EC 1.14.99.5) is an enzyme system consisting of cytochrome b5 reductase (EC 1.6.2.2), cytochrome b5, and the terminal desaturase. We have developed a simple method for routine assay of the terminal enzyme based on complementation of the enzyme with chick embryo liver microsomes lacking desaturase activity. Desaturation of [1-14C]stearoyl CoA by the enzyme-microsome mixture is then assayed by thin-layer chromatography of the reaction products and determination of the amount of oleate formed. Microsomes from the livers of starved-refed rats were used as the source of the stearoyl CoA desaturase. The enzyme alone, solubilized and free from cytocrome b5 reductase and cytochrome b5, was unable to catalyze the desaturation of stearoyl CoA. However, after preincubation with chick embryo liver microsomes in the presence of 1% Triton X-100, the enzyme was active. The enzyme activity was linear with time and desaturase protein under the conditions described and depended on the concentrations of Triton X-100 present in the preincubation and the assay. The optimum concentrations of Triton X-100 were 1% for the preincubation and 0.1-0.15% in the assay. The desaturation activity was dependent on NADH and O2, and was inhibited 95% by 1 mM KCN. The use of chick embryo liver microsomes in this method eliminates the need to use purified cytochrome b5 reductase, cytochrome b5, and liposomes for routine assays and greatly reduces the complexities of timing and order of addition encountered in the existing assays.     

The effects of experimental inflammation on adaptive synthesis of rat liver fatty acid synthetase

The effects of experimental inflammation, induced by subcutaneous injection of oil of turpentine, on adaptive synthesis of rat liver fatty acid synthetase were investigated. Liver levels of α₁-acid glycoprotein, an “acute-phase” protein known to be synthesized at an accelerated rate as a result of inflammation, were also measured. The increase in fatty acid synthetase activity in livers of rats which were starved and then fed a fat-free diet was suppressed to an extent dependent on the periods between fat-free feeding and inflammation and inflammation and sacrifice. Inflammation induced 2 h after refeeding gave complete suppression, whereas inflammation after 10 h of fat-free feeding had no suppressive effect. When induced 2.5 or 7.5 h after refeeding, inflammation led to partial suppression of the increase in fatty acid synthetase activity. The increase in liver α₁,-acid glycoprotein levels characteristic of inflammation was reduced in animals inflamed 7.5 or 10 h after fat-free feeding, but was unaffected when inflammation was induced 2.5 h after refeeding. The ratio of free to membrane-bound polyribosomes in liver increased from 0.77 in rats which were neither starved nor fed a fat-free diet to 3.31 in rats which were starved and then fed a fat-free diet for 15 h. When inflammation was induced 2.5 h after refeeding, the ratio increased to only 1.74 after 15 h of refeeding. Inflammation resulted in a marked reduction in the level of glycogen in the liver, regardless of the time of induction of inflammation and the dietary status of the animal.     

The effect of dietary fat on lipogenesis in mammary gland and liver from lactating and virgin mice

1. Virgin and lactating C(3)H mice maintained on laboratory chow were transferred to a high-fat (15% corn oil) or a fat-free diet 3 days before being killed. 2. The linoleate content of liver, mammary gland and milk was decreased in lactating mice given the fat-free diet but was increased in those fed on the high-fat diet. Changes in linoleate content and mammary gland followed a similar but much less marked trend in virgin animals. 3. Hepatic fatty acid synthesis in lactating and virgin mice fed on the fat-free diet was higher than in corresponding animals fed on either the chow or the high-fat diet. The lipogenic capacity of livers from mice fed on either the chow or the high-fat diet was greater in lactating than in virgin animals. These changes in hepatic lipogenic capacity were accompanied by alterations in the specific activities of certain enzymes involved in fat synthesis. 4. Mammary gland from virgin and lactating animals showed no such adaptation to dietary fat. Results indicate that fatty acid synthesis in neither mammary-gland parenchymal cells nor mammary-gland adipose cells can be influenced by dietary fat in the same way as in the hepatocyte.     

Hepatic acetoacetyl-CoA deacylase activity in rats fed ethyl chlorophenoxyisobutyrate (CPIB)

Intact or sonicated mitochondria from the livers of rats fed a diet containing 0.2% ethyl chlorophenoxyisobutyrate (CPIB) for 3 wk showed acetoacetyl-CoA deacylase activity enhanced 26 and 39%, respectively, over that shown by comparable fractions from rats fed the same diet without CPIB. The corresponding supernatant fractions did not differ in activity. The enhanced activity of mitochondrial acetoacetyl-CoA deacylase in the livers of the CPIB-treated rats could effectively decrease the amount of acetoacetyl CoA available within the cell for synthetic processes.     

Regulation of hepatic betaine-homocysteine methyltransferase by dietary methionine

The hepatic activity of betaine-homocysteine methyltransferase is a complex function of the content of methionine in the diet. Enzyme levels are lower in the livers of rats fed a 0.3% methionine diet than in livers of animals maintained on either methionine-free or excessivemethionine (1.0%) rations. The finding that activities are increased at both extremes of the spectrum of dietary methionine intake suggests the possibility that the betaine-homocysteine methyltransferase reaction may function both to maintain tissue concentrations of methionine when intake of this amino acid is limited and to remove homocysteine when methionine intake is excessive.     

Effect of exercise on response of liver lipogenic enzymes to a high fructose diet.

Meal-fed Long-Evans rats fed a high fructose diet and exercised for 2-hr daily on a treadmill for three days had lower levels of several hepatic lipogenic enzymes (acetyl CoA carboxylase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, malic enzyme and citrate cleavage enzyme) than did sedentary rats pair-fed the diet. Accumulation of triglycerides in plasma following ingestion of a fat-free, high fructose meal and injection of Triton WR-1339, an inhibitor of plasma triglyceride clearance, was not significantly different in the two groups of animals. All of the hepatic lipogenic enzymes measured, with the exception of citrate cleavage enzyme, attained similar levels in the runners as in the controls after 5 days on the high fructose diet. Thus the exercise appeared to affect the time course of the increase in the levels of activity of most of the lipogenic enzymes but not the final steady state levels attained.     

Abnormal hepatic lipid accumulation following treatment of diabetic rats with insulin and a high-carbohydrate, fat-free diet

This study correlates the morphological and biochemical events during the accumulation of hepatic lipids in diabetic rats in response to insulin treatment and a high-carbohydrate, fat-free diet. Alloxan-diabetic rats were fed a high-carbohydrate, fat-free diet and treated with insulin for 12, 36, or 60 hr or 4.5 or 6.5 days. Samples of livers were obtained for determination of malic enzyme activity and the histochemical demonstration of lipids. An increased accumulation of hepatic lipids, although delayed, was observed following insulin treatment of diabetic rats fed the special diet. Small lipid droplets were visible after 36 hr of treatment, which later increased and coalesced into larger droplets present in all hepatocytes. Maximal accumulation was observed at 4.5 days of treatment. These changes were paralleled by an increase in the activity of hepatic malic enzyme. By 6.5 days of treatment, the lipid content of the hepatocytes had decreased and a periportal pattern was discernible. In contrast, malic enzyme activity continued to increase through 6.5 days of treatment. By comparison, no hepatic lipid accumulation occurred in regular chow-fed diabetic rats receiving insulin treatment or in diabetic rats placed on the special diet alone. These results suggest that the combination of insulin treatment and a high-carbohydrate, fat-free diet caused an imbalance in the production and mobilization of hepatic lipids.     

The in vitro stability of rat liver glucose-6-phosphate dehydrogenase as a function of diet

Rat liver glucose-6-phosphate dehydrogenase (G6PD) activity was studied in starved-refed rats given diets containing three levels of fat (35%, high-fat; 5%, low-fat; 0%, fat-free). Elution characteristics from DEAE-cellulose, Km for glucose-6-phosphate, pH optimum, and molecular weight appeared to be similar. During storage or heat denaturation, stability apparently was lowest of G6PD of livers from rats refed the high-fat diet. Heat stability was enhanced by the addition of NADP, but some differences due to diet persisted. Titration of a constant amount of enzyme with heating gave inconsistent results: in two of four experiments rats refed the high-fat diet had an equivalence point twice that of rats refed the fat-free diet. This difference disappeared if the antibody titration was carried out in the cold. The diet-induced instability of the G6PD, as measured in vitro, was reversible by changing the diet of the rats.