Early effects of feeding excess vitamin A: mechanism of fatty liver production in rats

Oral administration of vitamin A (30,000 IU daily for 2 days) to young rats caused a marked increase in hepatic glycogen, cholesterol, and glycerides, while hepatic phospholipid content remained almost unaltered. In an examination of the pathogenesis of the lipid accumulation, it was found that more glucose-(14)C was incorporated into liver lipids in vitamin A-fed rats, whereas incorporation of glucose-(14)C and dl-glycine-(14)C into liver protein remained unaltered. The increase in glucose-(14)C incorporation was confined to the glyceride-glycerol portion of the lipids; incorporation into liver fatty acids was inhibited. Plasma free fatty acid concentrations were elevated. It is postulated that in the vitamin A-fed rats, increased accumulation of lipids in the liver is caused by a stimulation of fatty acid mobilization from adipose tissue and enhanced formation of glycerophosphate through glycolysis, with consequent increase in the glyceride synthesis in the liver. The weight of the adrenals was increased, whereas cholesterol concentration in the gland was decreased, after administration of vitamin A to rats. This indicates adrenocortical stimulation. Interestingly enough, vitamin A feeding did not affect either the level of liver lipids or of plasma FFA in adrenalectomized rats.     

The effects of vitamin A deficiency on hepatic folate metabolism in rats

The effects of severe vitamin A deficiency (liver retinol less than 2 micrograms/g) on hepatic folate metabolism in rats were studied. The oxidation of a [ring-2-14C] histidine load or a [14C]formate load to 14CO2 was significantly depressed in vitamin A-deficient rats and those given histidine also excreted more urinary formiminoglutamic acid (FiGlu) than pair-fed controls. The increase in FiGlu excretion was not due to augmented production from histidine, implicating an impairment of FiGlu catabolism. FiGlu formiminotransferase activity was unaltered in vitamin A-deficient rats, but hepatic tetrahydrofolic acid (THF) concentration was decreased by 58% in vitamin A-deficient rats given a histidine load while 5-methyl-THF concentration was increased by 39%. Formyl-THF and total folate levels were similar to controls. A redistribution of folate coenzymes was not found in vitamin A-deficient rats not force fed histidine. A 43% decrease in 10-formyl-THF dehydrogenase activity, which generates both THF and the 14CO2 from the labeled substrates, and an 81% increase in 5,10-methylene-THF reductase activity, which generates 5-methyl-THF, were found in vitamin A-deficient rats. It appears that the production of severe vitamin A deficiency results in selective changes in the activities of hepatic folate-dependent enzymes, so that when a load of a one-carbon donor is given, THF concentration decreases and metabolism of the load is impaired.     

Biosynthetic studies on mannolipids and mannoproteins of normal and vitamin A-depleted hamster livers.

The incorporation of [1-14C]mannose into hamster liver glycolipids and glycoproteins was studied in normal and vitamin A-depleted hamsters. Severly (25% weight loss) and mildly (no weight loss) deficient animals were compared to vitamin A-fed controls. The incorporation of [14C]mannose into glycolipids and glycoproteins decreased in mild and severe vitamin A deficiency by 63-90% compared to vitamin A-fed animals. These results were essentially the same whether expressed per g of wet liver, per DNA or per protein. The size of the pools of mannose, glucose and galactose and their specific radioactivity in liver were determined by gas-liquid chromatography of the boronates of the hexitols (Eisenberg, Jr, F. (1972) Methods Enzymol. XXVIIIB, 168-178) in normal and vitamin A-deficient conditions. It was found that the amount of free hexoses per g of liver was very similar in normal and vitamin A-deficient conditions. The specific radioactivities for mannose and glucose were greater in vitamin A deficiency, thus excluding the possibility that the observed severe decrease in glycopeptide and glycolipid synthesis is a reflection of a similar decrease in the specific radioactivity of the precursor pools. Quantitation of mannose in glycoprotein showed a 79% decrease in vitamin A deficiency. Specific radioactivity of mannose in glycoproteins, 20 min after injection of the label, was 187 dpm/mug of mannose in the normal and 48 kpm/mug of mannose in the vitamin A-deficient livers. It is concluded that vitamin A is necessary for the biosynthesis of liver mannose-containing glycoproteins and glycolipids.     

Quantitative aspects of free fatty acid metabolism in the fasted rat

Palmitate-1-(14)C was injected intravenously into unanesthetized, fasted rats. Disappearance of tracer from plasma free fatty acids was studied. A large component of free fatty acid (FFA) recycling was directly demonstrated by reinjection experiments. The latter studies also indicated the existence of an unidentified, rapidly turning over polar lipid in plasma which was synthesized from palmitate-(14)C. The appearance of (14)C in hepatic and extrahepatic triglycerides, in other esters, and in respired CO(2) was also followed. The data were analyzed using a multicompartmental model and a digital computer. Only a small fraction of the triglycerides formed in liver was derived directly from plasma free fatty acids. The major portion of net triglyceride formation appeared to be by way of an intermediate nontriglyceride ester pool which turned over relatively slowly compared to plasma free fatty acids. Initial approximations are as follows ( micromoles of fatty acid per min per 100 g body weight): net free fatty acid mobilization (irreversible disposal) = 2.4; hepatic triglyceride formation directly from plasma free fatty acid = 0.1; total hepatic lipid formation from plasma free fatty acids = 0.5; oxidation of free fatty acids to CO(2) = 0.8; percentage of respired CO(2) from direct oxidation of fatty acids = 12%; extrahepatic triglyceride formation directly from fatty acids = 0.4; total extrahepatic lipid formed directly from fatty acids = 1.2.     

Accumulation and release of triglycerides by rat liver following partial hepatectomy

Regenerating liver accumulates lipid for about 20 hr following partial hepatectomy. During this time incorporation of intravenously administered palmitate-9, 10-(3)H into beta-lipoprotein increased. 13 hr after partial hepatectomy, there was no change in the level of serum beta-lipoproteins, but the specific activities of the triglycerides in the liver and beta-lipoproteins were significantly diminished. Extension of these studies to the isolated perfused liver system demonstrated that 13 hr after partial hepatectomy the regenerating liver is capable of secreting greater quantities of the lipid, but not the protein, moiety of the beta-lipoproteins in comparison with liver taken immediately from a partially hepatectomized animal, although there was no difference between the weights of the livers. However following addition of palmitate-(3)H and (14)C-labeled amino acids to the perfusate, the specific activity of the hepatic and beta-lipoprotein triglycerides of the liver excised 13 hr after partial hepatectomy was diminished, but that of the protein was not affected. Prelabeling of the accumulated triglyceride with palmitate-1-(14)C in vivo revealed that the proportions of the accumulated triglyceride secreted as beta-lipoproteins by perfused livers excised immediately and 13 hr after partial hepatectomy were identical. It is concluded that regenerating liver rapidly acquires the ability to mobilize triglycerides at a rate equal to that of the much larger normal liver, so that it can handle all free fatty acids presented to it.     

Choline-deficiency fatty liver: impaired release of hepatic triglycerides

After intravenous injection of palmitate-1-(14)C to rats fed a choline-deficient (CD) or choline-supplemented (CS) diet for 15-18 hr, liver triglycerides became labeled very rapidly. In CS, but not in CD rats, there was a considerable loss, with time, of radioactivity from liver triglycerides. At the same time, significantly less radioactivity appeared in plasma triglycerides of CD rats than of CS animals. No difference was seen in the triglyceride content of microsomes isolated from the liver of rats fed the two diets. The lower radioactivity in plasma triglycerides of CD rats was essentially due to a lower level and specific activity of very low density lipoprotein triglycerides. After intravenous injection of Triton and labeled palmitate, considerably less radioactivity accumulated in plasma triglycerides and phospholipids of CD rats than of CS animals. Post-Triton hyperphospholipidemia was also less pronounced in CD rats. It was concluded that the fatty liver observed in CD rats results from an impaired release of hepatic triglycerides into plasma.     

The effect of vitamin A deficiency on hepatic, renal and pulmonary glutathione S-transferase activities in the rat.

Feeding male weanling rats on a vitamin A-deficient diet for 6 weeks resulted in significant increases (44-57%) in glutathione S-aryl-, S-aralkyl- S-alkyl- and S-epoxidetransferase activities in the liver cytosol. Only the S-aralkyl- (27%) and S-alkyltransferase (14%) activities were significantly increased in the kidney as a result of deficiency. There was no effect on any of the pulmonary glutathione S-transferase activities. The increases in hepatic transferase activities were due primarily to increases (25-96%) in the apparent Vmax. There were no changes in the apparant Km of any of the four drug substrates employed. With 3,4-dichloronitrobenzene as the second substrate, the apparent Km for glutathione was increased by over 2-fold in vitamin A-deficient livers as compared with controls. The relationship between these results and enhanced susceptibility to chemical carcinogens in vitamin A deficiency is briefly discussed, and comparison is made between the effects of this nutritional state and pretreatment with drug inducers on the glutathione S-transferases.     

Liver microsomal membrane fluidity and lipid characteristics in vitamin A-deficient rats.

Rat liver microsomes (microsomal fraction) were isolated from vitamin A-deficient and -sufficient rats and analysed for membrane lipid characteristics. Membrane fluidity was found to be significantly decreased in microsomes from the vitamin A-deficient rats, but not in liposomes prepared from lipid extracts. Microsomes from vitamin A-deficient animals showed a significant decrease in C18:2, omega 6 and an increase in C22:5, omega 6 fatty acids.     

Metabolism of hepatic and plasma triglycerides in rabbits given ethanol or ethionine

The formation and transport of hepatic triglyceride fatty acids (TGFA) were studied after intravenous administration of palmitate-1-(14)C or palmitate-9,10-(3)H in rabbits pretreated with ethanol or ethionine. Administration of ethanol produced significant hypertriglyceridemia without consistent accumulation of hepatic fat. The isotopic studies suggest that plasma free fatty acids were the major precursors of TGFA in d < 1.006 lipoproteins and that fatty acids synthesized in the liver were not the source of the hypertriglyceridemia in the ethanol-treated animals. Administration of ethionine resulted in an increased concentration of TGFA in the liver, a decreased level of TGFA in d < 1.006 lipoproteins and a very low specific activity in this plasma fraction. These findings suggest that the development of fatty liver after administration of ethionine is in part accompanied by impaired release of TGFA from the liver.     

Effects of long-term feeding of high-protein or high-fat diets on the response to exercise in the rat

The aim of this work was to find by which mechanisms an increased availability of plasma free fatty acids (FFA) reduced carbohydrate utilization during exercise. Rats were fed high-protein medium-chain triglycerides (MCT), high-protein long-chain triglycerides (LCT), carbohydrate (CHO) or high-protein low-fat (HP) diets for 5 weeks, and liver and muscle glycogen, gluconeogenesis and FFA oxidation were studied in rested and trained runner rats. In the rested state the hepatic glycogen store was decreased by fat and protein feeding, whereas soleus muscle glycogen concentration was only affected by high-protein diets. The percentage decrease in liver and muscle glycogen stores, after running, was similar in fat-fed, high-protein and CHO-fed rats. The fact that plasma glucose did not drastically change during exercise could be explained by a stimulation of hepatic gluconeogenesis: the activity of phosphoenolpyruvate carboxykinase (PEPCK) and liver phosphoenolpyruvate (PEP) concentration increased as well as cyclic adenosine monophosphate (AMPc) while liver fructose 2,6-bisphosphate decreased and plasma FFA rose. In contrast, the stimulation of gluconeogenesis in rested HP-, MCT- and LCT-fed rats appears to be independent of cyclic AMP.     

Changes in rat liver mitochondrial lipids in vitamin A deficiency

The alterations in the lipid profiles of rat liver mitochondria due to vitamin A deficiency were studied. The amount of total lipids and phospholipids were decreased with a concomitant increase in triglycerides and cholesterol levels in mitochondria, isolated from vitamin A-deficient animals. Of particular significance was the observation that the content of lysolecithin, a potent cytolytic agent, was increased. An analysis of individual fatty acids showed that the percentage of polyunsaturated fatty acids was decreased significantly in vitamin A deficiency. Further, mitochondria from vitamin A-deficient animals, when incubated in 0.1 M Tris-HCl buffer (pH 7.4)in vitro, produced increased amounts of malondialdehyde and lipofuchsin pigments indicating increased susceptibility of the mitochondrial membrane to peroxidative damage. These results suggest a possible role of vitamin A in the prevention of the decomposition of structural lipids.     

Disruption of the lecithin:retinol acyltransferase gene makes mice more susceptible to vitamin A deficiency

Lecithin:retinol acyltransferase (LRAT) catalyzes the esterification of retinol (vitamin A) in the liver and in some extrahepatic tissues, including the lung. We produced an LRAT gene knock-out mouse strain and assessed whether LRAT-/- mice were more susceptible to vitamin A deficiency than wild type (WT) mice. After maintenance on a vitamin A-deficient diet for 6 weeks, the serum retinol level was 1.34 +/- 0.32 microM in WT mice versus 0.13 +/- 0.06 microM in LRAT-/- mice (p < 0.05). In liver, lung, eye, kidney, brain, tongue, adipose tissue, skeletal muscle, and pancreas, the retinol levels ranged from 0.05 pmol/mg (muscle and tongue) to 17.35 +/- 2.66 pmol/mg (liver) in WT mice. In contrast, retinol was not detectable (<0.007 pmol/mg) in most tissues from LRAT-/- mice after maintenance on a vitamin A-deficient diet for 6 weeks. Cyp26A1 mRNA was not detected in hepatic tissue samples from LRAT-/- mice but was detected in WT mice fed the vitamin A-deficient diet. These data indicate that LRAT-/- mice are much more susceptible to vitamin A deficiency and should be an excellent animal model of vitamin A deficiency. In addition, the retinol levels in serum rapidly increased in the LRAT-/- mice upon re-addition of vitamin A to the diet, indicating that serum retinol levels in LRAT-/- mice can be conveniently modulated by the quantitative manipulation of dietary retinol.     

Regulation of lipid flux between liver and adipose tissue during transient hepatic steatosis in carnitine-depleted rats

Rats with carnitine deficiency due to trimethylhydrazinium propionate (mildronate) administered at 80 mg/100 g body weight per day for 10 days developed liver steatosis only upon fasting. This study aimed to determine whether the transient steatosis resulted from triglyceride accumulation due to the amount of fatty acids preserved through impaired fatty acid oxidation and/or from up-regulation of lipid exchange between liver and adipose tissue. In liver, mildronate decreased the carnitine content by approximately 13-fold and, in fasted rats, lowered the palmitate oxidation rate by 50% in the perfused organ, increased 9-fold the triglyceride content, and doubled the hepatic very low density lipoprotein secretion rate. Concomitantly, triglyceridemia was 13-fold greater than in controls. Hepatic carnitine palmitoyltransferase I activity and palmitate oxidation capacities measured in vitro were increased after treatment. Gene expression of hepatic proteins involved in fatty acid oxidation, triglyceride formation, and lipid uptake were all increased and were associated with increased hepatic free fatty acid content in treated rats. In periepididymal adipose tissue, mildronate markedly increased lipoprotein lipase and hormone-sensitive lipase activities in fed and fasted rats, respectively. On refeeding, carnitine-depleted rats exhibited a rapid decrease in blood triglycerides and free fatty acids, then after approximately 2 h, a marked drop of liver triglycerides and a progressive decrease in liver free fatty acids. Data show that up-regulation of liver activities, peripheral lipolysis, and lipoprotein lipase activity were likely essential factors for excess fat deposit and release alternately occurring in liver and adipose tissue of carnitine-depleted rats during the fed/fasted transition.     

Vitamin E deficiency and vitamin C supplements: exercise and mitochondrial oxidation

The effects of dietary antioxidant vitamins E and C on exercise endurance capacity and mitochondrial oxidation were investigated in rats. The endurance capacity of both vitamin E-deficient and vitamin C-supplemented, E-deficient rats was significantly (P less than 0.05) lower (38.1 and 33.6%, respectively) than control animals. Compared with the normal and vitamin E-deficient rats, there was a significant (P less than 0.05) increase in the concentration of vitamin C in blood and liver of the vitamin E-deficient, C-supplemented animals. Hence dietary vitamin C supplementation does not prevent the inhibition of exercise endurance capacity or increased hemolysis seen in vitamin E deficiency. The mitochondrial activities for the oxidation of palmitoyl carnitine and alpha-ketoglutarate were significantly (P less than 0.05) decreased by a single bout of exercise in brown adipose tissue but not in muscle, heart, or liver from vitamin C-supplemented, E-deficient groups of rats when compared with the activities in the tissue from the same group of rats killed at rest. Similar results were also seen in brown adipose tissue from vitamin E-deficient rats. The results suggest a tissue-specific role for vitamins E and C in substrate oxidation and show that the poor endurance capacity of vitamin E-deficient rats cannot be attributed to any changes in the mitochondrial activity in skeletal or cardiac muscles. It is also concluded that vitamin C supplementation, at least at the dose employed in the present study, cannot counteract the detrimental effects associated with vitamin E deficiency.     

Microsomal fatty acid desaturase activities in vitamin A-deficient rat liver

The activity of microsomal fatty acid delta 9-desaturase was significantly higher in liver microsomes of vitamin A-deficient rats as compared with their controls. Feeding of vitamin A-supplemented control diet to the deficient rats restored the delta 9-desaturase activity to the control values. The activity of delta 6-desaturase was not affected by vitamin A deficiency.     

Age-associated changes in fat metabolism in the rat and its relation to sympathetic activity

In this study, to determine if age associated changes in fat metabolism in skeletal muscle and liver were related with sympathetic activity, we measured sympathetic activity and palmitate oxidation rate, carnitine palmitoyltransferase-1 (CPT-1) activity, and triglyceride concentration in skeletal muscle and liver of rats at 8, 30 and 60 weeks of age. Body weight, intra-abdominal percent of fat mass, and plasma level of insulin, leptin, and triglyceride were all significantly increased with age. Tissue triglyceride concentration was increased with age in liver and skeletal muscle. The palmitate oxidation rate in liver and skeletal muscle was reduced with age in rats and inversely correlated with tissue triglyceride concentration. CPT-1 activity was not altered with age. Plasma catecholamine concentration and sympathetic activity, as measured by spectral analysis of heart rate variability, were increased with age. Plasma norepinephrine or epinephrine and tissue triglyceride had a positive correlation in liver and skeletal muscle. Plasma norepinephrine or epinephrine to tissue triglyceride ratio was similar according to age. In summary, in spite of increased sympathetic activity with age, the tissue triglyceride concentration was increased. Increased sympathetic activity may be the compensatory response and the reduced capacity of fatty acid oxidation is a main cause of obesity.     

Metabolism of [11-3H]retinyl acetate in liver tissues of vitamin A-sufficient, -deficient and retinoic acid-supplemented rats

A study was conducted on the incorporation of [11-3H]retinyl acetate into various retinyl esters in liver tissues of rats either vitamin A-sufficient, vitamin A-deficient or vitamin A-deficient and maintained on retinoic acid. Further, the metabolism of [11-3H]retinyl acetate to polar metabolites in liver tissues of these three groups of animals was investigated. Retinol metabolites were analyzed by high-performance liquid chromatography. In vitamin A-sufficient rat liver, the incorporation of radioactivity into retinyl palmitate and stearate was observed at 0.25 h after the injection of the label. The label was further detected in retinyl laurate, myristate, palmitoleate, linoleate, pentadecanoate and heptadecanoate 3 h after the injection. The specific radioactivities (dpm/nmol) of all retinyl esters increased with time. However, the rate of increase in the specific radioactivity of retinyl laurate was found to be significantly higher (66-fold) than that of retinyl palmitate 24 h after the injection of the label. 7 days after the injection of the label, the specific radioactivity between different retinyl esters were found to be similar, indicating that newly dosed labelled vitamin A had now mixed uniformly with the endogenous pool of vitamin A in the liver. The esterification of labelled retinol was not detected in liver tissues of vitamin A-deficient or retinoic acid-supplemented rats at any of the time point studied. Among the polar metabolites analyzed, the formation of [3H]retinoic acid from [3H]retinyl acetate was found only in vitamin A-deficient rat liver 24 h after the injection of the label. A new polar metabolite of retinol (RM) was detected in liver of the three groups of animals. The formation of 3H-labelled metabolite RM from [3H]retinyl acetate was not detected until 7 days after the injection of the label in the vitamin A-sufficient rat liver, suggesting that metabolite RM could be derived from a more stable pool of vitamin A.     

Metabolic adaptations in skeletal muscle of streptozotocin-diabetic rats following exercise training

Compensatory metabolic adaptations induced in streptozotocin-diabetic rat skeletal muscle by submaximal endurance training have been investigated. The gastrocnemius muscles of sedentary streptozotocin-diabetic rats were found to have a lower than normal myoglobin content, succinate dehydrogenase activity, and capacity to oxidize pyruvate and palmitate-1-[¹⁴C]. The values of these parameters were significantly increased in the diabetic skeletal muscle by the training program, obtaining levels similar to those of normal sedentary animals.     

Utilization of exogenous free fatty acids for the production of very low density lipoprotein triglyceride by livers of carbohydrate-fed rats

High carbohydrate diets enhance the hepatic output of very low density lipoprotein triglycerides. The fatty acids of these triglycerides could come from exogenous sources (i.e., diet or adipose tissue) or from de novo fatty acid synthesis in the liver. The role of exogenous free fatty acids was evaluated in rats fed Purina Chow or diets containing 10% fructose for up to 14 wk. In carbohydrate-fed rats, serum triglycerides were twice normal, and VLDL accounted for about 60% of the increases. Pre-beta-lipoprotein was increased and alpha- and beta-lipoprotein were decreased. Phospholipid and cholesterol levels were unchanged. Livers were perfused with glucose and free fatty acids. Perfusate free fatty acids rose from 180 to 1800 micro eq/liter as the infused acids increased from 0 to 992 micro eq/3 hr; simultaneously, net free fatty acid uptake rose from < 1 to 18 micro eq/g/hr and triglyceride output by the liver doubled. However, rates of secretion of triglyceride became constant, and triglyceride accumulated in liver at uptakes of free fatty acids > 13 micro eq/g/hr. More lauric and myristic acid appeared in the perfusate than was infused, suggesting the hepatic discharge of free fatty acids. Livers of fructose-fed rats secreted twice as much oleate-(14)C-labeled triglyceride as controls at all levels of free fatty acid uptake. The ratios of the specific activities of perfusate triglyceride to free oleate-(14)C were unaffected by diet and were about 0.6 and 1.0 at low and high triglyceride secretion rates, respectively. Thus, carbohydrate feeding did not result in altered uptakes of free fatty acids or preferential secretion of triglycerides containing endogenously synthesized fatty acid. Instead, the increased secretion of triglyceride was accomplished by enhanced formation of VLDL triglyceride from exogenous free fatty acids.     

Fatty acid oxidation by liver and muscle preparations of exhaustively exercised rats.

The influence of exhaustive exercise on the capacity of liver and muscle of rats to oxidize fatty acids was investigated in vitro. The rate of oxidation of fatty acids by liver preparations was significantly elevated as a result of exhaustion. Concurrently, the concentrations of beta-hydroxybutyrate were elevated in the plasma of the exhausted rats, suggesting that oxidation of fatty acids was also elevated in vivo. These findings are analogous to the findings of increased oxidation of fatty acids that results from training. In muscle, oxidation of palmitate, palmitoylcarnitine and beta-hydroxybutyrate by homogenates and isolated mitochondria was depressed with exercise. Despite the decrease in the oxidative capacity of the muscle preparations, the activities of several enzymes of beta-oxidation were either increased or unchanged as a result of exercise, suggesting that the depression in fatty acid oxidation may not be related to alterations in the process of beta-oxidation. Further studies showed that oxidation of [2-(14)C]pyruvate by muscle was depressed, whereas oxidation of [1-(14)C]pyruvate was not changed as a result of exercise. These results suggest that the decrease in fatty acid oxidation may be related to aberrations in the oxidation of acetyl-CoA. The changes in fatty acid oxidation that were observed, which are at variance with what is reported to occur with training, may have resulted from increased fragility of muscle mitochondria as a result of exercise. This increased fragility may render the mitochondria more susceptible to experimental manipulations in vitro and a subsequent loss of normal function.