Mitochondrial glycerol-3-phosphate acyltransferase-deficient mice have reduced weight and liver triacylglycerol content and altered glycerolipid fatty acid composition

Microsomal and mitochondrial isoforms of glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyze the committed step in glycerolipid synthesis. The mitochondrial isoform, mtGPAT, was believed to control the positioning of saturated fatty acids at the sn-1 position of phospholipids, and nutritional, hormonal, and overexpression studies suggested that mtGPAT activity is important for the synthesis of triacylglycerol. To determine whether these purported functions were true, we constructed mice deficient in mtGPAT. mtGPAT(-/-) mice weighed less than controls and had reduced gonadal fat pad weights and lower hepatic triacylglycerol content, plasma triacylglycerol, and very low density lipoprotein triacylglycerol secretion. As predicted, in mtGPAT(-/-) liver, the palmitate content was lower in triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine. Positional analysis revealed that mtGPAT(-/-) liver phosphatidylethanolamine and phosphatidylcholine had about 21% less palmitate in the sn-1 position and 36 and 40%, respectively, more arachidonate in the sn-2 position. These data confirm the important role of mtGPAT in the synthesis of triacylglycerol, in the fatty acid content of triacylglycerol and cholesterol esters, and in the positioning of specific fatty acids, particularly palmitate and arachidonate, in phospholipids. The increase in arachidonate may be functionally significant in terms of eicosanoid production.     

The role of mitochondrial glycerol-3-phosphate acyltransferase-1 in regulating lipid and glucose homeostasis in high-fat diet fed mice

Glycerol-3-phosphate acyltransferase (GPAT) is involved in triacylglycerol (TAG) and phospholipid synthesis, catalyzing the first committed step. In order to further investigate the in vivo importance of the dominating mitochondrial variant, GPAT1, a novel GPAT1^−/− mouse model was generated and studied. Female GPAT1^−/− mice had reduced body weight-gain and adiposity when fed chow diet compared with littermate wild-type controls. Furthermore, GPAT1^−/− females on chow diet showed decreased liver TAG content, plasma cholesterol and TAG levels and increased ex vivo liver fatty acid oxidation and plasma ketone bodies. However, these beneficial effects were abolished and the glucose tolerance tended to be impaired when GPAT1^−/− females were fed a long-term high-fat diet (HFD). GPAT1-deficiency was not associated with altered whole body energy expenditure or respiratory exchange ratio. In addition, there were no changes in male GPAT1^−/− mice fed either diet except for increased plasma ketone bodies on chow diet, indicating a gender-specific phenotype. Thus, GPAT1-deficiency does not protect against HFD-induced obesity, hepatic steatosis or whole body glucose intolerance.     

Mitochondrial glycerol-3-phosphate acyltransferase-1 directs the metabolic fate of exogenous fatty acids in hepatocytes

Because excess triacylglycerol (TAG) in nonadipose tissues is closely associated with the development of insulin resistance, interest has increased in the metabolism of long-chain acyl-CoAs toward beta-oxidation or the synthesis and storage of TAG. To learn whether a mitochondrial isoform of glycerol-3-phosphate acyltransferase (mtGPAT1) competes with carnitine palmitoyltransferase I (CPT I) for acyl-CoAs and whether it contributes to the formation of TAG, we overexpressed rat mtGPAT1 13-fold in primary hepatocytes obtained from fasted rats. When 100, 250, or 750 microM oleate was present, both TAG mass and the incorporation of [14C]oleate into TAG increased more than twofold in hepatocytes overexpressing mtGPAT1 compared with vector controls. Although the incorporation of [14C]oleate into CO2 and acid-soluble metabolites increased with increasing amounts of oleate in the media, these metabolites were approximately 40% lower in the Ad-mtGPAT1 infected cells, consistent with competition for acyl-CoAs between CPT I and mtGPAT1. A 50-60% decrease was also observed in [14C]oleate incorporation into cholesteryl ester. With increasing amounts of exogenous oleate, [14C]TAG secretion increased appropriately in vector control-infected hepatocytes, suggesting that the machinery for VLDL-TAG biogenesis and secretion was unaffected. Despite the marked increases in TAG synthesis and storage in the Ad-mtGPAT1 cells, however, the Ad-mtGPAT1 cells secreted the same amount of [14C]TAG as the vector control cells. Thus, in isolated hepatocytes, mtGPAT1 may synthesize a cytosolic pool of TAG that cannot be secreted.     

The C-terminal region of mitochondrial glycerol-3-phosphate acyltransferase-1 interacts with the active site region and is required for activity

Glycerol phosphate acyltransferase (GPAT) catalyzes the formation of 1-acyl-sn-glycerol-3-phosphate from glycerol-3-phosphate and long chain fatty acyl-CoA substrates. We previously determined the topography of the mitochondrial GPAT1 isoform (mtGPAT1, 828 amino acids). mtGPAT1 has two transmembrane domains (TMDs) (aa 472-493 and aa 576-592) with both the N- and C-termini facing the cytosol and a loop (aa 494-575) facing the intermembrane space. Alignment of amino acid sequences from mtGPAT1 and other acyltransferases and site directed mutagenesis studies have demonstrated that the active site of the enzyme resides in the N-terminal domain of the protein. In this study, we sequentially truncated the C-terminal domain and characterized the properties of the resulting mutants expressed in CHO cells. Although the mutants were overexpressed, none of them conferred GPAT activity. The loss of activity was not due to the miss-targeting of the proteins since immunofluorescence experiments demonstrated their mitochondrial localization. Instead, chemical crosslinking and protein cleavage studies demonstrated that the N- and C-termini of the protein interact. These results suggest that the C-terminal domain is necessary for mtGPAT1 activity, and probably contributes to catalysis or substrate binding.     

Mitochondrial glycerol-3-phosphate acyltransferase-1 is essential in liver for the metabolism of excess acyl-CoAs

In vitro studies suggest that the mitochondrial glycerol-3-phosphate acyltransferase-1 (mtGPAT1) isoform catalyzes the initial and rate-controlling step in glycerolipid synthesis and aids in partitioning acyl-CoAs toward triacylglycerol synthesis and away from degradative pathways. To determine whether the absence of mtGPAT1 would increase oxidation of acyl-CoAs and restrict the development of hepatic steatosis, we fed wild type and mtGPAT1-/- mice a diet high in fat and sucrose (HH) for 4 months to induce the development of obesity and a fatty liver. Control mice were fed a diet low in fat and sucrose (LL). With the HH diet, absence of mtGPAT1 resulted in increased partitioning of acyl-CoAs toward oxidative pathways, demonstrated by 60% lower hepatic triacylglycerol content and 2-fold increases in plasma beta-hydroxybutyrate, acylcarnitines, and hepatic mRNA expression of mitochondrial HMG-CoA synthase. Despite the increase in fatty acid oxidation, liver acyl-CoA levels were 3-fold higher in the mtGPAT1-/- mice fed both diets. A lack of difference in CPT1 and FAS mRNA expression between genotypes suggested that the increased acyl-CoA content was not because of increased de novo synthesis, but instead, to an impaired ability to use long-chain acyl-CoAs derived from the diet, even when the dietary fat content was low. Hyperinsulinemia and reduced glucose tolerance on the HH diet was greater in the mtGPAT1-/- mice, which did not suppress the expression of the gluconeogenic genes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. This study demonstrates that mtGPAT1 is essential for normal acyl-CoA metabolism, and that the absence of hepatic mtGPAT1 results in the partitioning of fatty acids away from triacylglycerol synthesis and toward oxidation and ketogenesis.     

Relationship between glycerol-3-phosphate dehydrogenase,fatty acid synthase and fatty acid binding proteins in developing human placenta

The activities of the enzymes glycerol-3-phosphate dehydrogenase and fatty acid synthase are inhibited by palmitoyl-coenzyme A and oleate. The two isoforms of fatty acid binding proteins (PI 6.9 and PI 5.4) enhance the activities of glycerol-3-phosphate dehydrogenase and fatty acid synthase in the absence of palmitoyl-coenzyme A or oleate and also protect them against palmitoyl-coenzyme A or oleate inhibition. Levels of fatty acid binding proteins, the activities of the enzymes fatty acid synthase and glycerol-3-phosphate dehydrogenase increase with gestation showing a peak at term. However, the activity of fatty acid synthase showed the same trend up to the 30th week of gestation and then declined slightly at term. With the advancement of pregnancy when more lipids are required for the developing placenta, fatty acid binding proteins supply more fatty acids and glycerol-3-phosphate for the synthesis of lipids. Thus a correlation exists between glycerol-3-phosphate dehydrogenase, fatty acid synthase and fatty acid binding proteins in developing human placenta.     

Brain phospholipid arachidonic acid half-lives are not altered following 15 weeks of N-3 polyunsaturated fatty acid adequate or deprived diet

Previous studies have infused radiolabeled arachidonic acid (AA) into rat brains and followed AA esterification into phospholipids for up to 24 h; however, the half-life of AA in rat brain phospholipids is unknown. Eighteen day old rats were fed either an n-3 PUFA adequate or deprived diet for 15 weeks. Following the 15 weeks, 40 µCi of [³H] AA was injected intracerebroventricularly into the right lateral ventricle using stereotaxic surgery and returned to their dietary treatment. From 4–120 days after [³H] AA administration, brains were collected for chemical analyses. The half-life of AA in rat brain phospholipids was 44 ± 4 days for the n-3 PUFA adequate group and 46 ± 4 days for the n-3 PUFA deprived group, which closely approximates the predicted half-life previously reported, based on the rate of entry from the plasma unesterified pool, suggesting the plasma unesterified pool is a major contributor to brain uptake of AA. Furthermore, unlike a previous report in which the half-life of brain phospholipid docosahexaenoic acid (DHA) was increased in n-3 PUFA deprived rats, n-3 PUFA deprivation did not significantly alter the AA half-life, suggesting different mechanisms exist to maintain brain concentrations of AA and DHA.     

A low-protein, high-carbohydrate diet increases the adipose lipid content without increasing the glycerol-3-phosphate or fatty acid content in growing rats

The amount of triacylglycerol (TAG) that accumulates in adipose tissue depends on 2 opposing processes: lipogenesis and lipolysis. We have previously shown that the weight and lipid content of epididymal (EPI) adipose tissue increases in growing rats fed a low-protein, high-carbohydrate (LPHC) diet for 15 days. The aim of this work was to study the pathways involved in lipogenesis and lipolysis, which ultimately regulate lipid accumulation in the tissue. De novo fatty acid synthesis was evaluated in vivo and was similar for rats fed an LPHC diet or a control diet; however, the LPHC-fed rats had decreased lipoprotein lipase activity in the EPI adipose tissue, which suggests that there was a decreased uptake of fatty acids from the circulating lipoproteins. The LPHC diet did not affect synthesis of glycerol-3-phosphate (G3P) via glycolysis or glyceroneogenesis. Glycerokinase activity - i.e., the phosphorylation of glycerol from the hydrolysis of endogenous TAG to form G3P - was also not affected in LPHC-fed rats. In contrast, adipocytes from LPHC animals had a reduced lipolytic response when stimulated by norepinephrine, even though the basal adipocyte lipolytic rate was similar for both of the groups. Thus, the results suggest that the reduction of lipolytic activity stimulated by norepinephrine seems essential for the TAG increase observed in the EPI adipose tissue of LPHC animals, probably by impairment of the process of activation of lipolysis by norepinephrine.     

1-Acyl-sn-glycerol-3-phosphate acyltransferase in maturing safflower seeds and its contribution to the non-random fatty acid distribution of triacylglycerol

A 20,000 X g particulate preparation isolated from maturing safflower seeds catalyzed the acylation of 1-acyl-sn-glycerol 3-phosphate with acyl-CoA to form phosphatidate. The specific activity of the reaction exceeded 200 nmol min-1 mg protein-1. Although this preparation was also capable of catalyzing the acylation of sn-glycerol 3-phosphate with acyl-CoA, the hydrolysis of phosphatidate, and the acylation of 1,2-diacylglycerol, phosphatidate was the only major product when the preparation was incubated with 1-acyl-glycerol-3-P and acyl-CoA. The enzyme responsible for this phosphatidate synthesis, 1-acyl-glycerol-3-P acyltransferase, showed a strict acyl-CoA specificity. The relative order of specificity for acyl-CoA was linoleoyl = oleoyl greater than palmitoleoyl greater than elaidoyl greater than cis-vaccenoyl greater than stearoyl = palmitoyl. This observation strongly suggests that the fatty acid composition of position 2 in phosphatidate synthesized in vivo primarily depends on both the acyl-CoA specificity of the 1-acyl-glycerol-3-P acyltransferase and the fatty acid composition of the acyl-CoA pool in the cell. Thus, the absence of saturated fatty acids at position 2 of safflower triacylglycerol may be explained in terms of the acyl-CoA specificity of the 1-acyl-glycerol-3-P acyltransferase. The fatty acid moiety esterified at position 1 of glycerol-3-P also affected the effectiveness of the reaction. The 1-acyl-glycerol-3-P acyltransferase utilized 1-acyl-glycerol-3-P molecular species in the following order of effectiveness: linoleoyl = oleoyl greater than palmitoyl. With a rise in incubation temperature, the initial rates of acylation with unsaturated acyl-CoA species increased more rapidly than those for saturated acyl-CoA species. A similar tendency was observed for saturated and unsaturated acyl acceptors. These data suggest that affinity of the acyltransferase for substrates may vary in response to changes in temperature, and that 1-acyl-glycerol-3-P acyltransferase may be involved in the alteration of the individual fatty acid compositions at positions 1 and 2 of glycerolipids in tissues grown at different temperatures. Based on these findings, further metabolism of 1-acyl-glycerol-3-P acyltransferase products could be the major factor determining the non-random distribution of fatty acids in safflower triacylglycerol.     

Specific phospholipid fatty acid composition of brain regions in mice. Effects of n-3 polyunsaturated fatty acid deficiency and phospholipid supplementation

This study examined the effects of dietary alpha-linolenic acid deficiency followed or not by supplementation with phospholipids rich in n;-3 polyunsaturated fatty acid (PUFA) on the fatty acid composition of total phospholipids in 11 brain regions. Three weeks before mating, mice were fed a semisynthetic diet containing both linoleic and alpha-linolenic acid or deficient in alpha-linolenic acid. Pups were fed the same diet as their dams. At the age of 7 weeks, a part of the deficient group were supplemented with n;-3 polyunsaturated fatty acids (PUFA) from either egg yolk or pig brain phospholipids for 2 months. Saturated and monounsaturated fatty acid levels varied among brain regions and were not significantly affected by the diet. In control mice, the level of 22:6 n-3 was significantly higher in the frontal cortex compared to all regions. alpha-Linolenic acid deficiency decreased the level of 22:6 n-3 and was compensated by an increase in 22:5 n-6 in all regions. However, the brain regions were affected differently. After the pituitary gland, the frontal cortex, and the striatum were the most markedly affected with 40% reduction of 22:6 n-3. Supplementation with egg yolk or cerebral phospholipids in deficient mice restored a normal fatty acid composition in brain regions except for the frontal cortex. There was a regional distribution of the fatty acids in the brain and the impact of deficiency in alpha-linolenic acid was region-specific. Dietary egg yolk or cerebral phospholipids are an effective source of n-3 PUFA for the recovery of altered fatty acid composition induced by a diet deficient in n-3 PUFA.     

Fatty acid synthesis and generation of glycerol-3-phosphate in brown adipose tissue from rats fed a cafeteria diet

In vivo fatty acid synthesis and the pathways of glycerol-3-phosphate (G3P) production were investigated in brown adipose tissue (BAT) from rats fed a cafeteria diet for 3 weeks. In spite of BAT activation, the diet promoted an increase in the carcass fatty acid content. Plasma insulin levels were markedly increased in cafeteria diet-fed rats. Two insulin-sensitive processes, in vivo fatty acid synthesis and in vivo glucose uptake (which was used to evaluate G3P generation via glycolysis) were increased in BAT from rats fed the cafeteria diet. Direct glycerol phosphorylation, evaluated by glycerokinase (GyK) activity and incorporation of [U-14C]glycerol into triacylglycerol (TAG)-glycerol, was also markedly increased in BAT from these rats. In contrast, the cafeteria diet induced a marked reduction of BAT glyceroneogenesis, evaluated by phosphoenolpyruvate carboxykinase-C activity and incorporation of [1-14C]pyruvate into TAG-glycerol. BAT denervation resulted in an approximately 50% reduction of GyK activity, but did not significantly affect BAT in vivo fatty acid synthesis, in vivo glucose uptake, or glyceroneogenesis. The data suggest that the supply of G3P for BAT TAG synthesis can be adjusted independently from the sympathetic nervous system and solely by reciprocal changes in the generation of G3P via glycolysis and via glyceroneogenesis, with no participation of direct phosphorylation of glycerol by GyK.     

Deficit of didocosahexaenoyl phospholipid in the eyes of larval sea bass fed an essential fatty acid deficient diet

Larval sea bass Dicentrarchus labrax of 27 days old were reared on Artemia enriched with Super Selco©, Tuna Orbital Oil or Yeast. The first diet is commonly used in mariculture for larval rearing, the second diet was designed to deliver an optimal docosahexaenoic acid (22: 6n-3) to eicosapentaenoic acid (20: 5n-3) ratio, and the third diet was deficient in docosahexaenoic acid (22: 6n-3). The eyes of these larvae were analysed after 28 days and the molecular species of the three main phospholipid classes, phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) determined. Eyes from larvae fed Artemia enriched with yeast showed large decreases in molecular species containing 22: 6n-3 compared to those supplemented with tuna orbital oil, most notably in 16: 0/22: 6n-3 PC which fell from 10.6 to 0.4%, in 22: 6n-3/22: 6n-3 and 18: 1/22: 6n-3 PE which fell from 29.6 to 0.3% and from 10.8 to 1.1% respectively, and in 22: 6n-3/22: 6n-3 PS which fell from 34.3 to 1.7%. Molecular species containing all other fatty acids, and especially 20: 5n-3, were elevated in eyes from the yeast-supplemented fish. In larvae fed Artemia enriched with Super Selco, amounts of eye 22: 6n-3/22: 6n-3 phospholipid were slightly lower in all three phospholipid classes compared to eyes from the tuna orbital oil-supplemented larvae. There was also a trend of decreased saturated fatty acid/22: 6n-3 and monounsaturated fatty acid/22: 6n-3 molecular species in all classes from the Super Selco-supplemented fish, the deficits being made up with molecular species containing 20: 5n-3 and 22: 5n-3. These results are discussed in relation to larval viability with particular respect to visual function.     

Modification of fatty acid composition of membrane phospholipid in hepatocyte monolayer with n-3, n-6 and n-9 fatty acids and its relationship to triacylglycerol production

The objective of these studies with rat hepatocytes in primary culture was to establish that: (a) membrane phospholipids would become enriched with the specific fatty acid supplemented to the media and (b) hepatocyte monolayer triacylglycerol synthetic rates were dependent on the type of fatty acid enrichment of the membrane phospholipids. Hepatocytes cultured in the absence of media lipid developed a phospholipid fatty acid composition which is indicative of an essential fatty acid deficiency. The extensive rise in 18:1(n - 9) content indicated that delta 9-desaturase was active. The fatty acid composition of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol in the microsomal- and mitochondrial-enriched fractions was highly dependent upon the type of fatty acid supplemented to the medium. Incorporation of fatty acids into phospholipids was rapid, and a new steady-state in fatty acid composition was achieved within approx. 36 h. Changes in the fatty acid composition of these hepatocyte phospholipid subclasses resulting from media supplementation with 18:2/20:4(n-6) or 20:5(n-3) were similar, but not identical, to changes which occurred in vivo as a result of consuming diets rich in 18:2(n-6) or 20:5(n-3). Hepatocyte lipogenesis was highly dependent upon the type of fatty acid supplemented to the medium. Prior conditioning with 16:0 increased triacylglycerol synthesis and secretion. Secretion of triacylglycerol was reduced by polyenoic fatty acid enrichment with 20:5(n-3) greater than 20:4/18:2(n-6). The suppression of triacylglycerol synthesis by 20:5(n-3) was due to an increased (P less than 0.05) diacylglycerol specific activity, which indicates that 20:5(n-3) suppression of hepatic triacylglycerol production may be caused in part by the inhibition of diacylglycerol acyltransferase.     

Influence of chronically altered thyroid status on the activity of liver mitochondrial glycerol-3-phosphate dehydrogenase in female inbred lewis rats.

The activity of liver mitochondrial flavoprotein-dependent glycerol-3-phosphate dehydrogenase (GPDH) is considered a reliable marker of thyroid status in acute and short-lasting experiments. The aim of this study was to ascertain whether GPDH activity could also be used as an index of thyroid status during chronic experiments over several months. We therefore analyzed GPDH activity in liver mitochondria of female inbred Lewis rats with thyroid status altered for 2 to 12 months. Hyperthyroid state was maintained by triiodothyronine (T (3)) or thyroxine (T (4)) administration, while methimazole was employed for inducing hypothyroidism. We found a seven- and three-fold increase of GPDH activity in female rats after T (3) or T (4) administration, respectively, compared to euthyroid females (8.9 +/- 2.3 nmol/min/mg protein), whereas administration of methimazole reduced the enzyme activity almost to one-third of the euthyroid values. These changes were not significantly influenced by the duration of hyperthyroid or hypothyroid treatment. We conclude that the level of the rat liver GPDH activity could serve as a useful marker for evaluation of hyperthyroid and hypothyroid status in chronic long-lasting experiments on female inbred Lewis rats.     

Polyhydroxybenzoates inhibit ascorbic acid activation of mitochondrial glycerol-3-phosphate dehydrogenase: implications for glucose metabolism and insulin secretion

Glycerol-3-phosphate dehydrogenase from pig brain mitochondria was stimulated 2.2-fold by the addition of 50 microm l-ascorbic acid. Enzyme activity, dependent upon the presence of l-ascorbic acid, was inhibited by lauryl gallate, propyl gallate, protocatechuic acid ethyl ester, and salicylhydroxamic acid. Homogeneous pig brain mitochondrial glycerol-3-phosphate dehydrogenase was activated by either 150 microm L-ascorbic acid (56%) or 300 microm iron (Fe(2+) or Fe(3+) (62%)) and 2.6-fold by the addition of both L-ascorbic acid and iron. The addition of L-ascorbic acid and iron resulted in a significant increase of k(cat) from 21.1 to 64.1 s(-1), without significantly increasing the K(m) of L-glycerol-3-phosphate (10.0-14.5 mm). The activation of pure glycerol-3-phosphate dehydrogenase by either L-ascorbic acid or iron or its combination could be totally inhibited by 200 microm propyl gallate. The metabolism of [5-(3)H]glucose and the glucose-stimulated insulin secretion from rat insulinoma cells, INS-1, were effectively inhibited by 500 microm or 1 mm propyl gallate and to a lesser extent by 5 mm aminooxyacetate, a potent malate-aspartate shuttle inhibitor. The combined data support the conclusion that l-ascorbic acid is a physiological activator of mitochondrial glycerol-3-phosphate dehydrogenase, that the enzyme is potently inhibited by agents that specifically inhibit certain classes of di-iron metalloenzymes, and that the enzyme is chiefly responsible for the proximal signal events in INS-1 cell glucose-stimulated insulin release.     

Effect of long chain fatty acids on triacylglycerol accumulation,fatty acid composition and related gene expression in primary cultured bovine satellite cells

Abstract

This study was conducted to determine the effects of long chain fatty acids (LCFAs) on triacylglycerol (TAG) content, as well as on genes associated with lipid synthesis and fatty acid composition in bovine satellite cells. Both saturated (palmitic and stearic) and unsaturated (oleic and linoleic) fatty acids stimulated the TAG accumulation at a concentration of 100?µM and oleate increased it significantly more than stearate and palmitate. The results revealed that the lipid droplet formation was markedly stimulated by linoleate and oleate at 100?µM. Compared to control, the expressions of adipose triglyceride lipase, carnitine acyltransferase 1 and the fatty acid translocase 36 were upregulated by LCFAs. All the fatty acids also significantly increased diacylglycerol acyltransferase 2 than the untreated control (p?<?0.05). The monounsaturated fatty acids significantly increased (p?<?0.05) in response to oleate and linoleate compared to the control as did the polyunsaturated fatty acids (p?<?0.05), in addition to stearate, linoleate and oleate. In contrast, saturated fatty acids were significantly decreased in the oleate and linoleate-treated groups. The study results contribute to our enhanced understanding of LCFAs’ regulatory roles on the bovine cell lipid metabolism.