Effect of dietary fat sources on fatty acid deposition and lipid metabolism in broiler chickens.

The hypothesis tested was that dietary vegetable fats rich in saturated fatty acids, when compared with a vegetable oil rich in linoleic acid, increase fat deposition in broiler chickens and affect synthesis or oxidation, or both, of individual fatty acids. Diets with native sunflower oil (SO), a 50:50 mix of hydrogenated and native SO, palm oil, and randomized palm oil were fed to broiler chickens. Intake of digestible fat and fatty acids, whole body fatty acid deposition, hepatic fatty acid profile, and hepatic enzyme activities involved in fatty acid oxidation and synthesis were measured. The fat deposition:digestible fat intake ratio was significantly lower for the SO group in comparison with the groups fed the vegetable fats rich in saturated fatty acids. The difference between digestible intake and deposition of C18:2, reflecting its maximum disappearance rate, was highest for the SO group and lowest for the palm oil- and randomized palm oil-fed birds. The calculated minimal rate of de novo synthesis of monounsaturated fatty acids (MUFA), calculated as deposition minus digestible intake, was more than 50% lower for the SO group than for the other 3 dietary groups. Based on the fatty acid profiles in the liver, it would appear that increasing contents of C18:2 decrease the desaturation of saturated fatty acids into MUFA. It is concluded that a diet rich in C18:2 in comparison with different kinds of vegetable saturated fatty acids decreases the deposition of fat, especially of MUFA. It appears to be caused by a higher ß-oxidation and a reduced de novo synthesis of MUFA, but this conclusion is not fully supported by the measured activities of enzymes involved in fatty acid synthesis and oxidation.

The autophagic and endocytic pathways converge at the nascent autophagic vacuoles.

We used an improved cryosectioning technique in combination with immunogold cytochemistry and morphometric analysis to study the convergence of the autophagic and endocytic pathways in isolated rat hepatocytes. The endocytic pathway was traced by continuous uptake of gold tracer for various time periods, up to 45 min, while the cells were incubated in serum-free medium to induce autophagy. Endocytic structures involved in fusion with autophagic vacuoles (AV) were categorized into multivesicular endosomes (MVE) and vesicular endosomes (VE). Three types of AV--initial (AVi), intermediate (AVi/d), and degradative (AVd)--were defined by morphological criteria and immunogold labeling characteristics of marker enzymes. The entry of tracer into AV, manifested as either tracer-containing AV profiles (AV+) or fusion profiles (FP+) between AV and tracer-positive endosomal vesicles/vacuoles, was detected as early as 10 min after endocytosis. The number of AV+ exhibited an exponential increase with time. FP+ between MVE or VE and all three types of AV were observed. Among the 112 FP+ scored, 36% involved VE. Of the AV types, AVi and AVi/d were found five to six times more likely to be involved in fusions than AVd. These fusion patterns did not significantly change during the period of endocytosis (15-45 min). We conclude that the autophagic and endocytic pathways converge in a multistage fashion starting within 10 min of endocytosis. The nascent AV is the most upstream and preferred fusion partner for endosomes.

Fatty acid digestion and deposition in broiler chickens fed diets containing either native or randomized palm oil.

The hypothesis tested was that randomization of palm oil would increase its digestibility, especially that of its palmitic acid (C16:0) component, with subsequent changes in the fatty acid composition in body tissues. Broiler chickens were fed diets containing either native or randomized palm oil. Diets with either native or a 50/50 mix of native and hydrogenated sunflower oil were also fed. Randomization of palm oil raised the fraction of C16:0 at the sn-2 position of the glycerol molecule from 14 to 32%. Hydrogenation of sunflower oil reduced fat and total saturated fatty acid digestibility, whereas no change in digestibility of total unsaturated fatty acids was found. Randomization of palm oil raised the group mean apparent digestibility of C16:0 by 2.6 and 5.8% units during the starter and grower-finisher phase, respectively. On the basis of the observed digestibilities in the grower-finisher period, it was calculated that the digestibility for C16:0 at the sn-2 and sn-1,3 position was 90 and 51%, respectively. The feeding of randomized instead of native palm oil significantly raised the palmitic acid content of breast meat and abdominal fat and lowered the ratio of unsaturated to saturated fatty acids. It is concluded that randomized palm oil may be used as vegetable oil in broiler nutrition with positive effect on saturated fatty acid digestibility when compared with native palm oil and positive effect on firmness of meat when compared with vegetable oils rich in unsaturated fatty acids.

Body composition and heat expenditure in broiler chickens fed diets with or without trans fatty acids.

The effect of a diet containing trans fatty acids (TFA) on the fatty acid composition and fat accumulation was investigated in broiler chickens. Female broilers were fed a control or a TFA-containing diet. The difference between the diets was that a part of cis 18:1 in the control diet was replaced by the TFA. Body composition, energy balance and the fatty acid composition were examined. Over the time-period studied (15 days), the body fat content and the protein content did not differ significantly between the TFA-fed group and the control. In energy balance studies, total energy intake, energy loss in excreta, energy expenditure and energy storage did not differ between the treatments. Compared to the control diet, the TFA diet resulted in significantly higher amounts of 14:0 and 18:1n-7 and lower amounts of 18:1n-9 and 20:4n-6 in the body. In conclusion, the data suggest that feeding TFA for 15 days to female broilers had no effect on energy retention, energy expenditure and energy loss in excreta or in body composition in terms of fat and protein. Only the fatty acid composition in the body was affected by the treatment with TFA. In addition, 50% of ingested TFA was incorporated into the body fat. This may have a negative effect on the dietetic value of chicken meat.

Studies on hepatic lipidosis and coinciding health and fertility problems of high-producing dairy cows using the "Utrecht fatty liver model of dairy cows". A review.

Fatty liver or hepatic lipidosis is a major metabolic disorder of high-producing dairy cows that occurs rather frequently in early lactation and is associated with decreased health, production and fertility. A background section of the review explores reasons why high-producing dairy cows are prone to develop fatty liver post partum. Hepatic lipidosis and coinciding health and fertility problems seriously endanger profitability and longevity of the dairy cow. Results from a great number of earlier epidemiological and clinical studies made it clear that a different approach was needed for elucidation of pathogenesis and etiology of this complex of health problems. There was a need for an adequate animal model in which hepatic lipidosis and production, health and fertility problems could be provoked under controlled conditions. It was hypothesized that overconditioning ante partum and feed restriction post partum might induce lipolysis in adipose tissue and triacylglycerol accumulation in the liver following calving. This consideration formed the basis for the experiments, which resulted in the "Utrecht fatty liver model of dairy cows". In this model, post partum triacylglycerol-lipidosis as well as the whole complex of health and fertility problems are induced under well-controlled conditions. The experimental protocol based on this hypothesis produced in all cases (10 feeding trials with over 150 dairy cattle) the intended result, i.e. all experimental cows developed post partum higher hepatic triacylglycerol concentrations than did control cows. The model was evaluated in biochemical, clinical pathology, immunological, clinical and fertility terms. It turned out that in this model, post partum triacylglycerol-lipidosis as well as the whole complex of health and fertility problems were induced under well-controlled conditions.

Measurement of acetyl-CoA carboxylase activity in isolated hepatocytes.

An assay is described for acetyl-CoA carboxylase activity in isolated hepatocytes. The assay is based on two principles: The hepatocytes are made permeable by digitonin. 64 micrograms of digitonin per mg of cellular protein were most effective in exposing enzyme activity without a significant effect on mitochondrial permeability. Enzyme activity is measured by coupling the carboxylase reaction to the fatty acid synthase reaction. The advantages offered by this procedure over existing assays are: rapidity, no need to prepare cell extracts, absence of product inhibition, no interference by mitochondrial enzymes, useful in systems with bicarbonate buffers, and simple separation of radioactive substrate from labelled products. Using this coupled enzyme assay a good correlation was observed between changes in the activity of acetyl-CoA carboxylase and changes in the rate of fatty acid synthesis in hepatocytes as effected by short-term modulators.

Effects of dietary conditions on the pool sizes of precursors of phosphatidylcholine and phosphatidylethanolamine synthesis in rat liver.

We developed a new method for the determination of choline- and ethanolamine-containing precursors of phosphatidylcholine and phosphatidylethanolamine after their separation by HPLC and we have studied the effects of different dietary conditions on the pool sizes of these metabolites in rat liver. Fasting for 48 h induced only a small decrease in the amounts of phosphatidylethanolamine and its water-soluble precursors. Upon refeeding with a high-sucrose, fat-free diet for 72 h, the levels of ethanolamine-containing compounds were only slowly restored. The effects of various dietary conditions on the amounts of phosphatidylcholine and its water-soluble precursors were much more pronounced. Fasting induced a sharp decrease, especially of the amount of cholinephosphate. However, the levels of phosphatidylcholine and the choline-containing precursors were rapidly restored upon refeeding for 24 h. Continued refeeding for an additional 48 h enhanced the cholinephosphate pool size to a level more than double that found in normally fed rats. The latter effect was accompanied by an inhibition of the enzyme CTP:choline-phosphate cytidylyltransferase. The results are discussed in view of a possible regulatory mechanism that may balance the amounts of phosphatidylcholine and phosphatidylethanolamine.

On the mechanism of the so-called uncoupling effect of medium- and short-chain fatty acids.

Octanoate applied to rat liver mitochondria respiring with glutamate plus malate or succinate (plus rotenone) under resting-state (State 4) conditions stimulates oxygen uptake and decreases the membrane potential, both effects being sensitive to oligomycin but not to carboxyatractyloside. Octanoate also decreases the rate of pyruvate carboxylation under the same conditions, this effect being correlated with the decrease of intramitochondrial content of ATP and increase of AMP. The decrease of pyruvate carboxylation and the change of mitochondrial adenine nucleotides are both reversed by 2-oxoglutarate. Fatty acids of shorter chain length have similar effects, though at higher concentrations. Addition of octanoate in the presence of fluoride (inhibitor of pyrophosphatase) produces intramitochondrial accumulation of pyrophosphate, even under conditions when oxidation of octanoate is prevented by rotenone. In isolated hepatocytes incubated with lactate plus pyruvate, octanoate also increases oxygen uptake and produces a shift in the profile of adenine nucleotides similar to that observed in isolated mitochondria. It decreases the 'efficiency' of gluconeogenesis, as expressed by the ratio between an increase of glucose production and an increase of oxygen uptake upon addition of gluconeogenic substrates (lactate plus pyruvate), and increases the reduction state of mitochondrial NAD. These effects taken together are not compatible with uncoupling, but point to intramitochondrial hydrolysis of octanoyl-CoA and probably also shorter chain-length acyl-CoAs. This mechanism probably functions as a 'safety valve' preventing a drastic decrease of intramitochondrial free CoA under a large supply of medium- and short-chain fatty acids.

Accumulation of pyruvate by isolated rat liver mitochondria.

1. Various methods to measure the rate of accumulation of [3-14C]pyruvate in the sucrose-impermeable space of isolated rat liver mitochondria are tested and compared with respect to their ability to distinguish between carrier-linked pyruvate transport and non-carrier-linked processes (adsorption and diffusion). 2. Evidence is presented that the cinnamic acid derivatives commonly used as specific inhibitors of the pyruvate carrier (i) do not completely abolish all carrier-mediated pyruvate transport; (ii) inhibit pyruvate adsorption, and (iii) at higher concentrations lead to a removal of previously accumulated pyruvate from the mitochondria. It is concluded that procedures which avoid the use of transport inhibitors allow more reliable estimates of carrier-linked pyruvate transport. 3. It is proposed to measure pyruvate adsorption as the accumulation of pyruvate in the presence of an uncoupler. Using this procedure, it could be shown that, with 1 mM pyruvate, adsorption represents only a small part of the total pyruvate accumulation, the main part being carrier-linked transport driven by the pH gradient across the mitochondrial inner membrane.

Reversible translocation of CTP:phosphocholine cytidylyltransferase from cytosol to membranes in the adult bovine liver around parturition.

The key regulatory enzyme of phosphatidylcholine (PC) synthesis, CTP:phosphocholine cytidylyltransferase (CT), is known to be activated in vitro by translocation from soluble to particulate fractions of the cell. In the present study the periparturient cow was chosen as a model to investigate whether translocation of CT can contribute to the regulation of PC synthesis in vivo. Between parturition and 1.5 weeks post-partum, the cytosolic CT activity in the liver of the adult animal decreased 1.9-fold, and this correlated with a 1.8-fold increase in microsomal CT activity. At that time, microsomal CT activity started to decline again whereas the cytosolic activity rose concomitantly until both activities reached their pre-partum values at 8 weeks post-partum. The activities of soluble and membrane-bound CTP:phosphoethanolamine cytidylyltransferase (ET), the analogous enzyme in the CDP-ethanolamine pathway, did not change significantly throughout this period. Whereas hepatic PC concentrations declined until about 2 weeks post-partum and thereafter gradually returned to pre-partum levels, the PC levels in very-low-density-lipoproteins, started to rise 2 weeks after the partus reaching a maximum of 219% of the original value at 8 weeks post-partum. These results strongly suggest that there is a reversible redistribution of CT between cytosol and membranes in a physiologically relevant animal model, supporting the concept that translocation of CT is occurring in vivo.

Immunochemical quantitation of fatty-acid-binding proteins. I. Tissue and intracellular distribution, postnatal development and influence of physiological conditions on rat heart and liver FABP.

Antisera against rat heart and liver fatty acid-binding protein (FABP) were applied in Western blotting analysis and ELISA to assess their tissue and intracellular distribution, and the influence of development, physiological conditions and several agents on the FABP content of tissue cytosols. The data obtained are compared with the oleic acid-binding capacity. Heart FABP is found in high concentrations in heart, skeletal muscles, diaphragm and lung, and in lower concentrations in kidney, brain and spleen, whereas liver FABP is limited to liver and intestine. In heart and liver, FABP is only present in the cytosol. The FABP content of both heart and liver shows a progressive increase during the first weeks of postnatal development, in contrast to their constant oleic acid-binding capacity. The reciprocally declining alpha-fetoprotein content of both tissues may partially account for the complementary fraction of the fatty acid-binding capacity. The FABP content and the fatty acid-binding capacity of adult heart and liver were in good accordance under various physiological conditions. Addition of clofibrate to the diet induces an increase of liver FABP content, whereas feeding of cholesterol, cholestyramine, mevinolin or cholate caused a marked decrease. The significance of the combined determination of fatty acid-binding capacity and FABP content (by immunochemical quantitation and blotting analysis) is indicated.

Substrate specificity of CTP: phosphoethanolamine cytidylyltransferase purified from rat liver.

CTP: phosphoethanolamine cytidylyltransferase was recently purified to homogeneity from rat liver (Vermeulen, P.S. Tijburg, L.B.M., Geelen, M.J.H. and van Golde, L.M.G. (1993) J. Biol. Chem. 268, 7458-7464). The present study focuses on the specificity of this enzyme for phosphorylated bases with a varying degree of N-methylation. The apparent Km for phosphoethanolamine was 0.072 mM. As the number of N-methylated substituents on phosphoethanolamine increased, the apparent Km increased: 0.11 mM for phosphomonomethylethanolamine and 6.8 mM for phosphodimethylethanolamine. Introduction of a third N-methyl group did not further increase the Km value. The effect of N-methyl groups on the reaction velocity was far more pronounced. A decreased Vmax for the reaction was found as the number of N-methyl substituents increased: 1.52 and 0.24 mumol/min per mg protein for phosphoethanolamine and phosphomonomethylethanolamine, and 44 and 0.69 nmol/min per mg protein for phosphodimethylethanolamine and phosphocholine, respectively. Phosphomonomethylethanolamine, phosphodimethylethanolamine and phosphocholine were weak competitive inhibitors of the cytidylyltransferase catalyzed reaction when phosphoethanolamine was used as a substrate, with Ki values of 7.0, 6.8 and 52.9 mM, respectively. The results show that this cytidylyltransferase is highly specific for phosphoethanolamine. Comparison of these data with previously reported information on the substrate specificity of CTP: phosphocholine cytidylyltransferase endorses the view that the two cytidylyltransferases are functionally different.

The synthesis and esterification of cholesterol by hepatocytes and H35 hepatoma cells are independent of the level of nonspecific lipid transfer protein.

The level of the nonspecific lipid transfer protein (i.e., sterol carrier protein 2) is 16-fold lower in the Reuber H35 hepatoma cells as compared to the hepatocytes in culture (49 and 810 ng of protein per mg of 105000 X g supernatant protein, respectively). In order to establish whether there is a relationship between the level of nonspecific transfer protein and intracellular cholesterol metabolism, we have determined the biosynthesis and esterification of cholesterol in these hepatoma cells and hepatocytes. Both types of cells incorporated [3H]mevalonate into cholesterol and cholesterol ester. Incubation of both cell types with [3H]cholesterol in the medium resulted in a time-dependent uptake and subsequent conversion into cholesterol ester. In both instances, the amount of 3H label incorporated into cholesterol per mg of cellular protein was about 2-fold higher for the hepatoma cells. The kinetics of esterification of endogenously synthesized cholesterol were similar for both hepatoma cells and hepatocytes. Esterification of cholesterol derived from the medium proceeded 2-times faster in the hepatoma cells than in the hepatocytes. From the kinetics of cholesterol esterification we conclude that cells do not discriminate between cholesterol synthesized de novo and cholesterol derived from the medium. In addition, the proposition that the nonspecific lipid transfer protein is involved in cholesterol synthesis and esterification is not substantiated by this study.

Effects of vasopressin on the synthesis of phosphatidylethanolamines and phosphatidylcholines by isolated rat hepatocytes.

The effect of vasopressin on the biosynthesis of phosphatidylcholines and phosphatidylethanolamines was investigated in freshly isolated rat hepatocytes in suspension. Treatment of hepatocytes with vasopressin inhibits the incorporation of [Me-14C]choline into phosphatidylcholines in a dose-dependent manner. The hormone does not affect the uptake, phosphorylation or oxidation of choline. Pulse-chase studies indicate that CTP:cholinephosphate cytidylyltransferase might be subject to hormonal regulation by vasopressin. In contrast with the inhibitory effect of vasopressin on the synthesis of phosphatidylcholines, this hormone stimulates the incorporation of [1,2-14C]ethanolamine into phosphatidylethanolamines in a dose-dependent manner. Pulse and pulse-chase studies with labelled ethanolamine show that the conversion of ethanolaminephosphate to CDPethanolamine as well as the formation of phosphatidylethanolamines from CDPethanolamine and diacylglycerol are enhanced. Determination of the effect of vasopressin on the activity of the enzymes of the synthesis de novo of phosphatidylethanolamines demonstrates an increase of the activity of ethanolaminephosphotransferase, probably as a result of the increased amount of diacylglycerol in vasopressin-treated cells.

Persistent changes in the initial rate of pyruvate transport by isolated rat liver mitochondria after preincubation with adenine nucleotides and calcium ions.

1. Preincubation of isolated rat-liver mitochondria in the presence of adenine nucleotides or Ca2+ results in definite and persistent changes in the initial rate of pyruvate transport. 2. These changes in the rate of pyruvate transport are accompanied by equally persistent changes in the opposite direction of the activity of pyruvate dehydrogenase (EC 1.2.4.1). 3. Changes of the transmembrane pH gradient and of the membrane potential, brought about by the pretreatments of the mitochondria, cannot account for the observed changes in the rate of pyruvate transport. 4. It is proposed that the pretreatment of the mitochondria directly modulates the activity of the mitochondrial pyruvate carrier. The possible regulatory role of such a modulation system is discussed.

Regulation of triacylglycerol synthesis in the liver: a decrease in diacylglycerol acyltransferase activity after treatment of isolated rat hepatocytes with glucagon.

Isolated rat hepatocytes were used to investigate the possibility of a short-term effect of glucagon on the synthesis of triacylglycerols in the liver. Incubation of hepatocytes in the presence of glucagon, followed by homogenization in a buffer containing F- (50 mM) and EDTA (2.5 mM), resulted in a 53% decrease in activity of microsomal diacylglycerol acyltransferase (EC 2.3.1.20), the only enzyme that is exclusively involved in the synthesis of triacylglycerols. The activity of cholinephosphotransferase (EC 2.7.8.2), which also uses diacylglycerols as substrate, was not decreased after exposure of the hepatocytes to glucagon. This may imply that triacylglycerol synthesis can be regulated independently of phosphatidylcholine synthesis. The activity of diacylglycerol acyltransferase in microsomes isolated from a homogenate of whole liver could be reduced by preincubating the microsomes with Mg2+ (5 mM), ATP (1 mM) and 105 000 X g supernatant. The enzyme could be reactivated by incubation of the washed microsomes with a 105 000 X g supernatant in the presence of dithiothreitol (5 mM). Fluoride (50 mM) inhibited this reactivation. It is concluded that the activity of diacylglycerol acyltransferase is subject to hormonal short-term control, possibly via a phosphorylation-dephosphorylation mechanism.

The effects of insulin and glucagon on the release of triacylglycerols by isolated rat hepatocytes are mere reflections of the hormonal effects on the rate of triacylglycerol synthesis.

1. Isolated hepatocytes from meal-fed donor rats secrete newly synthesized very-low-density lipoproteins (VLDL) when incubated in a simple bicarbonate buffer. When incubated with 3H2O for 2 h, 72-81% of the 3H-labelled triacylglycerols secreted by the hepatocytes were recovered in VLDL. The secretion of newly synthesized triacylglycerols shows a lag phase of about 30 min. 2. Insulin stimulates the secretion of newly synthesized VLDL triacylglycerols, whereas glucagon has an inhibitory effect on this process. 3. When hepatocytes triacylglycerols were labelled by preincubating the cells with 3H2O or [1-14C]oleate and the cells were subsequently washed and further incubated in radioisotope-free buffer containing hormones, it was observed that the release of the pre-labelled triacylglycerols is not hormone-sensitive. This suggests that insulin and glucagon do not affect the release of triacylglycerols per se. 4. It is concluded that the effects of insulin and glucagon on the overall process of triacylglycerol secretion are reflections of the hormone-determined rate of triacylglycerol synthesis.

Opposite effects of insulin and glucagon in acute hormonal control of hepatic lipogenesis.

Conditions for the isolation of rat hepatocytes that are responsive to insulin with regard to fatty acid synthesis were explored. Cells prepared according to the procedure of Ingebretsen and Wagle require the presence of fetal calf serum for insulin expression. Cells isolated by the Seglen method are the preparation of choice, since they respond to insulin in a simple, well-defined medium and, moreover, show much higher basal rates of fatty acid synthesis. In the latter cells isolated from fed male rats, the rate of fatty acid synthesis, as determined by tritium incorporation from [3H]H2O at 37 degrees C, is enhanced within 30 min after addition of insulin to the incubation medium; with glucagon, it is depressed. In the presence of insulin, the cellular content of malonyl coenzyme A is noticeably increased, whereas the concentrations of pyruvate, lactate, and citrate are not markedly affected. Glucagon, on the other hand, decreases the concentrations of all four intermediates. The activity of acetyl-CoA carboxylase is stimulated and depressed after addition of insulin and glucagon, respectively. In all conditions tested, the activity of acetyl-CoA carboxylase correlates with the rate of fatty acid synthesis, which in turn correlates with the cellular level of malonyl-CoA.

Do cytoskeletal components control fatty acid translocation into liver mitochondria?

For two decades it has been assumed that inhibition of carnitine palmitoyltransferase I (CPT-I) by malonyl-CoA represents the main regulatory mechanism of liver ketogenesis. However, recent evidence indicates that CPT-I activity is also controlled by interactions between mitochondria and cytoskeletal components. This newly recognized mechanism emphasizes the emerging role of the cytoskeleton in the regulation of metabolic pathways.

Studies on the substrate for hepatic lipogenesis in the rat.

The contribution of hepatic glycogen to lipogenesis was studied in isolated, intact rat hepatocytes. To establish its importance as a substrate for lipogenesis, the glycogen of isolated hepatocytes was prelabelled with 14C from glucose. Evidence is presented that neither glucose nor glycogen constitute major sources of carbon for de novo synthesis of fatty acids and that less than 1% of glycogen is converted into fatty acids.