Triacylglycerol metabolism in isolated rat kidney cortex tubules

Triacylglycerol metabolism has been studied in kidney cortex tubules from starved rats, prepared by collagenase treatment. Triacylglycerol was determined by a newly developed fully enzymic method. Incubation of tubules in the absence of fatty acids led to a decrease of endogenous triacylglycerol by about 50% in 1h. Addition of albuminbound oleate or palmitate resulted in a steady increase of tissue triacylglycerol over 2h. The rate of triacylglycerol synthesis was linearly dependent on oleate concentration up to 0.8mm, reaching a saturation at higher concentrations. Triacylglycerol formation from palmitate was less than that from oleate. This difference was qualitatively the same when net synthesis was compared with incorporation of labelled fatty acids. Quantitatively, however, the difference was less with the incorporation technique. Gluconeogenic substrates, which by themselves had no effect on triacylglycerol concentrations, stimulated neutral lipid formation from fatty acids. Glucose and lysine did not have such a stimulatory effect. Inhibition of gluconeogenesis from lactate by mercaptopicolinic acid likewise inhibited triacylglycerol formation. This inhibitory effect was seen with oleate as well as with oleate plus lactate. When [2-¹⁴C]lactate was used the incorporation of label into triacylglycerol was found in the glycerol moiety exclusively. Addition of dl-β-hydroxybutyrate (5mm) to the incubation medium in the presence of oleate or oleate plus lactate led to a significant increase in triacylglycerol formation. In contrast with the gluconeogenic substrates, dl-β-hydroxybutyrate had no stimulatory effect on fatty acid uptake. The results suggest that renal triacylglycerol formation is a quantitatively important metabolic process. The finding that gluconeogenic substrates, but not glucose, increase lipid formation, indicates that the glycerol moiety is formed by glyceroneogenesis in the proximal tubules. The effect of ketone bodies seems to be caused by the sparing action of these substrates on fatty acid oxidation. The decrease of triacylglycerol in the absence of exogenous substrates confirms previous conclusions that endogenous lipids provide fatty acids for renal energy metabolism.     

Triacylglycerol metabolism in rat skeletal muscle after exercise

The temporal relationships between triacylglycerol (TG) content and TG lipase activity in slow-twitch (STR) and fast-twitch red (FTR) muscles were determined in rats during recovery from a 2-h swim. Immediately after the exercise, plasma free fatty acid (FFA) was elevated and glycogen concentrations were decreased. TG content was decreased 40% in STR muscle and reduced 45% in FTR muscle. The TG concentration of STR muscle increased in a linear fashion throughout recovery so that control levels were reached within the first 24 h after exercise. TG lipase activity of STR muscle was elevated 36% above control immediately after the swim and continued to increase to 84% above control 24 h after the work. In STR muscle there was a net synthesis of TG, while lipase activity was elevated above that measured in muscle of control rats. TG content of FTR muscle remained 45% below control throughout the first 24 h of recovery, and TG lipase activity increased from 26% (P greater than 0.05) greater than control immediately after exercise to threefold above control 24 h after work. All parameters returned to control levels by 48 h of recovery. These data indicated that a net TG synthesis occurs in STR muscle when lipolytic activity is elevated. In FTR muscle, however, a gradual increase in TG lipase activity that occurs during the first 24 h of recovery accompanies a TG concentration well below the control level throughout this same time frame.(ABSTRACT TRUNCATED AT 250 WORDS)     

Triacylglycerol and polar lipid metabolism in germinating sea buckthorn seeds

Dark germination of sea buckthorn (Hippophal rhamnoides L.) seeds was characterized by an initial 3-day-long lag-period, when the contents of triacylglycerols (TAGs) and polar lipids (PLs) remained nearly the same due to a retardation in lipid metabolization. Subsequently, TAG content declined rapidly, and by the 10th day of germination, it did not exceed 5% of total lipids. In this case, total saturated (S) and total unsaturated (U) fatty acids (FAs), as well as various TAG types such as S₂U, SU₂, and U₃, were consumed at nearly similar relative rates. At the same time, separate TAG groups, which included one of the individual FAs, such as palmitic (P), stearic (St), oleic (O), linoleic (L), or linolenic (Le), differed from each other in the intensity of degradation. For L- and Le-TAGs, initial and final concentrations were similar, while initial concentrations of St- and O-TAGs by the 10th day of germination increased 2.3- and 1.5-fold, respectively, and as regards P-TAGs, this value decreased 3.5-fold. Thus, P-TAGs considerably exceeded other TAG groups in their consumption rate in seedlings, while St- and O-TAGs ranked below them in this respect. By the 10th day, the absolute level of PLs increased 16-fold due to a de novo formation of lipid membranes of the cells in the course of growth and differentiation of seedling tissues; this increase was accompanied by an increase in the S-FA concentration in PLs and a decrease in the amount of U-FAs.     

Triacylglycerol biosynthesis in everted sacs of rat intestinal mucosa.

The molecular specificity of the biosynthesis of triacylglycerols by rat intestinal mucosa was examined by means of radioactive and mass tracers, and thin-layer chromatography with silver nitrate and gas-liquid chromatography with radioactivity monitoring. Bile salt micelles of alternately labeled monoacylglycerols and free fatty acids were incubated with everted sacs of intestinal mucosa for various periods of time and the triacylglycerols isolated by solvent extraction and thin-layer chromatography. Analyses of the molecular species of the triacylglycerols labeled from monoacylglycerols showed that the 2-monoacylglycerol pathway was responsible for the biosynthesis of a maximum of 90% and the X-1-monoacylglycerol pathway for about 10% of the total radioactive triacylglycerols. Detailed analyses of the molecular species of triacylglycerols labeled fro free fatty acids showed that the phosphatidic acid pathway contributed a minimum of 20-30% of the total labeled triacylglycerol formed. There was a preferential utilization in triacylglycerol biosynthesis of the more unsaturated diacylglycerols arising from the monoacylglycerol pathway and of the more saturated diacylglycerols originating from the phosphatidic acid pathway. The above experiments do not allow a demonstration of the utilization of the sn-2,3-diacylglycerols in triacylglycerol biosynthesis but are not inconsistent with it.     

Triacylglycerol lipase activities of cultured rat L6 myoblasts

The utilization of exogenous triacylglycerol by fusing and non-fusing rat L6 myoblasts grown in culture was investigated. Although small quantities of triacylglycerol were accumulated by both cell lines during an incubation of 2 h, no evidence could be found for the presence of lipoprotein lipase, either in the cells or released into the medium. Cell homogenate studies confirmed the absence of lipoprotein lipase but revealed the presence of an acid lipase having a pH optimum at 4.6. Acid lipase activity was mainly associated with a 15 000 g pellet and was capable of hydrolysing triolein at maximum velocity in the millimolar range. Unlike lipoprotein lipase, acid lipase was strongly inhibited by serum and preliminary investigations suggest that the inhibitory component of serum is located amongst the higher density lipoproteins. It is likely that the acid lipase is of lysosomal origin and is responsible for the hydrolysis of internalized triacylglycerol for subsequent utilization by the cell.     

Phospholipid asymmetry in rough- and smooth-endoplasmic-reticulum membranes of untreated and phenobarbital-treated rat liver.

Phospholipase C was used as a probe for the distribution of phospholipids about the membrane of rough and smooth microsomal fractions from normal and phenobarbital-treated rat liver. All membranes exhibited an asymmetric distribution, with phosphatidylethanolamine and phosphatidylserine concentrated in the inner leaflet of the bilayer and phosphatidylcholine and sphingomyelin concentrated in the outer leaflet. The only phospholipid showing a significant difference in distribution between fractions was phosphatidylcholine, which was shifted towards the outer leaflet in the smooth microsomal fraction compared with the rough microsomal fraction, and towards the outer leaflet in both rough and smooth microsomal fractions from phenobarbital-treated liver compared with the same preparations from untreated rat liver. Apart from this small change, the asymmetric distribution of phospholipids was conserved in microsomal fractions which had proliferated in response to phenobarbital and in which the protein composition had changed.     

Triacylglycerol hydrolysis by cells isolated from lactating rat mammary gland

The hydrolysis of exogenous trioleoylglycerol emulsions by suspensions of cells prepared from lactating rat mammary gland has been investigated.Cell integrity remains high throughout short (at least 30 min) incubations, during which extracellular hydrolysis of trioleoylglycerol proceeds at a mean rate (11 preparations) of 1.9 nmol oleate (and 0.6 nmol glycerol) released/min per mg protein. This hydrolysis shows partial dependence upon added serum and partial inhibition by protamine sulphate—both characteristic properties of lipoprotein lipase-catalyzed lipolysis. One or more monoacylglycerol hydrolase enzymes may also contribute to the measured lipolysis. Evidence is presented consistent with the hypothesis that a surface-located lipoprotein lipase is responsible for the observed lipolysis. Very little lipoprotein lipase activity is released from the cell surface by heparin.During trioleoylglycerol hydrolysis, non-esterified oleate does not accumulate in the cells or in the medium in quantities stoicheiometric with glycerol release. Analyses indicate that it passes into the cells without prior equilibration with the extracellular oleate pool(s). Once inside the cells, oleate is rapidly re-esterified into the triacylglycerol fraction.The possible relevance of these findings to the physiological mechanism of fatty acid uptake from triacylglycerol at the capillary endothelium is discussed.     

Triacylglycerol biosynthesis in yeast

Triacylglycerol (TAG) is the major storage component for fatty acids, and thus for energy, in eukaryotic cells. In this mini-review, we describe recent progress that has been made with the yeast Saccharomyces cerevisiae in understanding formation of TAG and its cell biological role. Formation of TAG involves the synthesis of phosphatidic acid (PA) and diacylglycerol (DAG), two key intermediates of lipid metabolism. De novo formation of PA in yeast as in other types of cells can occur either through the glycerol-3-phosphate- or dihydroxyacetone phosphate-pathways-each named after its respective precursor. PA, formed in two steps of acylation, is converted to DAG by phosphatidate phosphatase. Acylation of DAG to yield TAG is catalyzed mainly by the two yeast proteins Dga1p and Lro1p, which utilize acyl-CoA or phosphatidylcholine, respectively, as acyl donors. In addition, minor alternative routes of DAG acylation appear to exist. Endoplasmic reticulum and lipid particles (LP), the TAG storage compartment in yeast, are the major sites of TAG synthesis. The interplay of these organelles, formation of LP, and enzymatic properties of enzymes catalyzing the synthesis of PA, DAG, and TAG in yeast are discussed in this communication.     

Triacylglycerol turnover in the failing heart

No longer regarded as physiologically inert the endogenous triacylglyceride (TAG) pool within the cardiomyocyte is now recognized to play a dynamic role in metabolic regulation. Beyond static measures of content, the relative rates of interconversion among acyl intermediates are more closely linked to dynamic processes of physiological function in normal and diseased hearts, with the potential for both adaptive and maladaptive contributions. Indeed, multiple inefficiencies in cardiac metabolism have been identified in the decompensated, hypertrophied and failing heart. Among the intracellular responses to physiological, metabolic and pathological stresses, TAG plays a central role in the balance of lipid handling and signaling mechanisms. TAG dynamics are profoundly altered from normal in both diabetic and pathologically stressed hearts. More than just expansion or contraction of the stored lipid pool, the turnover rates of TAG are sensitive to and compete against other enzymatic pathways, anabolic and catabolic, for reactive acyl-CoA units. The rates of TAG synthesis and lipolysis thusly affect multiple components of cardiomyocyte function, including energy metabolism, cell signaling, and enzyme activation, as well as the regulation of gene expression in both normal and diseased states. This review examines the multiple etiologies and metabolic consequences of the failing heart and the central role of lipid storage dynamics in the pathogenic process. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.     

Inosine metabolism in the rat

1. Uptake and subsequent metabolism of purine and ribose moieties was monitored after intravenous administration of doubly labelled inosine. 2. More than 95% was cleared from the plasma within 5 min, and 99% within 20 min. 3. Approx. 50% of the 160 mumol total was rapidly incorporated into liver and kidney. Kidney removed the greatest amount (21 mumol/g wet wt.), about 10-fold more than heart, lung or liver. Lung and heart accounted for only 3%. These tissues then lost radioactivity during the remainder of the experiment. Radioactivity in the skeletal muscle, in contrast, increased from 8% of the injected dose at 5 min to 40% at 60 min. 4. In liver, kidney, heart and lung there was a significant difference in the fate of inosine. After initial incorporation of inosine, kidney predominantly lost inosine; heart preferentially lost purines; lung preferentially lost ribose radioactivity; and in liver the ribose radioactivity was rapidly lost, whereas purine was retained. Some of the ribose moiety was metabolized to glucose, presumably in the liver, and then released into the blood. Ribose radioactivity (probably as glucose) and radioactive hypoxanthine accumulated in skeletal muscle throughout the experiment. 5. Inosine caused a rapid and prolonged increase in the blood glucose content, from 6 to 15 mM in 60 min. This was accompanied by a small increase in plasma insulin. 6. It is concluded that the purine and ribose radioactivity lost from the kidney, liver and other tissues becomes incorporated into skeletal muscle.     

Triacylglycerol synthesis in cultured rat hepatocytes. The rate-limiting role of diacylglycerol acyltransferase

The limiting role of diacylglycerol acyltransferase with respect to triacylglycerol synthesis in cultured rat hepatocytes was evaluated by following the inhibition of the overall synthetic flux by 2-bromooctanoate acting as an inhibitor of the diacylglycerol acyltransferase step. The flux-control coefficient of diacylglycerol acyltransferase in intact cultured hepatocytes amounted to 0.76 in the presence of saturating glycerol and either palmitate or oleate as the fatty acyl substrates. The flux-control coefficient of diacylglycerol acyltransferase in lysolecithin-permeabilized cultured hepatocytes amounted to 0.80 and 0.99 in the presence of saturating glycerol 3-phosphate and either palmitate or oleate as the fatty acyl substrate, respectively. Hence, triacylglycerol synthesis in liver cells under the experimental conditions employed is rate-limited by the diacylglycerol acyltransferase.     

Triacylglycerol lipases of the yeast

All eukaryotes including the yeast contain a lipid storage compartment which is named lipid particle, lipid droplet or oil body. Lipids accumulating in this subcellular fraction serve as a depot of energy and building blocks for membrane lipid synthesis. In the yeast, the major storage lipids are triacylglycerols (TGs) and steryl esters (SEs). An important step in the life cycle of these non-polar lipids is their mobilization from their site of storage and channeling of their degradation components to the appropriate metabolic pathways. A key step in this mobilization process is hydrolysis of TG and SE which is accomplished by lipases and hydrolases. In this review, we describe our recent knowledge of TG lipases from the yeast based on biochemical, molecular biological and cell biological information. We report about recent findings addressing the versatile role of TG lipases in lipid metabolism, and discuss non-polar lipid homeostasis and its newly discovered links to various cell biological processes in the yeast.     

Triacylglycerol secretion in very low density lipoproteins by isolated rat liver parenchymal cells

Enzymatically isolated rat liver parenchymal cells secreted labeled triacylglycerols when incubated with [³H]glycerol or [³H]oleic acid. The presence of albumin or serum did not affect the secretion, but it was strongly inhibited by cycloheximide, colchicine, EDTA and by incubating at 4°C instead of at 37°C. Analyses of incubation media by agarose gel electrophoresis and by ultracentrifugation showed that the labeled triacylglycerols were in particles with the properties of very low density lipoproteins.     

Triacylglycerol lipase in bovine erythrocytes

Acid lipase activity was found in the bovine erythrocyte ghosts, but little neutral or alkaline lipase activity was observed in the erythrocytes. The membrane-bound lipase showed a remarkable activity in the ghosts only after hemolysis. The membrane-bound lipase showed its maximum activity at pH 4.5, 38 degrees C, and it was stable below 40 degrees C. The hydrolysis rate was linear with time up to 60 min, and was proportional to the amount of enzyme up to 0.4 mg protein. The bound lipase was activated markedly by bovine serum albumin and slightly by octyl-glycoside. The lipase was remarkably inhibited by bovine serum.     

Triacylglycerol accumulation and oxidative stress in Rhodococcus species: differential effects of pro-oxidants on lipid metabolism

In general, members of Rhodococcus genus are highly resistant to desiccation. Desiccation is a complex process which includes the formation of reactive oxygen species that results in significant damage to cells. In this study, we demonstrate that extremophile actinobacterial strains isolated from diverse environments, mainly belonging to Rhodococcus genus, exhibited high tolerance to the pro-oxidants hydrogen peroxide (H₂O₂) and methyl viologen (MV). In addition, we investigated the possible interconnections between the responses of the oleaginous Rhodococcus opacus PD630 to oxidative stress and lipid metabolism, since both processes demand a metabolic reorganization of cells. Experiments with metabolic inhibitors showed differential effects of both pro-oxidants on lipid metabolism in PD630 cells. The inhibition of carotenoid biosynthesis by the addition of diphenylamine to the media negatively affected the tolerance of cells to H₂O₂, but not to MV. The inhibition of triacylglycerol (TAG) biosynthesis and accumulation in PD630 did not affect the tolerance of cells to H₂O₂ and MV; whereas, the blockage of lipolysis decreased the tolerance of cells to H₂O₂ (but not MV) under carbon-starvation conditions. Interestingly, the addition of MV to the media (but not H₂O₂) induced a reduction of TAG accumulation by cells. Resuming, results of this study revealed metabolic connections between lipid metabolism and oxidative stress responses in R. opacus PD630, and probably in other extremophile TAG-accumulating rhodococci.