Triacylglycerol Turnover Research Articles

Triacylglycerol lipolysis is linked to sphingolipid and phospholipid metabolism of the yeast Saccharomyces cerevisiae

Previous work from our laboratory had demonstrated that deletion of TGL3 encoding the major yeast triacylglycerol (TAG) lipase resulted in decreased mobilization of TAG, a sporulation defect and a changed pattern of fatty acids, especially increased amounts of C22:0 and C26:0 very long chain fatty acids in the TAG fraction [K. Athenstaedt and G. Daum, J. Biol. Chem. 278 (2003) 23317–23323]. To study a possible link between TAG lipolysis and membrane lipid biosynthesis, we carried out metabolic labeling experiments with wild type and deletion strains bearing defects in the three major yeast TAG lipases, Tgl3p, Tgl4p and Tgl5p. Using [ 3H]inositol, [ 32P]orthophosphate, [ 3H]palmitate and [ 14C]acetate as precursors for complex lipids we demonstrated that tgl mutants had a lower level of sphingolipids and glycerophospholipids than wild type. ESI-MS/MS analyses confirmed that TAG accumulation in these mutant cells resulted in reduced amounts of phospholipids and sphingolipids. In vitro and in vivo experiments revealed that TAG lipolysis markedly affected the metabolic flux of long chain fatty acids and very long chain fatty acids required for sphingolipid and glycerophospholipid synthesis. Activity and expression level of fatty acid elongases, Elo1p and Elo2p were enhanced as a consequence of reduced TAG lipolysis. Finally, the pattern of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine molecular species was altered in tgl deletion strain underlining the important role of TAG turnover in maintaining the pool size of these compounds and the remodeling of complex membrane lipids.

Shedding light on the enigma of myocardial lipotoxicity: the involvement of known and putative regulators of fatty acid storage and mobilization

Excessive fatty acid (FA) uptake by cardiac myocytes is often associated with adverse changes in cardiac function. This is especially evident in diabetic individuals, where increased intramyocardial triacylglycerol (TG) resulting from the exposure to high levels of circulating FA has been proposed to be a major contributor to diabetic cardiomyopathy. At present, our knowledge of how the heart regulates FA storage in TG and the hydrolysis of this TG is limited. This review concentrates on what is known about TG turnover within the heart and how this is likely to be regulated by extrapolating results from other tissues. We also assess the evidence as to whether increased TG accumulation protects against FA-induced lipotoxicity through limiting the accumulations of ceramides and diacylglycerols versus whether it is a maladaptive response that contributes to cardiac dysfunction.

Identification of a novel splicing isoform of murine CGI-58

The comparative gene identification-58 (CGI-58) gene, mutations of which are linked to Chanarin-Dorfman syndrome, encodes a protein of the α/β hydrolase domain subfamily. We report here a new alternative splicing isoform of the murine CGI-58 gene, termed mCGI-58S. Sequence comparison indicates the lack of second and third exons in this cDNA variant. While the full-length protein displayed perilipin-dependent localization to lipid droplets, mCGI-58S showed a predominant cytoplasmic staining when expressed in cells. mCGI-58S was incapable of activating adipose triglyceride lipase but retained the capacity to acylate lysophosphatidic acid. Overexpression of mCGI-58S failed to promote lipid droplet turnover and loss of intracellular triacylglycerols. These results suggest that this splicing event may be involved in the regulation of lipid homeostasis.

Es-x/Ces1 prevents triacylglycerol accumulation in McArdle-RH7777 hepatocytes

Mouse esterase-x/carboxylesterase 1 (Es-x/Ces1) is a close homolog of triacylglycerol hydrolase/carboxylesterase 3 (TGH/Ces3). Es-x possesses a conserved esterase/lipase active site motif, suggesting that like TGH it could play a role in hepatic triacylglycerol (TG) metabolism. McArdle-RH7777 cells stably transfected with Es-x cDNA accumulated significantly less TG and had increased production of acid-soluble metabolites (an indicator of β-oxidation) during incubations with 0.4 mM oleic acid when compared to empty vector or TGH cDNA transfected cells. Reduction of cellular TG persisted in the presence of esterase/lipase inhibitor E600 indicating that Es-x-mediated TG lowering can be largely explained by reduced partitioning of exogenous fatty acids to TG and increased redirection to β-oxidation, rather than by increased TG turnover. Glycerol supplementation increased TG synthesis in both control and Es-x expressing cells to similar extent suggesting that Es-x expression did not reduce flux of metabolic intermediates through the glycerol-3-phosphate pathway. While Es-x expression reduced cellular TG levels, secretion of TG and apolipoprotein B remained unchanged when compared to control cells. Overall, these results suggest that Es-x limits hepatic TG accumulation by promoting β-oxidation.

Metabolic flexibility in the development of insulin resistance and type 2 diabetes: effects of lifestyle

Lipotoxicity in skeletal muscle plays a critical role in the aetiology of insulin resistance and type 2 diabetes mellitus by interference of lipid metabolites with insulin signalling and action. The dynamics of lipid oxidation and fine tuning with fatty acid uptake and intramyocellular triacylglycerol turnover may be very important to limit the accumulation of lipid intermediates. The use of metabolic inflexibility, defined as the impaired capacity to increase fat oxidation upon increased fatty acid availability and to switch between fat and glucose as the primary fuel source after a meal, does more justice to the complexity of changes in fuel oxidation during the day. Fatty acid availability, uptake and oxidation all play a role in metabolic flexibility and insulin resistance. During high fatty acid availability, fatty acid transporters may limit cellular and mitochondrial fatty acid uptake and thus limit fat oxidation. After a meal, when the demand for fatty acids as fuel is low, an increased fractional extraction of lipids from plasma may promote intramyocellular lipid accumulation and insulin resistance. Furthermore, defects in fuel switching cluster together with impaired mitochondrial content and/or function. Lifestyle changes in dietary fat intake, physical activity and weight loss may improve metabolic flexibility in skeletal muscle, and thereby contribute to the prevention of type 2 diabetes.

High resting triacylglycerol turnover of rainbow trout exceeds the energy requirements of endurance swimming

Fish may use lipoproteins instead of albumin-bound fatty acids to fuel endurance exercise, but lipoprotein kinetics have never been measured in ectotherms. In vivo bolus injections of labeled very-low-density lipoproteins ((3)H-VLDL labeled in vivo from donor fish) and continuous infusions of Intralipid (3H-labeled artificial emulsion) were used to investigate the effects of prolonged exercise (6 h at 1.5 body length/s) and heparin (600 U/kg) on the turnover rate of circulating triacylglycerol (TAG) in rainbow trout. We hypothesized that swimming would stimulate TAG turnover rate to fuel working muscles and that heparin would reduce flux by releasing lipoprotein lipase (LPL) from endothelial cells. Results from both tracer methods show that the baseline TAG turnover rate of trout ranges from 24 to 49 mumol TAG.kg(-1) x min(-1) and exceeds all values measured to date in endotherms. More important, this high resting turnover rate is not stimulated during swimming, because it can already cover several times the energy requirements of locomotion. The fact that heparin causes a 50% decrease in baseline TAG turnover rate suggests that fish LPL must be bound to the endothelium for normal tissue uptake of fatty acids supplied by lipoproteins, as in mammals. We propose that the high resting TAG turnover rate of rainbow trout could be needed by ectotherms for rapid restructuring of membrane phospholipids. The continuous tracer infusion method implemented here could be a versatile tool to investigate the potential role of lipoproteins in providing fatty acids for rapid homeoviscous adaptation.

Fatty acids derived from triacylglycerol hydrolysis are a significant source of ligands for peroxisome proliferator‐activated receptor‐α (PPAR‐α) in rat primary hepatocytes

Fatty acids (FAs), especially polyunsaturated fatty acids, have gained a lot of attention for their important roles in regulating transcription factors involved in hepatic energy metabolism. Intracellular FAs are derived from multiple sources including exogenous, de novo lipogenesis, and triacylglycerol (TAG) hydrolysis. However, little is known regarding how FAs from different sources affect activity of transcription factors such as PPAR‐α. Thus, the aim of this study was to determine if FAs generated from TAG hydrolysis influence PPAR‐α activity in rat primary hepatocytes. To manipulate TAG hydrolysis, we overexpressed adipose differentiated‐related protein (ADRP), which resulted in a 50% decrease in TAG turnover. Results from PPAR‐α reporter activity assays demonstrated that 250 μM eicosapentaenoic acid (EPA) alone or in combination with 250 μM oleic acid robustly increased PPAR‐α activity while overexpression of ADRP resulted in an inhibition of TAG hydrolysis and a decline in PPAR‐α activity. These results indicate that FAs generated from TAG hydrolysis are a significant source of ligands for PPAR‐α activation.

Adipocyte differentiation-related protein reduces the lipid droplet association of adipose triglyceride lipase and slows triacylglycerol turnover

Although neutral lipid storage droplets are ubiquitous in eukaryotic cells, very little is known about how their synthesis and turnover are controlled. Adipocyte differentiation-related protein (ADRP; also known as adipophilin) is found on the surface of lipid droplets in most mammalian cell types. To learn how ADRP affects lipid storage, we stably expressed the protein in human embryonic kidney 293 (HEK 293) cells, which express little endogenous ADRP. As expected, ADRP was targeted to the surface of lipid droplets and caused an increase in triacylglycerol (TAG) mass under both basal and oleate-supplemented conditions. At least part of the increased mass resulted from a 50% decrease in the rate of TAG hydrolysis in ADRP-expressing cells. Furthermore, ADRP expression increased the fraction of total cellular TAG that was stored in lipid droplets. ADRP expression induced a striking decrease in the association of adipose triglyceride lipase (ATGL) and mannose-6-phosphate receptor tail-interacting protein of 47 kDa with lipid droplets and also decreased the lipid droplet association of several other unknown proteins. Transient expression of ADRP in two other cell lines also reduced the lipid droplet association of catalytically inactive ATGL. We conclude that the reduced lipid droplet association of ATGL and/or other lipases may explain the decrease in TAG turnover observed in ADRP-expressing HEK 293 cells.

A single bout of brisk walking increases basal very low-density lipoprotein triacylglycerol clearance in young men

Very low-density lipoprotein triacylglycerol (VLDL-TG) turnover rate was evaluated in the morning, 12 hours after a single bout of brisk walking (90 minutes at approximately 60% of V̇ o 2max; EXE), compared to a resting control period (CON) in 10 recreationally active men. VLDL-TG fractional catabolic rate was calculated from the decay in isotopic enrichment after a bolus injection of [ 2H 5]glycerol. Plasma VLDL-TG concentration was 24% lower in the morning after the EXE trial compared to control (0.47 ± 0.04 and 0.36 ± 0.04 mmol L −1, for CON and EXE, respectively; P < .01). Serum insulin (7.4 ± 0.7 and 5.6 ± 0.4 mIU L −1, CON and EXE, respectively; P < .05) and plasma glucose (5.6 ± 0.1 and 5.4 ± 0.1 mmol/L, CON and EXE, respectively; P < .05) concentrations were also significantly lower in the EXE trial. Insulin sensitivity (homeostasis model assessment [HOMA] index) was improved by 27% in EXE compared with the CON trial ( P < .05).VLDL–apolipoprotein B-100 and plasma fatty acid concentrations were similar in the two trials. Hepatic VLDL-TG secretion rates were not significantly affected by exercise (13.1 ± 1.2 and 13.2 ± 1.6 μmol .min −1 for the CON and EXE trials, respectively), whereas VLDL-TG clearance rate increased by 36% (28.1 ± 1.3 and 38.1 ± 3.5 mL .min −1 for the CON and EXE trials, respectively; P < .05). It is concluded that the decrease in fasting plasma VLDL-TG concentration observed 12 hours after brisk walking is related mainly to increased VLDL-TG clearance from plasma.

Adipocyte differentiation‐related protein reduces lipid droplet association of adipose triglyceride lipase and slows triacylglycerol turnover

Despite the ubiquity of neutral lipid storage droplets and their importance in obesity, very little is known about how their synthesis and turnover are controlled. A major candidate for regulating these processes is adipocyte differentiation-related protein (ADRP; also known as adipophilin). To determine the mechanism through which ADRP affects lipid storage, we stably expressed the protein in HEK 293 cells. ADRP was targeted to the surface of lipid droplets in 293 cells and increased triacylglycerol (TAG) mass under both unsupplemented and oleate-supplemented conditions. At least part of the increased mass resulted from a 2-fold decrease in the rate of TAG hydrolysis in ADRP-expressing cells. Furthermore, ADRP expression induced a significant decrease in association of adipose triglyceride lipase (ATGL) with lipid droplets. Reduced lipid droplet association of ATGL and/or other lipases may explain the decrease in TAG turnover observed in ADRP-expressing cells. This work was supported by a National Institutes of Health (NIH) Ruth L. Kirschstein National Research Service Award (1 F32 GM074453-01, to L. L. L.), and NIH grant GM47897 (to D. A. B.).

Accelerated triacylglycerol turnover kinetics in hearts of diabetic rats include evidence for compartmented lipid storage

Triacylglycerol (TAG) storage and turnover rates in the intact, beating rat heart were determined for the first time using dynamic mode (13)C- NMR spectroscopy to elucidate profound differences between hearts from diabetic rats (DR, streptozotocin treatment) and normal rats (NR). The incorporation of [2,4,6,8,10,12,14,16-(13)C(8)]palmitate into the TAG pool was monitored in isolated hearts perfused with physiological (0.5 mM palmitate, 5 mM glucose) and elevated substrate levels (1.2 mM palmitate, 11 mM glucose) characteristic of the diabetic condition. Surprisingly, although the normal hearts were enriched at a near-linear profile for >or=2 h before exponential characterization, exponential enrichment of TAG in diabetic hearts reached steady state after only 45 min. Consequently, TAG turnover rate was determined by fitting an exponential model to enrichment data rather than conventional two-point linear analysis. In the high-substrate group, both turnover rate (DR 820+/- 330, NR 190 +/-150 nmol.min(-1).g(-1) dry wt; P< 0.001) and [TAG] content (DR 78 +/-10, NR 32+/- 4 micromol/g dry wt; P< 0.001) were greater in the diabetic group. At lower substrate concentrations, turnover was greater in diabetics (DR 530+/-300, NR 160+/- 30; P<0.05). However, this could not be explained by simple mass action, because [TAG] content was similar between groups [DR 34+/- 7, NR 39+/- 9 micromol/g dry wt; not significant (NS)]. Consistent with exponential enrichment data, (13)C fractional enrichment of TAG was lower in diabetics (low- substrate groups: DR 4+/-1%, NR 10+/- 4%, P<0.05; high-substrate groups: DR 8+/- 3%, NR 14+/- 9%, NS), thereby supporting earlier speculation that TAG is compartmentalized in the diabetic heart.

Differences in labelled triolein turnover after oral administration between liver and adipose tissue of rats

To investigate exogenous triacylglycerol turnover, the time courses for labelled triolein in the liver, plasma and epididymal adipose tissue (adipose tissue) after oral administration to rats fed a fat-free or 10 % corn oil diet for 3 d after fasting overnight were examined for 10 d. After the administration of labelled triolein to rats fed the fat-free diet, the incorporation (dpm/g) into total lipids of the liver and adipose tissue each reached the maximum in 8 h and was seven times higher in the adipose tissue than in the liver. The half-lives of total lipid radioactivities during the decreasing phases were 0.39 and 2.58 d, respectively, in the rapid and slow phases of the decay curve in the liver, and 4.78 d in only one phase of the adipose tissue. Radioactivity after administration of labelled triolein was mostly found in the oleic acid in the tissues. The half-life of oleic acid was 3.92 d in the adipose tissues. These half-lives were similar in both dietary groups. Thus, although dietary corn oil reduced the triolein incorporation to cellular lipids in comparison to the fat-free diet, it did not affect these half-lives. The labelled triacylglycerol-oleic acid stayed abundantly intact for a long time in the adipose tissue and was scarcely changed to other fatty acids, whereas it was slightly incorporated into total lipids and quickly metabolized in the liver. Non-essential fatty acids may be mostly endogenous in the liver but may be exogenous and endogenous in adipose tissue.

High triacylglycerol turnover rate in human skeletal muscle

In the present study we investigated the relationship between plasma fatty acids (FA) and intramuscular triacylglycerol (IMTAG) kinetics of healthy volunteers. With this aim [U-(13)C]-palmitate was infused for 10 h and FA kinetics determined across the leg. In addition, the rate of FA incorporation into IMTAG in vastus lateralis muscle was determined during two consecutive 4-h periods (2-6 h and 6-10 h). Fifty to sixty per cent of the FA taken up from the circulation were esterified into IMTAG, whereas 32 and 42% were oxidized between 2-6 and 6-10 h, respectively. IMTAG fractional synthesis rate was 3.4 +/- 0.8% h(-1) and did not change between the two 4- h periods, despite an increase in arterial FA concentration (34%, P < 0.01). IMTAG concentration was also unchanged, implying that the IMTAG fractional synthesis rate was balanced by an equal rate of breakdown. FA oxidation increased over time, which could be due to the observed decline in plasma insulin concentration (-74%, P < 0.01). In conclusion, a substantial fraction of the fatty acids entering skeletal muscle in post-absorptive healthy individuals is esterified into IMTAG, due to its high turnover rate (29 h pool(-1)). An increase in FA level, as a consequence of short-term fasting, does not seem to increase IMTAG synthesis rate and pool size.

Development of leptin resistance in rat soleus muscle in response to high-fat diets.

Direct evidence for leptin resistance in peripheral tissues such as skeletal muscle does not exist. Therefore, we investigated the effects of different high-fat diets on lipid metabolism in isolated rat soleus muscle and specifically explored whether leptin's stimulatory effects on muscle lipid metabolism would be reduced after exposure to high-fat diets. Control (Cont, 12% kcal fat) and high-fat [60% kcal safflower oil (n-6) (HF-Saff); 48% kcal safflower oil plus 12% fish oil (n-3)] diets were fed to rats for 4 wk. After the dietary treatments, muscle lipid turnover and oxidation in the presence and absence of leptin was measured using pulse-chase procedures in incubated resting soleus muscle. In the absence of leptin, phospholipid, diacylglycerol, and triacylglycerol (TG) turnover were unaffected by the high-fat diets, but exogenous palmitate oxidation was significantly increased in the HF-Saff group. In Cont rats, leptin increased exogenous palmitate oxidation (21.4 +/- 5.7 vs. 11.9 +/- 1.61 nmol/g, P = 0.019) and TG breakdown (39.8 +/- 5.6 vs. 27.0 +/- 5.2 nmol/g, P = 0.043) and decreased TG esterification (132.5 +/- 14.6 vs. 177.7 +/- 29.6 nmol/g, P = 0.043). However, in both high-fat groups, the stimulatory effect of leptin on muscle lipid oxidation and hydrolysis was eliminated. Partial substitution of fish oil resulted only in the restoration of leptin's inhibition of TG esterification. Thus we hypothesize that, during the development of obesity, skeletal muscle becomes resistant to the effects of leptin, resulting in the accumulation of intramuscular TG. This may be an important initiating step in the development of insulin resistance common in obesity.

Inactivation of microsomal triglyceride transfer protein impairs the normal redistribution but not the turnover of newly synthesized glycerolipid in the cytosol, endoplasmic reticulum and Golgi of primary rat hepatocytes

The requirements for microsomal triglyceride transfer protein (MTP) during the turnover and transfer of glycerolipids from intracellular compartments into secretory very low-density lipoprotein (VLDL) were studied by pre-labelling lipids with [ 3H]glycerol and [ 14C]oleate in primary cultures of rat hepatocytes. The intracellular redistribution of pre-labelled glycerolipids was then compared at the end of subsequent chase periods during which the MTP inhibitor BMS-200150 was either present or absent in the medium. Inhibition of MTP resulted in a decreased output of VLDL triacylglycerol (TAG) and a delayed removal of labelled TAG from the cytosol and from the membranes of the smooth endoplasmic reticulum (SER), the cis- and the trans-Golgi. Inactivation of MTP did not decrease the bulk lipolytic turnover of cellular TAG as reflected by changes in its [ 3H]glycerol:[ 14C]oleate ratios. However, a larger proportion of the resultant TAG fatty acids was re-esterified and remained with the membranes of the various subcellular fractions rather than emerging as VLDL. The effects of BMS-200150 on the pattern of phospholipid (PL) mechanism and redistribution suggested that inhibition of MTP prevented the normal lipolytic transfer of PL-derived fatty acids out of the SER, cis- and trans-Golgi membrane pools. Finally, changes in the 14C specific radioactivities of the cytosolic and membrane pools of TAG suggested that inhibition of MTP prevented a normal influx of relatively unlabelled fatty acids into these pools during the chase period.

Glucose Phosphorylation Is Essential for the Turnover of Neutral Lipid and the Second Stage Assembly of Triacylglycerol-Rich ApoB-Containing Lipoproteins in Primary Hepatocyte Cultures

Primary hepatocytes cultured in a medium supplemented with amino acids and lipogenic substrates responded to increased extracellular glucose by increasing the secretion of VLDL apoB. This effect was accompanied by an increased secretion of VLDL triacylglycerol (TAG) derived from endogenous stores. Glucose also stimulated intracellular TAG mobilization via the TAG lipolysis/esterification cycle. All these effects were abolished in the presence of mannoheptulose (MH), an inhibitor of glucose phosphorylation. Glucose also gave rise to a modest (50% to 60%) increase in the incorporation of 35S methionine into newly synthesized apoB (P<0.05) and to a doubling of newly-synthesized apoB secretion as VLDL (P<0. 05). The magnitude of these effects was similar for apoB-48 and for apoB-100. MH inhibited apoB-48 and apoB-100 synthesis and VLDL secretion at all glucose concentrations. The effects of glucose and MH on the secretion of newly-synthesized apoB-48 or apoB-100 as small dense particles were less pronounced. Glucose had no effects on the posttranslational degradation of newly-synthesized apoB-100 or apoB-48. However, this process was significantly enhanced by MH. The results suggest that glucose stimulates TAG synthesis, turnover, and output as VLDL. These effects are associated with an increased VLDL output of apoB mediated mainly by an increase in the net synthesis of both apoB-48 and apoB-100. All these changes are prevented by interference with glucose phosphorylation. Output of small, dense, apoB-containing particles is relatively unaffected by the glucose and MH-induced changes in TAG synthesis and lipolysis, an observation which suggests that only the bulk lipid addition step of VLDL assembly is affected by changes in glucose metabolism.

Moderate protein restriction during pregnancy modifies the regulation of triacylglycerol turnover and leads to dysregulation of insulin’s anti-lipolytic action

Moderate protein restriction throughout pregnancy in the rat leads to relative hyperlipidaemia and blunted insulin responsiveness of lipid fuel supply, and impairs foetal growth. The present study examined the basis for these changes. Isocaloric 8% (vs 20%) protein diets were provided throughout pregnancy. Rats were sampled at 19–20 days of gestation. Protein restriction enhanced triacylglycerol (TAG) secretion rates (estimated using Triton WR 1339) 1.6-fold ( P<0.05) in the post-absorptive state. Insulin infusion (4.2 mU/kg per min) decreased plasma TAG concentrations by 33% ( P<0.05) and 48% ( P<0.05) in control (C) and protein-restricted (PR) pregnant groups, an effect associated with suppression of TAG secretion by 42% ( P<0.05) and 51% ( P<0.01) respectively, in the C and PR groups. Since TAG concentrations decline more rapidly, while TAG secretion is enhanced, TAG utilisation during hyperinsulinaemia is enhanced in the PR group. We evaluated whether these changes were associated with dysregulation of lipolysis using adipocytes from two abdominal depots (mesenteric and parametrial). Noradrenaline-stimulated glycerol release was enhanced in parametrial adipocytes (by 40%; P<0.05) from PR pregnant rats. The anti-lipolytic action of insulin at low concentrations (≤15 μU/ml) was impaired by protein restriction (adipocytes from both depots). There was no evidence for altered intra-hepatic regulation of fatty acid (FA) disposal at the level of carnitine palmitoyltransferase. Our results demonstrate increased post-absorptive production of non-carbohydrate energy substrates (TAG and FA) as a consequence of mild protein restriction during pregnancy. These adaptations contribute to a homeostatic strategy to reduce the maternal requirement for gluconeogenesis from available amino acids, optimising the foetal protein supply. Protein restriction also enhances TAG turnover during hyperinsulinaemia. This effect is not a consequence of abnormal regulation of hepatic lipid metabolism by insulin.

Decreased secretion of very-low-density lipoprotein triacylglycerol and apolipoprotein B is associated with decreased intracellular triacylglycerol lipolysis in hepatocytes derived from rats fed orotic acid or n-3 fatty acids.

Hepatocytes from rats fed a chow (control) diet or from rats fed a chow diet supplemented with either orotic acid (OA; 1%, w/w) or fish oil (FO; 20%, v/w) were maintained in culture for periods up to 48 h. during the first 24 h period, the low rates of output of very-low-density lipoprotein (VLDL)-associated triacylglycerol (TAG) and apolipoprotein B (apoB) in hepatocytes from the FO- and OA-fed animals were associated with significantly lower rates of intracellular TAG lipolysis and re-esterification. Most of the VLDL TAG secreted was mobilized via lipolysis of the intracellular TAG pool, but the proportion of VLDL TAG secreted via this route in cells from the FO-fed and OA-fed animals was decreased compared with that in the control-fed animals' cells. In the presence of exogenous oleate the inhibitory effect of OA feeding on VLDL apoB and TAG secretion persisted in the derived hepatocytes for up to 48 h following isolation. However, when oleate was absent no inhibitory effect on the secretion of TAG and apoB was observed between 24 and 48 h. Under these conditions the rate of intracellular TAG turnover returned to normal. The initial inhibitory effect of FO feeding on VLDL TAG and apoB secretion did not persist in the derived hepatocytes between 24 h and 48 h of culture in the presence of exogenous oleate. Although intracellular TAG lipolysis and VLDL TAG and apoB secretion rates appear to be positively correlated, a causal relationship has not been conclusively established.

The Turnover of Cytoplasmic Triacylglycerols in Human Fibroblasts Involves Two Separate Acyl Chain Length-dependent Degradation Pathways

Cultured fibroblasts from patients affected with the genetic metabolic disorder named neutral lipid storage disease (NLSD) exhibit a dramatic accumulation of cytoplasmic triacylglycerols (Radom, J., Salvayre, R., Nègre, A., Maret, A., and Douste-Blazy, L. (1987) Eur. J. Biochem. 164, 703-708). We compared here the metabolism of radiolabeled short-, medium- and long-chain fatty acids in these cells. Short/medium-chain fatty acids (C4-C10) were incorporated into polar lipids (60-80%) and triacylglycerols (20-40%) at a lower rate (5-10 times lower) than long-chain fatty acids. Pulse-chase experiments allowed to evaluate the degradation rate of cytoplasmic triacylglycerols in normal and NLSD fibroblasts and to discriminate between two catabolic pathways of cytoplasmic triacylglycerols. Short/medium-chain (C4-C10) triacylglycerols were degraded at a normal rate in NLSD fibroblasts, whereas long-chain (C12 and longer) triacylglycerols remained undegraded. These data are confirmed by mass analysis. The use of diethylparanitrophenyl phosphate (E600) and parachloromercuribenzoate (PCMB) inhibitors allows to discriminate between the two triacylglycerol degradation pathways. E600 inhibited selectively the in situ degradation of short/medium-chain triacylglycerols without inhibition of the degradation of long-chain triacylglycerols, whereas PCMB inhibited selectively the in situ hydrolysis of long-chain triacylglycerols without affecting the degradation of long-chain triacylglycerols. This was correlated with the in vitro properties of cellular triacylglycerol-hydrolyzing enzymes characterized by their substrate specificity and their susceptibility to inhibitors; the neutral lipase specific to long-chain triacylglycerols is inhibited by PCMB, but not by E600, in contrast to short/medium-chain lipase, which is inhibited by E600 but not by PCMB. The data of in vitro and in situ experiments suggest the existence in fibroblasts of two separate acyl chain length-dependent pathways involved in the degradation of cytoplasmic triacylglycerols, one mediated by a neutral long-chain lipase and another one mediated by a short/medium-chain lipase.

Triacylglycerol turnover in isolated working hearts of acutely diabetic rats

Although myocardial triacylglycerol may be a potentially important source of fatty acids for beta-oxidation in diabetes, few studies have measured triacylglycerol turnover directly in hearts from diabetic animals. In this study, myocardial triacylglycerol turnover was directly measured in isolated working hearts from streptozotocin-induced acutely diabetic rats. Hearts were initially perfused in the presence of 1.2 mM [14C]palmitate and 11 mM glucose for 1 h (pulse) to label the endogenous lipid pools, followed by a 10-min washout perfusion. Hearts were then perfused for another hour (chase) with buffer containing 11 mM glucose +/- 1.2 mM [3H]palmitate. During the chase, both 14CO2 and 3H2O production (measures of endogenous and exogenous fatty acid oxidation, respectively) were determined. A second series of hearts were perfused using the same protocol, except that unlabeled palmitate was used during the pulse and 11 mM [14C(U),5-3H]glucose +/- unlabeled palmitate was present during the chase. Both glycolysis (3H2O production) and glucose oxidation (14CO2 production) rates were measured in this series. Myocardial triacylglycerol levels were significantly higher in the diabetic rat hearts (77.5 +/- 4.6 vs. 33.7 +/- 4.1 mumol fatty acid/g dry mass in control hearts). In diabetic rat hearts chased with 1.2 mM palmitate, triacylglycerol lipolysis was increased, although endogenous [14C]palmitate oxidation rates were similar to control hearts and contributed 10.1% of overall ATP production. The majority of fatty acids derived from triacylglycerol lipolysis were released into the perfusate. In the absence of palmitate, both triacylglycerol lipolysis and endogenous [14C]palmitate oxidation rates were significantly increased in diabetic rat hearts, compared with control.(ABSTRACT TRUNCATED AT 250 WORDS)