Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism最新文献

To further test the hypothesis that newly synthesized cholesteryl esters regulate hepatic apolipoprotein B (apoB) secretion into plasma, apoB kinetic studies were carried out in seven control miniature pigs and in seven animals after 21 days intravenous administration of the acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor DuP 128 (2.2 mg/kg/day). Pigs were fed a fat (34% of calories; polyunsaturated/monounsaturated/saturated ratio, 1:1:1) and cholesterol (400 mg/day; 0.1%; 0.2 mg/kcal) containing pig chow based diet. DuP 128 significantly reduced total plasma triglyceride and very low density lipoprotein (VLDL) triglyceride concentrations by 36 and 31%, respectively (P<0.05). Autologous ¹³¹I-VLDL and ¹²⁵I-LDL were injected simultaneously into each pig and apoB kinetic data was analyzed using multicompartmental analysis (SAAM II). The VLDL apoB pool size decreased by 26% (0.443 vs. 0.599 mg/kg; P<0.001) which was due entirely to a 28% reduction in VLDL apoB production or secretion rate (1.831 vs. 2.548 mg/kg/h; P=0.006). The fractional catabolic rate (FCR) for VLDL apoB was unchanged. The LDL apoB pool size and production rate were unaffected by DuP 128 treatment. Hepatic microsomal ACAT activity decreased by 51% (0.44 vs. 0.90 nmol/min/mg; P<0.001). Although an increase in hepatic free cholesterol and subsequent decrease in both LDL receptor expression and LDL apoB FCR might be expected, this did not occur. The concentration of hepatic free cholesterol decreased 12% (P=0.008) and the LDL apoB FCR were unaffected by DuP 128 treatment. In addition, DuP 128 treatment did not alter the concentration of hepatic triglyceride or the activity of diacylglycerol acyltransferase, indicating a lack of effect of DuP 128 on hepatic triglyceride metabolism. In our previous studies, DuP 128 treatment of miniature pigs fed a low fat, cholesterol free diet, decreased VLDL apoB secretion by 65% resulting in a reduction in plasma apoB of 60%. We conclude that in miniature pigs fed a high fat, cholesterol containing diet, the inhibition of hepatic cholesteryl ester synthesis by DuP 128 decreases apoB secretion into plasma, but the effect is attenuated relative to a low fat, cholesterol free diet.

The present study investigates the effects of various glutathione (GSH) depleting agents on sn-glycerol-3-phosphate acyltransferase (GPAT) activity, the first committed step in adipose triacylglycerol formation. GPAT activity was measured in the presence of [¹⁴C]glycerol-3-phosphate and palmitoyl-CoA, using different subcellular fractions. Glutathione deficiency in animals was induced in the presence of diethylmaleate (DEM) or buthionine sulfoximine. In this respect, DEM (1.75 mmoles/kg) was more effective and caused over 75% decrease in GPAT activity within 4 h of DEM administration. Further studies indicated that this decrease in GPAT activity was mainly related to the microsomal form of GPAT, without any significant effect on mitochondrial GPAT activity. Adipocytes incubated with 2.5 mm DEM for 1 h at 37°C also showed a reduction in the adipocyte glutathione content, which was accompanied by decreases in GPAT activity. The effect of DEM on adipocyte GPAT activity was partially reversible in the presence of cell permeable glutathione ethyl ester. Preincubation of adipose tissue homogenates with 2.5 mM DEM at 30°C for 45 min also showed a significant loss of the GPAT activity. The presence of 5 mM dithiothreitol in the preincubation mixture offered a significant protection of the GPAT activity against DEM. However, glutathione was ineffective in this respect as it interfered with the utilization of palmitoyl-CoA in the GPAT assay. Therefore, on the basis of these three different approaches, the present studies suggest that the thiol environment offered by glutathione (in vivo and in vitro studies) or dithiothreitol (in a cell-free system) is critical for the maintenance of GPAT activity.

Stimulus-induced release of polyunsaturated fatty acids from membranes has been proposed to couple the processes of stimulus perception and oxylipin synthesis in the octadecanoid signaling pathway. This study investigated wound-induced changes in free fatty acids, diacylglycerol, and phospholipids at the site of wounding and at an unwounded area of the same wounded leaf in castor bean (Ricinus communis L.). Increases in free fatty acids and diacylglycerol and decreases in phospholipids were relatively large and continuous at the site of wounding. The changes at the unwounded area were selective and transient, suggesting a regulated activation of lipid turnover in response to wounding. In unwounded cells, the free fatty acids that increased in the early phase of wounding were linolenate and linoleate, which peaked within 5 min after wounding. Diacylglycerols that increased in unwounded cells were the species containing linolenate and linoleate, not those with oleate and stearate. Within 5 min of wounding, the levels of phosphatidylcholine and phosphatidylglycerol, but not other phospholipids, decreased in unwounded cells. These results provide evidence for the wound-induced selective increase in linolenate and linoleate in unwounded cells. The varied susceptibility of different phospholipids to hydrolysis after wounding indicates that phosphatidylcholine and phosphatidylglycerol may serve as substrates that lead to the increase in linolenate and linoleate in the early phase of wound response. The pattern of increases in polyunsaturated fatty acids, diacylglycerol, and phosphatidic acid and of decreases in phospholipids suggests the activation of a PLD-initiated signaling pathway in response to wounding in castor bean.

The effect of dietary linoleic (C18:2n-6) and palmitic acids (C16:0) on rate of hepatic de novo fatty acid synthesis was assessed in normal subjects. The diet was formulated to provide combinations of high and low levels of C18:2n-6 and C16:0. After 21 days of diet treatment, plasma triacylglycerol level and incorporation of deuterium into the plasma very low density lipoprotein triacylglycerol (VLDL-TG) pool over 24 hours was measured. Plasma triacylglycerol levels were within the normal range. Increasing dietary intake of linoleic acid decreased plasma triacylglycerol level when subjects consumed a low level of dietary palmitic acid. The relative and net amount of de novo synthesized fatty acid in the plasma VLDL-TG pool was not influenced by the diet treatments. A relationship between plasma triacylglycerol level and rate of hepatic de novo fatty acid synthesis was observed.

Oxidation of straight-chain fatty acids in mitochondria involves the complicated interaction between a large variety of different enzymes. So far four different mitochondrial straight-chain acyl-CoA dehydrogenases have been identified. The physiological function of three of the four acyl-CoA dehydrogenases has been resolved in recent years especially from studies on patients suffering from certain inborn errors of mitochondrial fatty acid β-oxidation. The physiological role of long-chain acyl-CoA dehydrogenase (LCAD) has remained obscure, however. The results described in this paper provide strong evidence suggesting that LCAD plays a central role in branched-chain fatty acid metabolism since it turns out to be the major acyl-CoA dehydrogenase reacting with 2,6-dimethylheptanoyl-CoA, a metabolite of pristanic acid, which itself is the α-oxidation product of phytanic acid.

We previously reported a transient increase in plasma lipoprotein(a) (Lp(a)) concentrations following acute myocardial infarction and surgical operations, and demonstrated Lp(a) accumulation in healing tissues. In the present study, the stimulatory effect of Lp(a) on migration and proliferation of human umbilical vein endothelial cells (HUVEC) was assessed by quantitative assay methods and compared it with that of LDL. Lp(a) stimulated both migration and proliferation of HUVEC in a dose-dependent manner and the stimulatory activities for migration and proliferation were two times higher than those of LDL in terms of moles of apoB. In addition, this stimulatory activity of Lp(a) was not affected by the difference of Lp(a) phenotype. Although each neutralizing antibody to hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF) and interleukin-1β (IL-1β) had no further effect on migration and proliferation of HUVEC treated with Lp(a), only antibody to fibroblast growth factor-2 (FGF-2) partially suppressed them. Moreover, pertussis toxin, which inhibits FGF-2-stimulated endothelial cell movement, also partially suppressed Lp(a)-induced HUVEC migration. FGF-2 concentrations in the medium of HUVEC treated with Lp(a) were constant in spite of the increase in FGF-2 mRNA levels in HUVEC. Taken together, it is suggest that Lp(a) stimulates HUVEC migration and proliferation, which is mediated, at least in part, by FGF-2 and may promote the angiogenesis during wound healing.

Triacylglycerol (TG) biosynthetic enzymes were characterized in subcellular fractions of an oleaginous fungus, Mortierella ramanniana var. angulispora. When the membrane or lipid body fraction of this fungus was incubated with [¹⁴C]oleoyl-CoA without adding exogenous acyl acceptors, radioactivity was incorporated predominantly into TG, indicating that diacylglycerol acyltransferase (DGAT) used endogenous diacylglycerol to incorporate [¹⁴C]oleoyl-CoA into TG. Adding glycerol 3-phosphate or lysophosphatidic acid increased radiolabeled phosphatidic acid (PA) in the membrane fraction, which reflected the presence of glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT). Label accumulation did not occur in lysophosphatidic acid when glycerol 3-phosphate was added, suggesting that GPAT was rate-limiting in sequential acylation. In the lipid body fraction, adding lysophosphatidic acid similarly increased radiolabeled PA, whereas adding glycerol 3-phosphate caused much lower increase in radiolabeled PA. Quantitative assays for GPAT, LPAAT, phosphatidic acid phosphatase (PAP), and DGAT essentially confirmed the results obtained from [1-¹⁴C]oleoyl-CoA incorporation; LPAAT had the highest activity in the membrane and lipid body fractions, GPAT was significantly lower in the lipid body fraction, and DGAT was much higher in the lipid body fraction. GPAT and LPAAT in the membrane fraction had a strong preference toward oleoyl-CoA as a substrate over palmitoyl-CoA. Results indicate that TG biosynthetic enzymes had different subcellular distribution with the sequence of enrichment in the lipid body fraction, i.e., GPAT<LPAAT≈PAP<DGAT. This may reflect a TG biosynthetic process from endoplasmic reticulum membranes to lipid bodies in the fungus.

Dihydroxyacetonephosphate acyltransferase (DHAP-acyltransferase) and alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase) are the first two enzymes involved in the biosynthesis of ether phospholipids. Both peroxisomal enzymes have recently been purified to homogeneity and their molecular weights under denaturing conditions were reported. To determine the in situ functional size of both enzymes, radiation inactivation experiments were performed. Alkyl-DHAP synthase showed single exponential decays, both when enzymatic activity and when immunoreactive protein levels were measured, from which target sizes of 79±2 kDa and 78±4 kDa, respectively, were calculated. DHAP-acyltransferase activity increased at lower doses and decayed upon further irradiation with an apparent target size of 62±7 kDa. We conclude from these data that the functional unit sizes for both enzymes in situ are represented by their single polypeptide chains.

Complementary DNAs homologous to a rat 42-kDa choline/ethanolamine kinase [C. Aoyama et al., Biochim. Biophys. Acta 1390 (1998) 1–7] and to a 50-kDa choline kinase [T. Uchida and S. Yamashita, J. Biol. Chem. 267 (1992) 10156–10162] were isolated from a 17-day post coitum mouse embryo cDNA library and their sequences were compared with the two murine species, respectively. The nucleotide sequence homology (within the coding sequence) between mouse and rat 50-kDa choline kinases (96.0%) was considerably higher than that between their 42-kDa choline/ethanolamine kinases (92.4%). Northern blot and RT-PCR studies on several rat tissues demonstrated that both isozymes are expressed ubiquitously with the highest level in testis.

A glycosyldiacylglycerol was isolated from the marine bacterium Deleya marina (ATCC 25374). The structure was determined, mainly by spectral data, to be 1,2-diacyl-3-O-[α-2-amino-2-deoxy-glucopyranose-(1→4)-O-α-iduronopyranuronic acid]-glycerol. This is, to our knowledge, the first isolation of diglycosyldiacylglycerol containing both iduronopyranuronic acid and 2-amino-2-deoxy-glucopyranose from Gram-negative bacteria.