Enzymology of long-chain base synthesis by aorta: induction of serine palmitoyltransferase activity in rabbit aorta during atherogenesis

Serine palmitoyltransferase [EC 2.3.1.50] initiates the biosynthesis of sphingolipids by catalyzing the condensation of a fatty acyl-CoA with serine to yield the committed intermediate 3-ketosphinganine or one of its homologues. The presence of serine palmitoyltransferase in aorta was established under optimal assay conditions using microsomes from New Zealand White rabbits. Its activity was dependent on microsomal protein, L-serine, pyridoxal 5'-phosphate, and palmitoyl-CoA. Although several different saturated and unsaturated fatty acyl-CoA thioesters were utilized as substrates, maximal activity was with palmitoyl-CoA, suggesting that this enzyme contributes to the predominance of 18-carbon long-chain bases in sphingolipids from aorta. Rabbits, fed a Purina lab chow supplemented with 2% cholesterol, were used to study serine palmitoyltransferase activity in aorta during experimental atherogenesis. An increase in activity from intimal-medial preparations was detectable prior to prominent lipid accumulation or cellular proliferation. Activity continued to elevate over the 12-week duration of feeding concurrent with the increase in serum cholesterol and in proportion to the development of plaques resulting in a 3.7-fold increase in activity (20.7 +/- 2.6 pmol per min per mg microsomal protein +/- SE in the cholesterol-fed group versus 5.6 +/- 1.9 in the pair-fed controls also matched for age and sex; P less than 0.005). Thus, the accumulation of sphingomyelin that occurs in aorta during experimental atherogenesis may be related to increased long-chain base synthesis by serine palmitoyltransferase.     

Cholesterol feeding impairs endothelium-dependent relaxation of rabbit aorta

Experiments were designed to assess the effect of cholesterol feeding on the endothelium-mediated relaxation of the rabbit aorta to acetylcholine. Age-matched male New Zealand white rabbits were fed either a 2% cholesterol diet or standard rabbit chow. The animals were anaesthetized with sodium pentobarbitone and sacrificed after 4 and 8 weeks on these diets. Rings were prepared from the proximal thoracic aorta and examined in tissue baths. These rings were contracted first with norepinephrine (-6 log mol/L) and acetylcholine was added to demonstrate the endothelium-mediated relaxation. The endothelium-dependent relaxation was significantly less in aortas from rabbits fed the 2% cholesterol diet than in aortas from animals fed the conventional diet. This impairment of relaxation was apparent after both 4 and 8 weeks of cholesterol feeding. In both groups of animals no relaxation was seen in rings from which the endothelium was removed. These results show that cholesterol feeding leads to an impairment of endothelium-mediated relaxation of the rabbit aorta to acetylcholine.     

Activities of serine palmitoyltransferase (3-ketosphinganine synthase) in microsomes from different rat tissues

Serine palmitoyltransferase [EC 2.3.1.50] catalyzes the first unique reaction of sphingolipid biosynthesis. To determine whether or not different rat tissues are capable of initiating this pathway, its activity was determined for microsomes from rat liver, lung, brain, kidney, intestine, spleen, muscle, heart, pancreas, testes, ovary, and stomach. Serine palmitoyltransferase was found in every tissue, and, when compared to the microsomal glycerol 3-phosphate acyltransferase, the activities correlated directly with their sphingomyelin levels as a percentage of total phospholipids. This suggests that the activities were comparable to expected cellular needs for long-chain bases, if the initial enzymes of glycerolipid and sphingolipid biosynthesis influence the phospholipid composition of cells by determining the relative partitioning of fatty acyl-CoA's toward these two lipid classes. Serine palmitoyltransferase activities were also determined using different fatty acyl-CoA's and were consistently greatest with CoA thioesters of saturated fatty acids with 16 +/- 1 carbon atoms. This suggests that the predominance of 18-carbon long-chain bases in vivo is due to the higher activity of this enzyme with palmitoyl-CoA. Together, these findings indicate a role for serine palmitoyltransferase in regulating both the type and amount of long-chain bases found in tissues.     

Upregulation of LOX-1 expression in aorta of hypercholesterolemic rabbits: modulation by losartan

Angiotensin-II (Ang-II) enhances the modification of LDL and the expression of its lectin-like receptor (LOX-1) by activating type 1 (AT(1)) receptors. This study was designed to determine the effect of hypercholesterolemia on LOX-1 expression in aorta and its modulation by the AT(1) receptor blocker losartan. Male New Zealand White rabbits were fed regular chow (Control group), chow with 1% cholesterol and 4% peanut oil (HC-diet group), or 1% cholesterol and 4% peanut oil diet plus losartan (25 mg/kg/day) (Losartan + HC-diet group) for 10 weeks. Animal body weight, serum cholesterol levels, and arterial blood pressure were measured. Aortic intimal thickening was quantitated in H&E-stained segments. LOX-1 expression in aortas was examined by immunohistochemistry and semi-quantitative RT-PCR. High-cholesterol diet did not affect body weight, but induced hypercholesterolemia and extensive intimal thickening. Aortas of rabbits in the control group showed a modest LOX-1 expression in the endothelium. Aortic intimal proliferation in HC-diet group was associated with a marked increase in LOX-1 expression (protein and mRNA) in the endothelium and neointima. Treatment with losartan attenuated aortic intimal proliferation and markedly decreased the enhanced LOX-1 expression. Thus high-cholesterol diet induces the upregulation of LOX-1 expression in neointima of aortas of rabbits. Treatment with losartan, an AT(1) blocker, markedly decreases this enhanced LOX-1 expression.     

The effect of beta-chloro D-alanine and L-cycloserine on the serine, phosphorus and palmitic acid uptake and metabolism of Tetrahymena lipids

The serine palmitoyltransferase inhibitors beta-chloro-D-alanine and L-cycloserine resulted in the uptake and metabolism of 3H-serine, 3H-palmitic acid and 32P significant alterations in the unicellular Tetrahymena pyriformis GL as compared to the untreated cells. In contrast with the higher eukariotic cells, by these treatments - except 5 mM L-cycloserine - the ceramide formation were not inhibited in Tetrahymena. L-cycloserine inhibited the conversion of phosphatidylserine (PS) to phosphatidyl-ethanolamine (PE) by decarboxylation, and the conversion of PE to phosphatidylcoline (PC) by methylation. The shorter L-cycloserine treatments caused lower, and the longer treatments higher label in glycerophospholipids. beta-chloro-D-alanine resulted in the glycerophospholids higher lipid precursor incorporation both in the shorter and longer treatments. Presumably beta-chloro-D-alanine treatments inhibit the transaminase activity, and the higher concentration (5 and 10 mM) proved to be toxic for Tetrahymena. We found differences between the metabolism of serine and palmitic acid labeled lipids in the beta-chloro-D-alanine and L-cycloserine treated groups. This phenomenon is probably due to a difference in the uptake of phospholipid head group component serine and hydrophobic tail precursor palmitic acid: the incorporation of palmitic acid in Tetrahymena is extremely quick, on the other hand, the uptake of serine is slower, a clear time dependence was measured.     

Attenuation of tissue P-selectin and MCP-1 expression and intimal proliferation by AT(1) receptor blockade in hyperlipidemic rabbits

Angiotensin-II (Ang-II) participates in the development and progression of atherosclerosis by activating type 1 (AT(1)) receptors. In vitro studies show that inflammatory factors, such as P-selectin and MCP-1, which can be upregulated by Ang-II, play an important role in atherogenesis. We examined the effect of AT(1) receptor blockade with losartan on the expression of P-selectin and MCP-1 in hypercholesterolemic rabbits. Since AT(1) receptor blockade is associated with feedback upregulation of renin-angiotensin system (RAS), we also examined alterations in plasma Ang-II levels by losartan therapy. Male NZW rabbits were fed regular chow (high cholesterol diet or high cholesterol diet + losartan 25 mg/kg/day). As expected, there was a marked intimal proliferation in association with increase in serum cholesterol (P < 0.001). In addition, there was a modest increase in plasma Ang-II levels (P < 0.05), and a significant increase in the expression of AT(1) receptors, P-selectin and MCP-1 in aortas of high cholesterol diet rabbits. Concurrent administration of losartan with high cholesterol diet attenuated aortic intimal proliferation induced a fivefold increase in plasma Ang-II levels and caused a marked decrease in expression of P-selectin and MCP-1 without change in serum lipid levels and aortic AT(1) receptor expression. These observations in hypercholesterolemic animal models show that AT(1) receptor blockade is associated with modulation of P-selectin and MCP-1 expression concurrent with reduction in intimal proliferation. The rise in plasma Ang-II does not appear to limit the potential beneficial effect of losartan.     

C3 deposition in cholesterol-induced atherosclerosis in rabbits: a possible etiologic role for complement in atherogenesis.

Hypercholesterolemia was induced in rabbits by feeding Purina Chow supplemented with cholesterol (5 g/kg body weight/day). The serum cholesterol levels of these rabbits increased progressively and after 3 to 5 months were 4 to 9-fold greater than those of the control animals. Decrease in total hemolytic complement was not apparent during the feeding regimen. Morphologic examination of aortae of these hypercholesterolemic rabbits showed typical atherosclerotic intimal plaques. Immunofluorescent microscopy with fluorescein (F)-labeled anti-rabbit C3 showed deposition of C3 in the intimal and inner medial layers as early as 3 months on high cholesterol diet. C3 deposits were also observed in the renal glomeruli and in the walls of coronary arteries. However, fluorescent studies failed to demonstrate the presence of IgG, IgM, and C4 at these sites. Tissues from control animals fed normal diets were negative for immunoglobulins, C3, and C4. These results suggest that the complement system may be implicated in the pathogenesis of cholesterol-induced atherosclerosis in rabbits.     

Adipophilin在动脉粥样硬化病变和脂质负荷细胞中的作用研究(英文)

Adipophilin是细胞内脂质聚集和与脂质聚集有关疾病的标志物 ,巨噬细胞源性泡沫细胞的形成是动脉粥样硬化性疾病发生的重要环节 .为了探讨adipophilin在动脉粥样硬化性疾病的作用 ,通过高胆固醇饲料喂养新西兰白兔 12周 ,复制动脉粥样硬化疾病模型 ,同时测定血脂的变化和动脉壁胆固醇 ,使用HE染色、苏丹Ⅳ染色观察动脉粥样硬化病变的形成 ,使用免疫组织化学的方法观察动脉粥样硬化病变处和动物肝脏中adipophilin的表达 .结果发现 ,高胆固醇饲料喂养组血清总胆固醇、低密度脂蛋白胆固醇和动脉壁胆固醇明显增高 ,动脉粥样硬化病变面积增加到 (40 0 6± 7 2 9) % ,动脉粥样硬化病变处adipophilin表达呈阳性 ;而adipophilin在肝脏中的表达无论是高胆固醇饲料喂养组或对照组均为阴性 .使用80mg/L OxLDL与小鼠腹膜巨噬细胞共孵育 ,复制脂质负荷细胞 ,然后把构建的 1mmol/Ladipophilin反义寡核苷酸与该细胞共孵育 .结果发现 ,使用油红O染色观察的细胞内脂滴明显减少 ,生化测定细胞内胆固醇酯显著降低 ,与对照组相比 ,差别有显著性 .说明adipophilin与动脉粥样硬化病变有密切的关系 ,控制adipophilin的表达能够减少巨噬细胞细胞内胆固醇酯的聚集     

Subcellular localization and membrane topology of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase in mouse liver.

Serine palmitoyltransferase, 3-dehydrosphinganine reductase and sphinganine N-acyltransferase are responsible for the first steps in sphingolipid biosynthesis forming 3-oxosphinganine, sphinganine, and dihydroceramide, respectively. We confirmed the localization of these enzymes in the endoplasmic reticulum (ER) using highly purified mouse liver ER and Golgi preparations. Mild digestion of sealed "right-side out" mouse liver ER derived vesicles with different proteolytic enzymes under conditions where latency of mannose-6-phosphatase was 90% produced approximately 60-80% inactivation of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase activities. These sphingolipid biosynthetic activities (serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase) are not latent, indicating that they face the cytosolic side of the ER, so that substrates have free access to their active sites. Moreover, the membrane-impermeable compound, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, which binds to a large number of ER proteins, inhibits serine palmitoyltransferase and sphinganine N-acyltransferase activities by 30-70%.     

Arterial fatty acid-binding protein activity associated with dietarily-induced and spontaneously occurring atherosclerosis in the rabbit (Oryctolagus cuniculus)

1. Adult WHHL rabbits, or New Zealand rabbits fed either a stock chow diet or a high cholesterol diet were evaluated to assess the relationship between the development of aortic atherosclerosis and arterial FABP activity. 2. Aortic FABP activity was significantly (P less than 0.05) lower in atherosclerotic New Zealand aortas (0.039 +/- 0.008 nmol palmitoyl CoA bound/mg soluble prot) which had developed macroscopic lesions on 80% of the aortic surface as compared to lesion-free New Zealand aortas (0.053 +/- 0.002 nmol palmitoyl CoA bound/mg soluble prot). 3. In spontaneously hyperlipidemic rabbit (WHHL) aortas, FABP activity (0.023 +/- 0.004 nmol palmitoyl CoA bound/mg soluble prot) was significantly lower (P less than 0.05) than in either the normal or atherosclerotic New Zealand aortas. 4. To our knowledge, this study is the first to report a change in arterial FABP with the atherogenic process.     

Induction of Ganglioside Biosynthesis and Neurite Outgrowth of Primary Cultured Neurons by l-threo-1-Phenyl-2-Decanoylamino-3-Morpholino-1-Propanol

Abstract: We reported previously that stereoisomers of 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), the d - threo and l - threo forms, exerted inhibitory and stimulatory effects on glycosphingolipid (GSL) biosynthesis in B16 melanoma cells, respectively. In the present study, the primary cultured rat neocortical explants were treated with l - or d - threo -PDMP. These isomers exhibited opposite effects on neurite outgrowth: d -PDMP was inhibitory at concentrations ranging from 5 to 20 µ M , whereas l -PDMP was stimulatory over the same concentration range, and the maximal effect was observed at 10–15 µ M . Rat neocortical explants were doubly labeled with [¹⁴C]serine and [³H]galactose at 15 µ M l - or d -PDMP. l -PDMP increased the incorporations of both labels into sphinganine, sphingosine, ceramide, sphingomyelin, neutral GSLs, and gangliosides, whereas d -PDMP inhibited the glucosylation of ceramide resulting in a reduction of ganglioside biosynthesis and accumulation of precursors of glucosylceramide, ceramide, and sphingomyelin. To clarify the stimulatory effect of l -PDMP on GSL biosynthesis, serine palmitoyltransferase, sphingosine N -acyltransferase, glucosylceramide synthase, lactosylceramide synthase, GM3 synthase, and GD3 synthase were quantified in cell lysates of explants pretreated with this agent. Serine palmitoyltransferase was fully activated up to 150% of the control. Furthermore, marked increases in the activities of lactosylceramide synthase (200%), GM3 synthase (240%), and GD3 synthase (300%) were observed. These results suggest that the neurotrophic action of l -PDMP may be ascribable to its stimulatory effect on the biosynthesis of GSLs, especially that of gangliosides.     

Alterations in biliary lipids of mice during dehydrocholic acid feeding

Mice were fed a lithogenic diet consisting of Purina chow and 0.5% dehydrocholic acid (DHA group). Controls received Purina chow. Every 2 wk for 20 wk animals were killed, and biliary phospholipid, cholesterol, and bile salt concentrations were determined, as well as the extent of gallstone formation. With time there was a gradual, significant decline in the concentration and the relative composition of phospholipid in both groups compared with initial values. There was a significant increase in biliary cholesterol concentration and relative amount in the DHA group compared with the control. No significant differences were found in the relative amounts of bile salt or phospholipid between the two groups. Feeding DHA resulted in an increased concentration of bile salts and the sum of measured lipid compared with controls. After 8 wk, gallstones were found in approximately 60% of autopsied animals and correlated with increased cholesterol concentration. Our data support the hypothesis that there is a component of cholesterol secretion that may not be bile salt- or phospholipid-dependent. Our data also suggest that biliary phospholipid secretion decreases with age.     

Kinetics of long-chain (sphingoid) base biosynthesis in intact LM cells: effects of varying the extracellular concentrations of serine and fatty acid precursors of this pathway

Serine palmitoyltransferase (EC 2.3.1.50) catalyzes the condensation of L-serine and palmitoyl-CoA to yield 3-ketosphinganine in the first unique reaction of long-chain (sphingoid) base biosynthesis. The kinetic effects of changing the extracellular concentrations of the precursors for this pathway were studied with LM cells by following the incorporation of L-[3-14C]serine into the long-chain base (i.e., sphinganine and sphingenine) backbones of complex sphingolipids. [14C]Serine was taken up by the cells and rapidly reached steady-state concentrations similar to those of the medium. From the cellular [14C]serine concentrations and specific activities, the apparent Vmax [14 pmol min-1 (10(6) cells)-1] and Km (0.23 mM) values for long-chain base synthesis were determined and found to be essentially identical with those for serine palmitoyltransferase assayed in vitro [i.e., 13 pmol min-1 (10(6) cells)-1 and 0.27 mM, respectively]. The other precursor, palmitic acid, was also taken up rapidly and increased long-chain base biosynthesis in a concentration-dependent manner. This effect was limited to palmitic acid and matched the known specificity of serine palmitoyltransferase for saturated fatty acyl-CoA's of 16 +/- 1 carbon atoms. These studies delineate the influence of extracellular precursors on the formation of the sphingolipid backbone and suggest that the kinetic properties of serine palmitoyltransferase govern this behavior of long-chain base synthesis in intact cells.     

Effects of cholesterol diets on vascular function and atherogenesis in rabbits

Vascular endothelial dysfunction is an important early event in atherogenesis. To evaluate the effects of different levels of cholesterol-containing diets on vascular function and atherogenesis, 17 New Zealand White male rabbits were randomized into four groups: Control with noncholesterol, 10-week 0.5% (0.5C-10) or 1% cholesterol (1C-10), and 14-week 0.5% cholesterol (0.5C-14) feedings. After 10 or 14 weeks, the aortas were harvested for studies of vascular endothelial function and percentage surface lipid lesions. The 0.5% and 1% cholesterol feedings resulted in the same degree of hypercholesterolemia independent of the level and period of cholesterol feeding. There was a decreased trend in vascular endothelial-dependent relaxation to acetylcholine in cholesterol-fed rabbits. Fourteen-week cholesterol feeding induced the least vascular dilation at a concentration of 10-7 M acetylcholine (-38 +/- 3%, -23 +/- 4%, -23 +/- 2%, and -15 +/- 5% in control, 0.5C-10, 1C-10, and 0.5C-14 groups, respectively, P = 0.003). More cumulative exposure of arterial walls to cholesterol induced more surface lipid lesions in the aorta (r = 0.877, P < 0.001). There was a negative relationship between aortic lesions and vasodilation (r = -0.557, P = 0.020 for calcium ionophore; r = -0.463, P = 0.062 for acetylcholine). We conclude that the 0.5% and 1% cholesterol feedings induce similar degrees of hypercholesterolemia. However, aortic lipid lesions and vascular reactivity are dependent on cumulative exposure to cholesterol rather than serum cholesterol level only. Furthermore, decreased vascular endothelial relaxation in cholesterol-fed rabbits was related to lipid plaques in the aorta.     

Inhibition of VCAM-1 expression in the arterial wall is shared by structurally different antioxidants that reduce early atherosclerosis in NZW rabbits.

We previously established that probucol decreases basal expression of VCAM-1 in the aorta of WHHL rabbits and inhibits the up-regulation of VCAM-1 expression that normally accompanies atherogenesis. To determine whether this effect is shared by other antioxidants in vivo, we now investigated whether a structurally unrelated antioxidant, vitamin E, also inhibits arterial VCAM-1 expression and whether the degree of VCAM-1 inhibition correlates with the reduction of atherosclerosis or the antioxidant protection of LDL. Atherogenesis and VCAM-1 mRNA and protein were determined in four groups of NZW rabbits (n = 6;-8) fed 0.5% cholesterol alone or supplemented with 0.1% vitamin E, a low dose (0.04;-0.075%) of probucol yielding the same degree of antioxidant protection of plasma LDL as vitamin E, or a high dose (0.5%) of probucol, and in normocholesterolemic rabbits. After 81 days, extensive atherosclerosis and a greater than 4-fold up-regulation of VCAM-1 mRNA was seen in rabbits on high cholesterol diet, mostly in the intima. Treatment with vitamin E, high-dose probucol, and low-dose probucol significantly decreased VCAM-1 mRNA by 49.0, 74.9, and 57. 5%, respectively, and reduced atherosclerosis in adjacent segments of the thoracic aorta to a similar degree as reported by previous studies. Immunocytochemistry confirmed that lesions of antioxidant-treated animals also contained less VCAM-1 protein. Neither the degree of VCAM-1 inhibition nor the extent of atherosclerosis correlated with the degree of antioxidant protection of plasma LDL.In summary, treatment with structurally unrelated antioxidants conveyed different degrees of antioxidant protection to plasma LDL but significantly reduced VCAM-1 expression in vivo and inhibited atherogenesis. This is consistent with the assumption that antiatherogenic effects of antioxidants may in part be mediated by interference with oxidation-dependent intracellular signaling.     

Sphingomyelin synthesis in Ascaridia galli

Adult Ascaridia galli incorporate label from [U-¹⁴C] serine into various intermediates of sphingomyelin synthesis (ketosphinganine, sphinganine, sphingosine, ceramide and sphingomyelin). From the results it is concluded that A. galli possesses the five enzymes involved in sphingomyelin synthesis, namely: serine palmitoyltransferase, 3-ketosphinganine reductase, flavoprotein sphinganine reductase, sphingosine acyltransferase and ceramide choline phosphotransferase.     

Inhibition of serine palmitoyltransferase in vitro and long-chain base biosynthesis in intact Chinese hamster ovary cells by beta-chloroalanine

The effects of beta-chloroalanine (beta-Cl-alanine) on serine palmitoyltransferase activity and the de novo biosynthesis of sphinganine and sphingenine were investigated in vitro with rat liver microsomes and in vivo with intact Chinese hamster ovary (CHO) cells. The inhibition in vitro was rapid (5 mM beta-Cl-alanine caused complete inactivation in 10 min), irreversible, and concentration and time dependent and apparently involved the active site because inactivation only occurred with beta-Cl-L-alanine (not beta-Cl-D-alanine) and was blocked by L-serine. These are characteristics of mechanism-based ("suicide") inhibition. Serine palmitoyltransferase (SPT) was also inhibited when intact CHO cells were incubated with beta-Cl-alanine (complete inhibition occurred in 15 min with 5 mM), and this treatment inhibited [14C]serine incorporation into long-chain bases by intact cells. The concentration dependence of the loss of SPT activity and of long-chain base synthesis was identical. The effects of beta-Cl-L-alanine appeared to occur with little perturbation of other cell functions: the cells exhibited no loss in cell viability, [14C]serine uptake was not blocked, total lipid biosynthesis from [14C]acetic acid was not decreased (nor was the appearance of radiolabel in cholesterol and phosphatidylcholine), and [3H]thymidine incorporation into DNA was not affected. There appeared to be little effect on protein synthesis based on the incorporation of [3H]leucine, which was only decreased by 14%. Although beta-Cl-L-alanine is known to inhibit other pyridoxal 5'-phosphate dependent enzymes, alanine and aspartate transaminases were not inhibited under these conditions. These results establish the close association between the activity of serine palmitoyltransferase and the cellular rate of long-chain base formation and indicate that beta-Cl-alanine and other mechanism-based inhibitors might be useful to study alterations in cellular long-chain base synthesis.     

PPARalpha agonist induces the accumulation of ceramide in the heart of rats fed high-fat diet.

It was shown that high-fat feeding of mice with cardiac-specific overexpression of peroxisome proliferator-activated receptor (PPAR) alpha but not wild type animals leads to the accumulation of ceramide (an important mediator of lipotoxicity) in the heart [Finck et al. 2003 Proc Natl Acad Sci USA]. To investigate the mechanism of this phenomenon we examined the effects of PPARalpha activation on ceramide metabolism in the myocardium. Male Wistar rats were fed either a standard chow or a high-fat diet. Each group was divided into two subgroups: control and treated with selective PPARalpha activator - WY-14643. In the rats fed on the standard diet WY-14643 did not affect the myocardial content of sphingomyelin and ceramide but reduced the content of sphinganine and sphingosine. It also inhibited the activity of neutral sphingomyelinase and increased the activity of acid sphingomyelinase, whereas the activity of ceramidases and serine palmitoyltransferase (SPT) remained stable. High-fat diet itself did not affect the content of the examined sphingolipids. However, it reduced the activity of sphingomyelinases and ceramidases having no effect on the activity of SPT. Administration of WY-14643 to this group significantly increased the content of myocardial free palmitate, ceramide, sphingomyelin and the activity of SPT. Our results demonstrated that PPARalpha activation modulates myocardial ceramide metabolism and leads to the accumulation of ceramide in the heart of the high-fat fed rats due to its increased synthesis de novo.     

Putative mechanisms of action of probucol on high-density lipoprotein apolipoprotein A-I and its isoproteins kinetics in rabbits

Probucol is a widely prescribed lipid-lowering agent, the major effects of which are to lower cholesterol in both low- and high-density lipoproteins (LDL and HDL, respectively). The mechanism of action of probucol on HDL apolipoprotein (apo) A-I kinetics was investigated in rabbits, with or without cholesterol feeding. 125I-labeled HDL was injected intravenously, and blood samples were taken periodically for 6 days. Kinetic parameters were calculated from the apo A-I-specific radioactivity decay curves. Fractional catabolic rate (FCR) and synthetic rate (SR) of apo A-I in rabbits fed a normal chow and normal chow with 1% probucol were similar. Apo A-I FCR of the rabbits fed 0.5% cholesterol was significantly increased but there were no changes in SR, compared to findings in the normal chow-fed group. Apo A-I FCR of the rabbits fed 1% probucol with 0.5% cholesterol (both 1 month and 2 months) was significantly increased compared to findings in rabbits fed the normal chow as well as 0.5% cholesterol diet group, while SR of apo A-I was significantly reduced in the former groups. Kinetics at 1 month after discontinuation of 1% probucol (under cholesterol feeding) showed a similar FCR of HDL-apo A-I to that of the rabbits fed 0.5% cholesterol, but the SR of apo A-I remained lower. Apo A-I isoproteins kinetics assessed by autoradiography of isoelectric focusing slab gels showed that the synthesis of proapo A-I was significantly reduced in the 1% probucol with 0.5% cholesterol administered, compared to the 0.5% cholesterol group. Thus, the action of probucol on HDL apo A-I kinetics was only prominent in case of higher serum cholesterol levels. The decreased HDL or apo A-I seen with probucol was apparently the result of an increase in FCR and a decrease in SR of HDL-apo A-I. A decreased synthesis of apo A-I remained evident even 1 month after discontinuing probucol. The action of probucol on the intracellular synthetic processes of apo A-I was revealed by the reduced synthesis of proapo A-I.     

The effect of standard chow and reduced hexokinase II on growth, cardiac and skeletal muscle hexokinase and low-flow cardiac ischaemia-reperfusion injury

In the present study, we examined whether standard chow (SDS versus Purina 5001; both low fat, high carbohydrate) and reductions in hexokinase (HK) II (wild-type versus HKII(+/-) mice) affect (1) growth parameters, (2) HK levels in cardiac and skeletal muscle and (3) low-flow cardiac ischaemia-reperfusion (IR) injury. Total HK activity and HKI and HKII expressions were determined, and low-flow IR injury was examined in isolated hearts subjected to 40 min 5% low-flow ischaemia and 120 min reperfusion. Standard chow, but not HKII reductions, significantly affected body weight, heart weight and cardiac hypertrophy. Both standard chow and reduced HKII diminished total cardiac and skeletal muscle HK activity. For the heart, the Purina chow-induced decrease in total HK activity was through decreases in HKI expression, whereas for skeletal muscle post-translational mechanisms are suggested. Both standard chow and reduced HKII demonstrated a non-significant trend for affecting cardiac IR damage. However, the low-flow ischaemia model was associated with mild sublethal injury only (～1% cell death). In conclusion, standard chow affects body weight, heart weight and HK activity and HKI expression in the heart, without altering HKII expression. This implicates standard chow as an important factor in genomic, physiological research models and demonstrates that large differences in fat or carbohydrates in the diet are not necessary to affect growth. In a cardiac low-flow IR model, resulting in only mild injury, standard chow or reduced HKII does not significantly affect IR damage.