The Effect of 2-Ketobutyrate on Mitochondrial Substrate-Level Phosphorylation.

The reaction catalyzed by succinate-CoA ligase in the mitochondrial matrix yields a high-energy phosphate when operating towards hydrolysis of the thioester bond of succinyl-CoA, known as mitochondrial substrate-level phosphorylation (mSLP). The catabolism of several metabolites converge to succinyl-CoA but through different biochemical pathways. Among them, threonine, serine and methionine catabolize to succinyl-CoA through the common intermediate, 2-ketobutyrate. During the course of this pathway 2-ketobutyrate will become succinyl-CoA through propionyl-CoA catabolism, obligatorily passing through an ATP-consuming step substantiated by propionyl-CoA carboxylase. Here, by recording the directionality of the adenine nucleotide translocase while measuring membrane potential we tested the hypothesis that catabolism of 2-ketobutyrate negates mSLP due to the ATP-consuming propionyl-CoA carboxylase step in rotenone-treated, isolated mouse liver and brain mitochondria. 2-Ketobutyrate produced a less negative membrane potential compared to NADH or FADH2-linked substrates, which was sensitive to inhibition by rotenone, atpenin and arsenate, implying the involvement of complex I, complex II and a dehydrogenase-most likely branched chain keto-acid dehydrogenase, respectively. Co-addition of 2-ketobutyrate with NADH- or FADH2-linked substrates yielded no greater membrane potential than in the presence of substrates alone. However, in the presence of NADH-linked substrates, 2-ketobutyrate prevented mSLP in a dose-dependent manner. Our results imply that despite that 2-ketobutyrate leads to succinyl-CoA formation, obligatory metabolism through propionyl-CoA carboxylase associated with ATP expenditure abolishes mSLP. The provision of metabolites converging to 2-ketobutyrate may be a useful way for manipulating mSLP without using pharmacological or genetic tools.

Isorhamnetin derivatives and piscidic acid for hypercholesterolemia: cholesterol permeability, HMG-CoA reductase inhibition, and docking studies.

Bioactive compounds, such as isorhamnetin and piscidic acid, were obtained from decoctions of cladodes (stem pads from Opuntia ficus-indica). The effect of these phenolic compounds, in a fiber-free extract, were evaluated as inhibitors of cholesterol permeation through a Caco-2 cell monolayer and as 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. A reduction of 38% in cholesterol permeation through the Caco-2 cell monolayer was obtained, and the phenolic compounds all permeated between 6 and 9%. A mixture of these compounds showed an IC50 of 20.3 µg/mL as an enzyme inhibitor, whereas piscidic acid alone showed an IC50 of 149.6 µg/mL; this was slightly outperformed by the isorhamnetin derivatives. Docking studies confirmed that both piscidic acid and isorhamnetin derivatives, present in the decoction, could adequately bind to the enzyme active site. These results reveal that O. ficus-indica, and cladodes derived there from, is a promising plant for use in the development of new functional foods and pharmaceutical products.

Acute Testosterone Deficiency Alters Adipose Tissue Fatty Acid Storage.

Context: Although the long-term effects of testosterone on adipose tissue lipid metabolism in men have been defined, the short-term regulation of these effects is not well understood. Objective: We examined the effects of acute testosterone withdrawal on subcutaneous abdominal and femoral adipose tissue fatty acid (FA) storage and cellular mechanisms. Design: This was a prospective, randomized trial. Setting: Mayo Clinic Clinical Research Unit. Patients or Participants: Thirty-two male volunteers ages 18 to 50 participated in these studies. Interventions: Volunteers were randomized to receive (1) no treatment (control), (2) injections (7.5 mg) of Lupron®, or (3) Lupron and testosterone (L+T) replacement for 49 days, resulting in 4 weeks of sex steroid suppression in the Lupron group. Main Outcome Measures: We measured body composition, fat cell size, adipose tissue meal FA and direct free FA storage, lipoprotein lipase (LPL), acyl coenzyme A synthetase (ACS), diacylglycerol acyltransferase activities, and CD36 content. Results: Compared with control and L+T groups, acute testosterone deficiency resulted in greater femoral adipose tissue meal FA storage rates, fasting and fed LPL activity, and ACS activity. Conclusions: These results suggest that in men, testosterone plays a tonic role in restraining FA storage in femoral adipose tissue via suppression of LPL and ACS activities. FA storage mechanisms in men appear sensitive to short-term changes in testosterone concentrations.

Novel remodeling of the mouse heart mitochondrial proteome in response to acute insulin stimulation.

BACKGROUND AND AIM: Mitochondrial dysfunction contributes to the pathophysiology of diabetic cardiomyopathy. The aim of this study was to investigate the acute changes in the mitochondrial proteome in response to insulin stimulation. METHODS AND RESULTS: Cardiac mitochondria from C57BL/6 mice after insulin stimulation were analyzed using two-dimensional fluorescence difference gel electrophoresis. MALDI-TOF MS/MS was utilized to identify differences. Two enzymes involved in metabolism and four structural proteins were identified. Succinyl-CoA ligase [ADP forming] subunit beta was identified as one of the differentially regulated proteins. Upon insulin stimulation, a relatively more acidic isoform of this protein was increased by 53% and its functional activity was decreased by ∼32%. CONCLUSIONS: This proteomic remodeling in response to insulin stimulation may play an important role in the normal and diabetic heart.

Lanostane Triterpenes from the Tibetan Medicinal Mushroom Ganoderma leucocontextum and Their Inhibitory Effects on HMG-CoA Reductase and α-Glucosidase.

Sixteen new lanostane triterpenes, ganoleucoins A-P (1-16), together with 10 known tripterpenes (17-26), were isolated from the cultivated fruiting bodies of Ganoderma leucocontextum, a new member of the Ganoderma lucidum complex. The structures of the new compounds were elucidated by extensive spectroscopic analysis and chemical transformation. The inhibitory effects of 1-26 on HMG-CoA reductase and α-glucosidase were tested in vitro. Compounds 1, 3, 6, 10-14, 17, 18, 23, 25, and 26 showed much stronger inhibitory activity against HMG-CoA reductase than the positive control atorvastatin. Compounds 13, 14, and 16 presented potent inhibitory activity against α-glucosidase from yeast with IC50 values of 13.6, 2.5, and 5.9 µM, respectively. In addition, the cytotoxicity of 1-26 was evaluated against the K562 and PC-3 cell lines by the MTT assay. Compounds 1, 2, 6, 7, 10, 12, 16, 18, and 25 exhibited cytotoxicity against K562 cells with IC50 values in the range 10-20 µM. Paclitaxel was used as the positive control with an IC50 value of 0.9 µM. This is the first report of secondary metabolites from this medicinal mushroom.

Influence of large molecular polymeric pigments isolated from fermented Zijuan tea on the activity of key enzymes involved in lipid metabolism in rat.

Influence of large molecular polymeric pigments (LMPP) isolated from fermented Zijuan tea on the activity and mRNA expression of key enzymes involved in lipid metabolism in rat was explored. The results show that intragastric infusion of high-dose LMPP (1.215 g/kg body weight) effectively suppressed the elevation in TC and LDL-C (p<0.05), and prevented the reduction in HDL-C (p<0.05), compared with the hyperlipidemia model group. LMPP significantly enhanced the activity of HL and HSL, and increased the HSL mRNA expression in the liver tissue and adipose tissue. High-LMPP treatment significantly reduced the HMG-CoA reductase expression by 56.5% in the liver compared with hyperlipidemia model group. In contrast, LDL-R expression was increased by 120% in the presence of high-LMPP treatment. These results suggest that LMPP have the hypolipidemic effect to some extent and significantly enhance HSL mRNA expression in the liver and adipose tissue, thereby increasing HSL activity in rat.

Regulation of stearoyl-coenzyme A desaturase and fatty acid delta-6 desaturase-2 expression by linoleic acid and arachidonic acid in human sebocytes leads to enhancement of proinflammatory activity but does not affect lipogenesis.

BACKGROUND: Treatment of SZ95 sebocytes with the essential fatty acid linoleic acid (LA) and the polyunsaturated fatty acid arachidonic acid (AA) leads to sebaceous lipogenesis. Animal data indicate that stearoyl-coenzyme A desaturase (SCD), a key enzyme in fatty acid biosynthesis, is involved in sebaceous lipogenesis and proinflammatory signalling in the sebaceous gland. On the other hand, fatty acid delta-6 desaturase-2 (FADS2) catalyses the conversion of LA to AA. OBJECTIVES: To identify the effects of LA and AA on the expression of SCD and FADS2 and to detect its biological relevance. METHODS: SZ95 sebocytes were treated with LA (10(-5) and 10(-4) mol L(-1) ), AA (10(-6) and 10(-5) mol L(-1) ) and the combination of LA (10(-4) mol L(-1) ) and testosterone (2 × 10(-8) mol L(-1) ), with or without addition of the SCD inhibitor FPCA (10(-8) and 10(-6) mol L(-1) ). Cytotoxicity was determined by the lactate dehydrogenase assay. SCD and FACS2 mRNA levels were assessed by semiquantitative reverse transcription-polymerase chain reaction and protein expression by Western blot analysis. SZ95 sebocyte lipid content and cell number were measured by the Nile red and the fluorescein diacetate microassays, respectively. Determination of interleukin (IL)-6 and IL-8 release was evaluated by enzyme-linked immunosorbent assay. RESULTS: LA treatment induced an increase of SCD and FADS2 at mRNA and protein levels in SZ95 sebocytes after 1·5 h. Treatment with AA led to an increase of SCD but to a decrease of FADS2 mRNA levels. LA/testosterone cotreatment stimulated lipogenesis in SZ95 sebocytes. A distinct proinflammatory pattern was registered: whereas LA strongly upregulated IL-6 secretion only, AA induced a mild level of IL-6 and IL-8 release from SZ95 sebocytes. Treatment with the SCD inhibitor FPCA reduced the LA/testosterone-upregulated SCD and FADS2 mRNA levels and resulted in an anti-inflammatory effect, but did not affect sebaceous lipogenesis. CONCLUSIONS: LA-induced sebaceous lipogenesis is likely to be an SCD-independent effect. Regulation of SCD and FADS2 expression by LA and AA leads to enhancement of proinflammatory activity but does not affect lipogenesis in human sebocytes.

Taurine suppresses development of atherosclerosis in Watanabe heritable hyperlipidemic (WHHL) rabbits.

While the hypocholesterolemic effects of taurine have extensively been studied using experimental animals, the anti-atherosclerotic effects of taurine have been given less attention. We examined the effect of taurine on atherosclerotic lesions in Watanabe heritable hyperlipidemic (WHHL) rabbits. Treatment of WHHL rabbits with taurine (0.3% in drinking tap water) for 24 weeks decreased aortic lesions by 31%, estimated as intimal thickening. Taurine significantly decreased cholesteryl ester content of aortic arch, thoracic aorta, and abdominal aorta by 35, 43, and 54%, respectively. Concomitantly, activity of acyl-CoA:cholesterol acyltransferase (ACAT), an enzyme responsible for cholesterol esterification, was also significantly decreased. Immunohistochemical analysis revealed decreased macrophages in the intima of taurine-treated rabbits. Taurine had no apparent effect on blood pressure and serum cholesterol levels. Contents of thiobarbituric acid reactive substances (TBARS), a marker of lipid peroxidation, was reduced in serum and aorta by 29 and 50%, respectively, when taurine was ingested. In addition, LDL from taurine-treated rabbits was resistant to copper-induced oxidative modification. These results revealed that taurine prevents development of atherosclerosis and that the anti-atherosclerotic effects of taurine are independent of serum cholesterol levels. The anti-oxidant action of taurine may be involved in inhibiting atherosclerosis in these rabbits.

Acyl-CoA oxidase is imported as a heteropentameric, cofactor-containing complex into peroxisomes of Yarrowia lipolytica.

Five isoforms of acyl-CoA oxidase (Aox), designated Aox1p to Aox5p, constitute a 443-kD heteropentameric complex containing one polypeptide chain of each isoform within the peroxisomal matrix of the yeast Yarrowia lipolytica. Assembly of the Aox complex occurs in the cytosol and precedes its import into peroxisomes. Peroxisomal targeting of the Aox complex is abolished in a mutant lacking the peroxin Pex5p, a component of the matrix protein targeting machinery. Import of the Aox complex into peroxisomes does not involve the cytosolic chaperone Pex20p, which mediates the oligomerization and import of peroxisomal thiolase. Aox2p and Aox3p play a pivotal role in the formation of the Aox complex in the cytosol and can substitute for one another in promoting assembly of the complex. In vitro, these subunits retard disassembly of the Aox complex and increase the efficiency of its reassembly. Neither Aox2p nor Aox3p is required for acquisition of the cofactor FAD by other components of the complex. We provide evidence that the Aox2p- and Aox3p-assisted assembly of the Aox complex in the cytosol is mandatory for its import into peroxisomes and that no component of the complex can penetrate the peroxisomal matrix as a monomer.

Effect of fatty acid chain length and thioesterification on the augmentation of expression of plasminogen activator inhibitor-1.

BACKGROUND AND AIM: Concentrations in blood of plasminogen activator inhibitor type 1 (PAI-1) and circulating free (non-esterified) fatty acids (FFA) are increased in diabetes and may accelerate atherosclerosis. We have shown that FFA increase expression of PAI-1 by activation of a transcription factor that binds to the repeated sequence 5'-TG(G/C) 1-2CTG-3'. This study was designed to determine whether FFA chain length, saturation, or both affect agonist properties and whether agonist properties are mediated by activated, thioesterified FFA (fatty acyl-CoA). METHODS AND RESULTS: Human hepatoma cells were exposed to selected FFA associated with bovine serum albumin (BSA). Triacsin C (5 microM) was used to inhibit production of fatty acyl-CoA. PAI-1 was assayed by enzyme linked immunosorbent assay. Maximal induction of PAI-1 was similar with medium and long chain FFA (fold induction of PAI-1 accumulated in conditioned media compared with control: C10 = 1.8 +/- 0.1, C12 = 2.0 +/- 0.1, C14 = 2.0 +/- 0.2, C16 = 1.4 +/- 0.1, C18 = 1.6 +/- 0.1, C20 = 1.32 +/- 0.1, p < 0.005 for each compared with control). Increased unsaturation did not alter the agonist properties of FFA (fold induction with C16: 0 = 1.4 +/- 0.1, C16: 1 = 1.4 +/- 0.1; C18: 0 = 1.6 +/- 0.1, C18: 1 = 1.5 +/- 0.1, C18: 2 = 1.6 +/- 0.1, C18: 3 = 1.4 +/- 0.1 and C20: 4 = 1.3 +/- 0.1, C20: 5 = 1.4 +/- 0.1, n = 6). However, maximal effects were seen with lower concentrations of longer chain FFA. Triacsin C consistently attenuated effects of FFA. CONCLUSIONS: Longer chain FFA exhibit maximal effects at lower concentrations. Augmented expression of PAI-1 is mediated by the fatty acyl-CoA derivative. These criteria identify targets for therapy designed to normalize expression of PAI-1 and retard progression of atherosclerosis in subjects with elevated concentrations of FFA in blood including those with insulin resistance.

Repeated analysis of semen parameters in beagle dogs during a 2-year study with the HMG-CoA reductase inhibitor, atorvastatin.

Sperm analyses are often incorporated into reproductive toxicity studies in rats. Due to the relative ease of collecting multiple samples throughout a study, semen analysis in non-rodents such as dogs offers the opportunity to assess potential development of functional effects of compounds on male reproduction over time. In the present study, semen parameters were evaluated in beagle dogs during and at termination of a chronic toxicity study with the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, atorvastatin. Male dogs received 0, 10, 40, or 120 mg/kg orally in gelatin capsules for up to 104 weeks (n = 10/group). After 52 weeks of dosing, 3 dogs/group were euthanized, and 2/group were withdrawn from treatment for a 12-week reversal period and euthanized at Week 64. The remaining 5/group continued treatment until Week 104. Semen was collected from all animals for 3 consecutive weeks prior to termination of the 52-week animals (Weeks 50, 51, 52) for analysis of sperm parameters, using manual methods of evaluation. Semen was collected from the remaining animals at Weeks 64, 78, 91, and 104, and was analyzed. At necropsy, testes, epididymides, and prostates were weighed and evaluated histologically, and epididymal sperm counts were determined. Serum cholesterol was decreased 25--60% at all doses during the study. There were no drug-related differences in semen volume and color, total sperm count, and sperm concentration, morphology, progressiveness, and percent motility during treatment with atorvastatin. There were also no effects on reproductive organ weights or histopathology, and no effects on epididymal sperm count. Thus, incorporation of semen analyses into this study allowed the evaluation of potential male reproductive effects in dogs at multiple time points during the study. Statistical power calculations demonstrated acceptable statistical power (> 80%) for semen sperm count, concentration, morphology, and motility with group sizes of 8--10 animals, and for semen sperm count and concentration or epididymal sperm count with group sizes of 3--5 animals, using the methodology described in this paper.

Dietary conjugated linoleic acid mixture affects the activity of intestinal acyl coenzyme A: cholesterol acyltransferase in hamsters.

The present study was designed to study the mechanisms by which dietary conjugated linoleic acids (CLA) decrease serum cholesterol. Hamsters were fed a semi-synthetic diet containing 1 g cholesterol/kg diet with or without supplementation with 20 g linoleic acid (LA) and 20 g CLA/kg diet. After 8 weeks, serum fasting total cholesterol (TC) and triacylglycerol (TG) were significantly lower in the LA-supplemented and CLA-supplemented groups compared with those of the control (CTL) hamsters. In contrast to LA, CLA significantly lowered hepatic cholesterol but it increased the level of adipose tissue cholesterol, suggesting that the hypocholesterolaemic mechanism of CLA is different from that of LA. CLA decreased the activity of intestinal acyl CoA:cholesterol acyltransferase (ACAT) whereas LA had no effect on this enzyme. Consequently, CLA supplementation increased the faecal excretion of total neutral sterols, but it had no or little effect on the faecal acidic sterols. If the ACAT is associated with cholesterol absorption, the part of mechanisms by which CLA decreases serum cholesterol may involve down-regulation of intestinal ACAT activity.

Different effect of simvastatin and atorvastatin on key enzymes involved in VLDL synthesis and catabolism in high fat/cholesterol fed rabbits.

The effects of atorvastatin (3 mg kg(-1)) and simvastatin (3 mg kg(-1)) on hepatic enzyme activities involved in very low density lipoprotein metabolism were studied in coconut oil/cholesterol fed rabbits. Plasma cholesterol and triglyceride levels increased 19 and 4 fold, respectively, after 7 weeks of feeding. Treatment with statins during the last 4 weeks of feeding abolished the progression of hypercholesterolaemia and reduced plasma triglyceride levels. 3-Hydroxy-3-methyl-glutaryl Coenzyme A reductase, acylcoenzyme A:cholesterol acyltransferase, phosphatidate phosphohydrolase and diacylglycerol acyltransferase activities were not affected by drug treatment. Accordingly, hepatic free cholesterol, cholesteryl ester and triglyceride content were not modified. Simvastatin treatment caused an increase (72%) in lipoprotein lipase activity without affecting hepatic lipase activity. Atorvastatin caused a reduction in hepatic phospholipid content and a compensatory increase in CTP:phosphocholine cytidylyl transferase activity. The results presented in this study suggest that, besides the inhibitory effect on 3-hydroxy-3-methyl-glutaryl Coenzyme A reductase, simvastatin and atorvastatin may have additional effects that contribute to their triglyceride-lowering ability.

GABA inhibits endozepine release from cultured rat astrocytes.

In the mammalian brain, the endogenous ligands for benzodiazepine receptors (also called endozepines) are predominantly synthesized by glial cells. It has recently been reported that rat astrocytes in primary culture release substantial amounts of endozepines. The aim of the present study was to investigate the possible involvement of GABA in the control of endozepine release. Exposure of cultured rat astrocytes to GABA (10(-7) to 10(-5) M) induced a dose-related inhibition of endozepine secretion. At higher doses (3 x 10(-5) to 10(-3) M), the effect of GABA gradually diminished. The inhibitory effect of GABA (10(-5) M) was mimicked by the GABA(B) receptor agonist baclofen (10(-5) M). In contrast, the GABA(A) receptor agonists 3APS and isoguvacine (10(-5) M each) did not modify endozepine release. The inhibition of endozepine secretion evoked by GABA and baclofen (10(-5) M each) was totally abrogated by the specific GABA(B) receptor antagonist phaclofen (10(-4) M). GABA and baclofen caused a significant inhibition of forskolin-evoked production of cAMP in astrocytes and this effect was abolished in the presence of phaclofen. In contrast, isoguvacine had no effect on cAMP production. Exposure of astrocytes to dbcAMP induced a time- and dose-dependent stimulation of endozepine release. These data indicate that GABA, acting through GABA(B) receptors negatively coupled to adenylyl cyclase, inhibits endozepine release from cultured rat astrocytes. The secretion of endozepines thus appears to be a valuable marker to monitor astrocyte activity.

Effect of sulphide on short chain acyl-CoA metabolism in rat colonocytes.

BACKGROUND: It has been proposed that the diminished n-butyrate oxidation observed in ulcerative colitis may be the result of sulphide induced inhibition of short chain acyl-coenzyme A (acyl-CoA) dehydrogenase activity. AIM: To examine the acyl-CoA ester profiles in isolated rat colonic epithelial cells treated in vitro with sodium hydrogen sulphide (NaHS). METHODS: Isolated rat colonic epithelial cell suspensions were incubated for 10 minutes in the presence of [1-14C] n-butyrate (5 mM), with and without NaHS (1.5 mM). Incubations were carried out both in the presence and the absence of exogenous CoA and ATP. Metabolic performance was assessed by 14CO2 production and by acyl-CoA ester production measured by HPLC with ultraviolet detection. RESULTS: Results are given as mean (SEM). For colonocytes incubated in the presence of exogenous CoA and ATP, treatment with NaHS significantly diminished 14CO2 production (control 0.97 (0.06) mumol/g dry weight cells/min, treated 0.26 (0.09) mumol/g dry weight cells/min, p = 0.0019), was associated with an increase in butyryl-CoA concentrations in the final reaction mixture at 10 minutes (control 2.55 (0.28) mumol/g dry weight cells, treated 3.32 (0.32) mumol/g dry weight cells, p = 0.002), and a reduction in crotonyl-CoA concentrations (control 0.274 (0.02) mumol/g dry weight cells, treated 0.120 (0.04) mumol/g dry weight cells, p = 0.008). The mean concentration of acetyl-CoA in the reaction mixture at 10 minutes was not significantly different between control and sulphide treated incubations. There were no significant differences in acyl-CoA ester profiles observed when cells were incubated in the absence of exogenous CoA and ATP. CONCLUSIONS: These results support the view that sulphides inhibit n-butyrate oxidation in colonic epithelial cells by inhibiting short chain acyl dehydrogenation of activated fatty acids.

Incorporation of [U-14C]palmitate into rat brain: effect of an inhibitor of beta-oxidation.

We examined the effect of a clinically therapeutic dose of methyl 2-tetradecylglycidate (McN-3716, methyl palmoxirate, MEP) (2.5 mg/kg), an inhibitor of beta-oxidation of fatty acids, on incorporation of radiolabeled palmitic acid ([U-14C]PAM) from plasma into brain lipids of awake rats. Four hour pretreatment with 2.5 mg/kg MEP significantly increased the incorporation of [U-14C]PAM into brain lipids and substantially decreased aqueous radiolabeled metabolites in brain that can constitute unwanted background signal when analyzed by quantitative autoradiography. MEP treatment increased the lipid to aqueous background radioactivity from 0.8 to 3.0. Net rate of incorporation, k*, was significantly increased (60%) by MEP and was attributed to incorporation of [U-14C]PAM into phospholipid and triglyceride brain compartments. MEP treatment did not affect the size of the fatty acyl-CoA pool or the distribution of the various molecular acyl-CoA species. These results indicate that MEP, at a dose of 2.5 mg/kg (per os), can be used to increase incorporation of [1-(11C)]PAM for studying brain lipid metabolism in humans by positron emission tomography (PET).

1,25-Dihydroxyvitamin D3-induced HL-60 macrophages: regulation of cholesterol and LDL metabolism.

Differentiation of human promyelocytic leukemic HL-60 cells with 1,25-dihydroxyvitamin D3 (D3) results in macrophages which exhibit specific and saturable receptor-mediated processing of both native and modified low density lipoprotein (LDL). Analysis of binding kinetics demonstrated that macrophages bind LDL and acetyl-LDL with similar affinities, yet possess significantly different numbers of receptors (55 +/- 6 x 10(3) LDL receptors/cell vs 79 +/- 7 x 10(3) acetyl-LDL receptors/cell). D3-induced HL-60 macrophages challenged with LDL or acetyl-LDL exhibited suppression of HMG-CoA reductase activity as well as a significant induction in the incorporation of [14C]oleate into cholesteryl ester compared with macrophages incubated with lipoprotein depleted serum. Maximum increases in ACAT activity were obtained in macrophages incubated with 25-hydroxycholesterol plus LDL or acetyl-LDL. The increase in ACAT activity in macrophages challenged with acetyl-LDL paralleled the increase in cellular cholesterol content and the increase of oil red O lipid stainable material, imparting the macrophages with a foamy appearance. The data indicate that D3-induced HL-60 macrophages are a useful model for the study of lipoprotein--macrophage interactions as related to foam cell development and atherogenesis.

Bioisosterism in drug design: identification of and structure-activity relationships in a series of glycine anilide ACAT inhibitors.

To examine the effects of bioisosteric replacement on the biological activity of our previously disclosed disubstituted urea inhibitors of the enzyme acyl-CoA:cholesterol acyltransferase (ACAT), we prepared a series of N'-substituted and N',N'-disubstituted glycine anilides. These compounds were tested for the ability to inhibit ACAT in vitro and lower plasma total cholesterol in cholesterol-fed rats given a single high-fat, high-cholesterol meal. ACAT inhibitory potency was greatest in compounds containing 2,6-diisopropyl substituents in the anilide portion with the glycine nitrogen substituted by a 1,1-diphenylmethyl moiety. Small improvements in potency in vitro were obtained by substitution of electron donating groups in the 2-, 3- or 5-positions of the aryl rings of the 1,1-diphenylmethyl moiety, but not by substitution in the 4-position. In vitro potency was maintained, but not improved by acylation of the glycine nitrogen. Through a QSAR analysis of in vitro ACAT inhibition for this set of compounds, an equation could be derived which accounted for 85% of the variance in the dataset. An optimal clogp of 6.65 was found, comparable to that found for other series of ACAT inhibitors. In general, compounds from this series displayed inhibitory potency against ACAT in vitro and hypocholesterolemic activity in the in vivo rat model of hypercholesterolemia comparable to that found with the ureas.

Effect of ursodeoxycholic acid on hepatic cholesterol metabolism.

Oral administration of ursodeoxycholic acid (UDCA) renders bile unsaturated with cholesterol by reducing the hepatic output of cholesterol. Theoretically, several mechanisms may be of importance. In the present overview, the effect of treatment with UDCA on hepatic cholesterol metabolism is evaluated, in particular the influence on hepatic cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity, bile acid synthesis, 7 alpha-hydroxylation of cholesterol, and esterification of cholesterol--acyl coenzyme A: cholesterol acetyltransferase (ACAT) activity. It is apparent that UDCA treatment does not inhibit the hepatic HMG CoA reductase activity. Neither is ACAT activity or the cholesteryl ester content changed by UDCA. The catabolism of cholesterol to bile acids is unaffected or slightly increased during administration of UDCA. It is concluded that a stimulated degradation of cholesterol to bile acids may partly explain the decrease in hepatic secretion of cholesterol obtained during UDCA administration. It is suggested that the reduction in cholesterol absorption from the intestine seen during UDCA therapy may also be of importance.

Total parenteral nutrition containing medium- vs. long-chain triglyceride emulsions elevates plasma cholesterol concentrations in rats.

Male Fischer 344 rats (235-246 g) were fed for 6-14 d by intravenous or intragastric infusion with total parenteral nutrition (TPN) solutions providing 40 or 65% of nonprotein energy as fat from long-chain triglyceride (LCT) or a 3:1 admixture of medium-chain triglyceride (MCT) and LCT emulsions. In three separate experiments, plasma cholesterol concentrations were significantly greater (24-32%) with intravenous infusion of TPN solutions containing MCT-LCT rather than LCT. Plasma cholesterol concentrations in rats were not significantly different with intragastric infusion of TPN solutions containing MCT-LCT rather than LCT. Hepatic total lipid and triglyceride concentrations were not significantly different. Hepatic total cholesterol and esterified cholesterol concentrations were significantly lower in animals given 65% of energy from MCT-LCT rather than LCT emulsions (main effects, two-way ANOVA). The concentration of individual hepatic acyl-CoA esters reflected the fatty acid profiles of the lipid emulsions infused. Total hepatic acyl-CoA concentrations suggested differences in utilization of acyl-CoA esters with intravenous infusion of MCT-LCT rather than LCT and were consistent with rapid oxidation of MCT. These data demonstrate that MCT-LCT elevates plasma cholesterol concentrations compared with LCT emulsions with intravenous, but not with intragastric, infusion of TPN solutions in rats.