- Email: [email protected]
Effect of glibenclamide on cholesterol metabolism in macrophages
Tuesday 11 October 1994: Poster Abstracts Metabolism
pJ Wolfow,
Cholesterol regulation In ras mutated rat fibroblasts Fuhrman B, Brook JG, Lipid Res. Lab., The Bruce
Rappaport Faculty of Med., Technion, Hatfa, Israel
Some cancer patients demonstrate low levels of plasma cholesterol. This has been explained as resulting from a defect in the cholesterol feedback control in tumor cells. The objective of this study was to determine the presence of a relationship between cholesterol metabolism and regulation, and between the occurrence of mutated ras protein activity. Total cholesterol mass in the mutated rat embryo fibroblasts was 2-4 times less than that in control cells (non-mutated rat embryo fibroblasts). Phospholipid content also fell, but to a significantly lesser extent than ihe cholesterol. LDL uotake. as determined bv 1251-labeled LDL demadation was twjce as high in the r& mutated cells. Downregulation of LDL uptake in both mutated and non-mutated cells was similar, It appears that in these mutated cells, over-utilization of the farnesyl pathway results in decreased accumulation of cholesterol in the cells. We suggest that a relationship between mutated ras protein activity and cholesterol metabolism is present and this has little influence on the intracellular cholesterol control. Characterization of S-AMP activated protein kinase
11061 in human liver using speciftc peptide substrates and the effects of S-AMP analogues on enzyme activity in vitro and in primary rat adipocytes Sullivan JE, Brocklehurst K, Marley AE, Carey F, Carling D*, J$&_K& Cardiovascular and Metabolism Res. Dept., ZENECA
119
artery disease (CAD) (minimal stenosis of 70%) and 11 women without CAD (maximal stenosis of 20%), matched for age and risk factors. Women with fasting plasma cholesterol (chol) values %.5 mmovl and triglycerides (TG) >2.5 mmolfl were excluded. The study was performed using oral fat tolerance tests (24 h; 50 g fat/m* body surface area). Fasting parameters of lipoprotein metabolism, including LDLchol, did not differ between the groups, with the exception of plasma-chol (5.7 rt 0.13 (SEM) in patients vs 5.1 f 0.19 mmoyl in controls; P < 0.05). Postprandial TG metabolism, expressed as area under the 8 and 24 h curves (AUCI, AUC24), was significantly impaired in the patient group in the Sf >lOOO-fraction, AUC24 (4.5 f0.38 vs 3.1 f0.51 mmol/hn; P< 0.05) and Sf
1
Pharmaceuticals, Alderley Park, UK; *MRC Molecular Med. Group, London, UK
Kobe Univ. Sch. of Med., Kusunoki cho 7-5-1, Chuo-ku. Kobe, 650 Japan
5’-AMP activated protein kinase (AMPK) is a recently defined multisubstrate enzyme that plays a key role in the regulation of fatty acid and cholesterol metabolism. Recently, an in vitro peptide (SAMS peptide) phosphorylation assay to specifically mea.ure rat liver AMPK activity has been developed. Hem, we report that the SAMS peptide and a peptide based on the sequence surrounding the site phosphorylated on HMG CoA-reductase by AMPK (HMG peptide) can be used to measure human liver AMPK. Our data demonstrate that both human and rat AMPKs have a higher affinity for the HMG peptide than the SAMS peptide in the presence and absence of S-AMP. In the presence of SAMP, the Km values (uM) for human AMPK were found to be 18 + 5 for the HMG peptide but 36 Z!Z 4 for the SAMS peptide. In the absence of S-AMP, the Kms were 52 f 5 (HMG peptide) compared to 143 + 50. Using these peptide phosphorylation assays, we have found that in addition to 5’-AMP, the structural analogues adenosine 5’-thiomonophosphate (ATMP) and adenosine 5’-phosphoramidate (APR) stimulate both rat and human AMPKs in vitro, with both compounds having a higher affinity than 5’-AMP (Kms for AMP, 4yM; for ATMP, 0.65pM; for APR. I .9pM). Several novel agents have been tested in primary adipocytes and shown to profoundly inhibit lipolysis and lipogenesis. Recent data demonstrating activation of AMPK in these isolated cells may explain the effects observed on lipid metabolism.
Glibenclamide is widely used for the treatment of diabetes mellitus. However, the effects of this drug on atherogenesis are controversial. In this study, we investigated the effects of glibenclamide on cellular cholesterol metabolism in THP- 1 human macrophages. The cells were cultured in RPMI- 1640 medium containing LDL in the presence or absence of various concentrations of glibenclamide (O.l-1OpM) and cellular cholesterol content was measured. Cellular cholesteryl ester content increased 36.6% on incubation with 5Opg/ml LDL for 48 h. However, glibenclamide inhibited cholesteryl ester accumulation in a dose-dependent manner and even decreased its content at 10pM compared with time 0 control. This effect was observed after 12-24 h incubation and sustained for up to 72 h. To elucidate the inhibitory mechanism of cholesteryl ester accumulation by gbbenclamide, the cells were incubated with ‘251-labeled LDL in the presence or absence of glibenclamide. There were no significant differences in association and degradation of ‘251-labeled LDL. Glibenclamide stimulated ACAT activity by 33.6% in 24 h. Cholesterol efflux was determined by measuring an amount of free cholesterol in the medium after incubation with [3H]mevalonolactone. Glibenclamide increased cholesterol efflux from macrophages by 22% in 24 h. Net cholesteryl ester hydrolysis was assessed by measuring the cellular cholesteryl ester by addition of ACAT inhibitor, compound 58-035, in the presence or absence of glibenclamide. Glibenclamide increased cholesteryl ester hydrolysis twofold. These results suggest that glibenclamide inhibits an accumulation of cholesteryl ester in THP-1 human macrophages by enhancing cholesteryl ester hydrolysis and cholesterol efflux.
Impaired clearance of postprandial triglyceride-rich lipoproteins in women with angiographically proven coronary artery disease I&&r-B, Heystek FC, Erkelens DW, Rienks R, de Bruin TWA,
1
Dept. of Med., Utrecht Univ., PO Box 85.500, 3508 GA Utrecht, The Netherlands
Remnants of triglyceride-rich-lipoproteins (TRLP) are considered to be atherogenic. Therefore, postprandial metabolism of TRLP was studied in 11 women with angiographically proven coronary
The mechanism of lack of hypocholesterolemic effect of pravastatin sodium, a 3-hydroxy-3-methylglutaryl coenxyme A reductase inhibitor, in rats w’, Nara F*, Tsujita Y’, Fukushige .I*, Fukami M2, Kuroda M3, ‘Pharmacology and Molecular Biology Res. Labs., 11091
‘Biological Res. Labs., 3R&D Planning Dept., Sankyo Co., Ltd., Hiromachi, Shinagawa-ku, Tokyo, Japan
Atherosclerosis X, Montreal, October I994
pJ Wolfow,
Cholesterol regulation In ras mutated rat fibroblasts Fuhrman B, Brook JG, Lipid Res. Lab., The Bruce
Rappaport Faculty of Med., Technion, Hatfa, Israel
Some cancer patients demonstrate low levels of plasma cholesterol. This has been explained as resulting from a defect in the cholesterol feedback control in tumor cells. The objective of this study was to determine the presence of a relationship between cholesterol metabolism and regulation, and between the occurrence of mutated ras protein activity. Total cholesterol mass in the mutated rat embryo fibroblasts was 2-4 times less than that in control cells (non-mutated rat embryo fibroblasts). Phospholipid content also fell, but to a significantly lesser extent than ihe cholesterol. LDL uotake. as determined bv 1251-labeled LDL demadation was twjce as high in the r& mutated cells. Downregulation of LDL uptake in both mutated and non-mutated cells was similar, It appears that in these mutated cells, over-utilization of the farnesyl pathway results in decreased accumulation of cholesterol in the cells. We suggest that a relationship between mutated ras protein activity and cholesterol metabolism is present and this has little influence on the intracellular cholesterol control. Characterization of S-AMP activated protein kinase
11061 in human liver using speciftc peptide substrates and the effects of S-AMP analogues on enzyme activity in vitro and in primary rat adipocytes Sullivan JE, Brocklehurst K, Marley AE, Carey F, Carling D*, J$&_K& Cardiovascular and Metabolism Res. Dept., ZENECA
119
artery disease (CAD) (minimal stenosis of 70%) and 11 women without CAD (maximal stenosis of 20%), matched for age and risk factors. Women with fasting plasma cholesterol (chol) values %.5 mmovl and triglycerides (TG) >2.5 mmolfl were excluded. The study was performed using oral fat tolerance tests (24 h; 50 g fat/m* body surface area). Fasting parameters of lipoprotein metabolism, including LDLchol, did not differ between the groups, with the exception of plasma-chol (5.7 rt 0.13 (SEM) in patients vs 5.1 f 0.19 mmoyl in controls; P < 0.05). Postprandial TG metabolism, expressed as area under the 8 and 24 h curves (AUCI, AUC24), was significantly impaired in the patient group in the Sf >lOOO-fraction, AUC24 (4.5 f0.38 vs 3.1 f0.51 mmol/hn; P< 0.05) and Sf
1
Pharmaceuticals, Alderley Park, UK; *MRC Molecular Med. Group, London, UK
Kobe Univ. Sch. of Med., Kusunoki cho 7-5-1, Chuo-ku. Kobe, 650 Japan
5’-AMP activated protein kinase (AMPK) is a recently defined multisubstrate enzyme that plays a key role in the regulation of fatty acid and cholesterol metabolism. Recently, an in vitro peptide (SAMS peptide) phosphorylation assay to specifically mea.ure rat liver AMPK activity has been developed. Hem, we report that the SAMS peptide and a peptide based on the sequence surrounding the site phosphorylated on HMG CoA-reductase by AMPK (HMG peptide) can be used to measure human liver AMPK. Our data demonstrate that both human and rat AMPKs have a higher affinity for the HMG peptide than the SAMS peptide in the presence and absence of S-AMP. In the presence of SAMP, the Km values (uM) for human AMPK were found to be 18 + 5 for the HMG peptide but 36 Z!Z 4 for the SAMS peptide. In the absence of S-AMP, the Kms were 52 f 5 (HMG peptide) compared to 143 + 50. Using these peptide phosphorylation assays, we have found that in addition to 5’-AMP, the structural analogues adenosine 5’-thiomonophosphate (ATMP) and adenosine 5’-phosphoramidate (APR) stimulate both rat and human AMPKs in vitro, with both compounds having a higher affinity than 5’-AMP (Kms for AMP, 4yM; for ATMP, 0.65pM; for APR. I .9pM). Several novel agents have been tested in primary adipocytes and shown to profoundly inhibit lipolysis and lipogenesis. Recent data demonstrating activation of AMPK in these isolated cells may explain the effects observed on lipid metabolism.
Glibenclamide is widely used for the treatment of diabetes mellitus. However, the effects of this drug on atherogenesis are controversial. In this study, we investigated the effects of glibenclamide on cellular cholesterol metabolism in THP- 1 human macrophages. The cells were cultured in RPMI- 1640 medium containing LDL in the presence or absence of various concentrations of glibenclamide (O.l-1OpM) and cellular cholesterol content was measured. Cellular cholesteryl ester content increased 36.6% on incubation with 5Opg/ml LDL for 48 h. However, glibenclamide inhibited cholesteryl ester accumulation in a dose-dependent manner and even decreased its content at 10pM compared with time 0 control. This effect was observed after 12-24 h incubation and sustained for up to 72 h. To elucidate the inhibitory mechanism of cholesteryl ester accumulation by gbbenclamide, the cells were incubated with ‘251-labeled LDL in the presence or absence of glibenclamide. There were no significant differences in association and degradation of ‘251-labeled LDL. Glibenclamide stimulated ACAT activity by 33.6% in 24 h. Cholesterol efflux was determined by measuring an amount of free cholesterol in the medium after incubation with [3H]mevalonolactone. Glibenclamide increased cholesterol efflux from macrophages by 22% in 24 h. Net cholesteryl ester hydrolysis was assessed by measuring the cellular cholesteryl ester by addition of ACAT inhibitor, compound 58-035, in the presence or absence of glibenclamide. Glibenclamide increased cholesteryl ester hydrolysis twofold. These results suggest that glibenclamide inhibits an accumulation of cholesteryl ester in THP-1 human macrophages by enhancing cholesteryl ester hydrolysis and cholesterol efflux.
Impaired clearance of postprandial triglyceride-rich lipoproteins in women with angiographically proven coronary artery disease I&&r-B, Heystek FC, Erkelens DW, Rienks R, de Bruin TWA,
1
Dept. of Med., Utrecht Univ., PO Box 85.500, 3508 GA Utrecht, The Netherlands
Remnants of triglyceride-rich-lipoproteins (TRLP) are considered to be atherogenic. Therefore, postprandial metabolism of TRLP was studied in 11 women with angiographically proven coronary
The mechanism of lack of hypocholesterolemic effect of pravastatin sodium, a 3-hydroxy-3-methylglutaryl coenxyme A reductase inhibitor, in rats w’, Nara F*, Tsujita Y’, Fukushige .I*, Fukami M2, Kuroda M3, ‘Pharmacology and Molecular Biology Res. Labs., 11091
‘Biological Res. Labs., 3R&D Planning Dept., Sankyo Co., Ltd., Hiromachi, Shinagawa-ku, Tokyo, Japan
Atherosclerosis X, Montreal, October I994