INHIBITION OF CHOLESTEROL, FATTY ACID, AND TRIGLYCERIDE BIOSYNTHESES BY ORGANOSULFUR COMPOUNDS DERIVED FROM GARLIC IN PRIMARY CULTURES OF RAT HEPATOCYTES
Open Access
- Author:
- Liu, Lijuan
- Graduate Program:
- Nutrition
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 17, 2001
- Committee Members:
- Madhu Reddy, Committee Member
Yu Yan Yeh, Committee Chair/Co-Chair
Mary Frances Picciano, Committee Member
Penny Margaret Kris Etherton, Committee Member
Michael Henry Green, Committee Member - Keywords:
- fatty acid
HMG-CoA reductase
triglyceride
cholesterol
organosulfur compounds
garlic - Abstract:
- The lipid-lowering effect of garlic has been shown in both animal and human studies. However, the active ingredients responsible for the lipid-lowering effect of garlic are poorly understood and the underlying mechanisms of garlic action are not fully known. Therefore, the present studies were conducted to identify the active compounds of garlic on cholesterol, fatty acid, and triglyceride syntheses and to elucidate the possible mechanisms underlying the inhibition of lipid synthesis by the compounds of garlic in primary cultures of rat hepatocytes. The first objective of the thesis was to establish the inhibitory potency of organosulfur compounds of garlic on cholesterol biosynthesis. Cultured rat hepatocytes were treated with [14C]acetate as substrate for cholesterol synthesis in the presence or absence of test compounds at 0.05 to 4.0 mmol/L. Eleven water-soluble and six lipid-soluble compounds of garlic were tested. Among water-soluble compounds, three S-alk(en)yl cysteines, i.e., S-allyl-cysteine (SAC), S-ethyl cysteine (SEC), and S-propyl cysteine (SPC), inhibited [14C]acetate incorporation into cholesterol in a concentration-dependent manner achieving 42% to 55% maximal inhibition. Three g-glutamyl S-alk(en)yl cysteines, i.e., g-Glutamyl-S-allyl cysteine (GSAC), g-glutamyl-S-methyl cysteine (GSMC), and g-glutamyl-S-propyl cysteine (GSPC) were less potent, exerting only 16% to 29% maximal inhibition. S-allyl mercaptocysteine (SAMC), a disulfur-containing compound, inhibited the [14C]acetate incorporation into cholesterol by 7% to 17% at 0.05 and 0.5 mmol/L, but diminished the incorporation into cholesterol at 2.0 and 4.0 mmol/L. Alliin, S-allyl-N-acetyl cysteine (SANC), S-allylsulfonyl alanine (SASA), and S-methyl cysteine (SMC) had no effect on cholesterol synthesis. Of the lipid-soluble compounds, diallyl disulfide (DADS), diallyl trisulfide (DATS), and dipropyl disulfide (DPDS) depressed cholesterol synthesis by 10% to 25% at low concentrations (£ 0.5 mmol/L), and abolished the synthesis at high concentrations (³ 1.0 mmol/L). Diallyl sulfide, dipropyl sulfide, and methyl allyl sulfide slightly inhibited [14C]acetate incorporation into cholesterol only at high concentrations. The complete inhibition of cholesterol synthesis by DADS, DATS, and DPDS was associated with extensive cytotoxicity as indicated by marked increase in cellular lactate dehydrogenase (LDH) release. There was no apparent increase in LDH release by water-soluble compounds except SAMC, which also abolished cholesterol synthesis. A comparison of cytotoxicity, maximal inhibition, and IC50 (concentration required for 50% of maximal inhibition) of various compounds suggests that water-soluble compounds of garlic may hold greater potential than lipid-soluble compounds in inhibiting cholesterol synthesis, which in turn may reduce plasma concentration of cholesterol. SAC, SEC, and SPC are the most potent inhibitors of cholesterol synthesis. The second objective of the thesis was to determine the effects of water-soluble organosulfur compounds on triglyceride synthesis, fatty acid synthesis, and the activities of important lipogenic enzymes including fatty acid synthase (FAS) and glucose-6-phosphate dehydrogenase (G6PDH). When incubated with cultured hepatocytes, SAC and SPC decreased [14C]acetate incorporation into triglyceride in a concentration-dependent fashion (from 0.05 to 4.0 mmol/L), achieving maximal inhibitions of 43% and 51% at 4.0 mmol/L, respectively. The rate of [14C]acetate incorporation into phospholipids was depressed to a similar extent by SAC and SPC. SPC, SAC, SEC, and GSMC decreased [14C]acetate incorporation into fatty acid synthesis by 81%, 59%, 35%, and 40%, respectively, at 2.0-4.0 mmol/L concentrations. Alliin, GSAC, GSPC, SANC, SASA, and SMC had no effect on fatty acid synthesis. The activities of lipogenic enzymes, i.e., FAS and G6PDH were measured in cultured hepatocytes treated with the inhibitors. The activity of FAS in cells treated with SAC and SPC at the concentration of 4.0 mmol/L was 32% and 27 % lower than that of non-treated cells, respectively. Neither SAC nor SPC affected G6PDH activity. The results indicate that SAC, SEC, and SPC inhibit triglyceride biosynthesis in cultured rat hepatocytes. More important, the study suggest that these S-alk(en)yl cysteines of garlic reduce triglyceride synthesis by decreasing de novo fatty acid synthesis resulting from the inhibition on FAS. The third objective of the thesis was to examine the regulatory mechanisms underlying the inhibition of cholesterol synthesis by S-alk(en)yl cysteines. The treatment of cultured rat hepatocytes with S-alk(en)yl cysteines (i.e., SAC, SEC, and SPC), inhibited cholesterol synthesis from [14C]acetate but not from [14C]mevalonate, suggesting that the point of regulation is at 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase. Consistent with this notion, the activity of HMG-CoA reductase in the cells treated with SAC, SEC, and SPC was 30% to 40% lower than that of non-treatment group. Abundance of HMG-CoA reductase mRNA and the amount of the enzyme protein were not altered by the sulfur compounds. The expressed (E) and total (T) activities of HMG-CoA reductase were determined and the ratios of E/T were used to estimate the phosphorylation state of the enzyme. SAC, SEC, and SPC reduced the ratios of E/T by 18% to 29% resulting primarily from decreased expressed activity. The results suggest that organosulfur compounds of garlic decrease the activity of HMG-CoA reductase by phosphorylation but not by alteration in gene expression of the enzyme. In addition, the activity of HMG-CoA reductase was measured at a lower dithiothreitol concentration and preincubation of microsomes with phosphatase to test the involvement of thiol redox status. Among three S-alk(en)yl cysteines, SAC was the only compound found to suppress the activity of the enzyme. Thus, SAC appears to inhibit HMG-CoA reductase activity by not only phosphorylating the enzyme, but also increasing sulfhydryl oxidation of the enzyme. The results strongly indicate that S-alk(en)yl cysteines modify the activity of HMG-CoA reductase at the posttranslational level. In summary, the results of the present studies indicate that water-soluble organosulfur compounds are the most important compounds in inhibiting hepatocyte cholesterol synthesis. Lipid-soluble sulfur compounds, on the other hand, may not be the major compounds in the reduction of cholesterol synthesis because of their cytotoxicity. Among the organosulfur compounds, water-soluble compounds SAC, SEC, and SPC are the most potent inhibitors of cholesterol synthesis. The inhibition of cholesterol synthesis by S-alk(en)yl cysteines results from the decreased activity of HMG-CoA reductase. The suppressed activity of HMG-CoA reductase by S-alk(en)yl cysteines is attributed to the inactivation of the enzyme by phosphorylation but not gene expression of the enzyme. SAC may also increase sulfhydryl oxidation of the enzyme. In addition, S-alk(en)yl cysteines inhibit triglyceride synthesis due in part to reduced de novo fatty acid synthesis. These results have added to our understanding of the active constituents of garlic responsible for the lipid-lowering effects and the underlying mechanisms.