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Institute of Human Nutrition and Department of Pediatrics, Columbia University, New York, New York 10032
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Introduction |
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Work from the laboratory of Brown and Goldstein (2) first demonstrated that cholesterol can regulate gene expression via sterol regulatory elements (SRE). SREs are found in the promoter regions of genes relating to cholesterol, fatty acid, triglyceride, and carbohydrate metabolism (3). Generally, excess sterol leads to inhibition of SRE-dependent gene expression, and depletion of cellular sterol will lead to activation of SRE-dependent gene expression. The effects of cholesterol on SREs are regulated through distinct proteins: sterol regulatory element binding proteins (SREBP) and the SREBP-cleavage-activating-protein (SCAP). SREBPs contain about 1150 amino acids and are located in the endoplasmic reticulum in an inactive form. Their orientation is similar to a hair pin; both the COOH- and the NH2- terminal ends protrude from the endoplasmic reticulum into the cytoplasm. Sterol depletion initiates a two-step proteolytic process that results in the release of the transcriptionally active N-terminal end of SREBP. First, SCAP and SREBP translocate to the Golgi where the protein is cleaved at the intraluminal loop. The second proteolysis at an intramembrane site releases the mature SREBP. This transcription factor (MW 68,000 kDa) translocates to the nucleus where it binds to the SRE region and initiates transcription of the respective gene (4). There are two major forms of SREBP: SREBP-1a/1c and SREBP-2. SREBP-1a/1c mainly regulate pathways related to fatty acid and triglyceride metabolism as well as carbohydrate metabolism. In contrast, SREBP-2, which is encoded on a different gene, mainly regulates expression of genes important in sterol or cholesterol metabolism. Nevertheless, there is overlap between the activities of the SREBPs in fatty acid and cholesterol metabolism (5).
Recognizing the frequent and close links between fatty acid and cholesterol metabolism, our recent work explored the possibility that free fatty acids, not only sterols, would regulate SRE expression. In recently published work we showed that mono- and polyunsaturated fatty acids do mediate SRE expression and that this occurs through changes in SREBP maturation (6). Three different cell lines were chosen, each differing in cell cholesterol homeostatic mechanisms so that we might demonstrate the commonality of regulatory pathways. The cells were either transiently or stably transfected with SRE-containing promoters linked to a luciferase reporter gene that originated from the HMG CoA synthase promoter or the fatty acid synthase promoter. In experiments with fatty acids complexed to albumin at physiologic concentrations, monounsaturated and polyunsaturated fatty acids (but not saturated fatty acids) dose-dependently downregulated SRE-mediated gene expression. Effects of fatty acids on SRE promoters originating from different genes in the different cell lines produced very similar results. The effects of the fatty acids were synergistic to those of cholesterol in suppressing SRE-mediated gene expression. Generally, increasing unsaturation led to higher suppression of SRE-mediated gene expression (e.g., linoleic acid had greater effects than did oleic acid, and the effects were most marked with arachidonate and docosahexanoate). Our experiments showed that free fatty acids alone and synergistic with cholesterol decreased intracellular levels of mature SREBP and decreased mRNA expression for HMG-CoA synthase. In experiments using sterol regulation–defective cell lines and cell lines transfected with mutant SREs, we showed that the effects of fatty acids are dependent upon cellular levels of mature SREBP and are not independent effects of fatty acids acting directly on SREs. As well, our experiments suggested that fatty acids can act upon SREBPs through changes in cell cholesterol partitioning. Recently, other groups also demonstrated that polyunsaturated fatty acids can decrease SREBP-mediated gene expression (7), and this may involve pathways relating to intracellular metabolism of SREBP mRNAs (8).
More recent work in our group has indicated that fatty acids may act to redistribute sterols between the plasma membrane and intracellular compartments by at least two different mechanisms, one physical, and one biochemical. In collaboration with James Hamilton at Boston University, we have shown that free fatty acids decrease the affinity of free cholesterol in model phospholipid membranes and increase its transfer to other membranes (9). We postulate that if such effects occur in vivo, then free fatty acids will facilitate the transfer of cholesterol from the plasma membrane to intracellular compartments to decrease SREBP cleavage and decrease cellular levels of mature SREBP. In separate experiments, we have shown that free fatty acids increase cell sphingomyelinase activity and decrease cell sphingomyelin levels (10). Since sphingomyelin has higher affinity and binding capacity to cholesterol than other cell membrane phospholipids, decreasing membrane sphingomyelin would also lead to transfer of free cholesterol toward intracellular membranes and decrease SRE-mediated gene expression through the SREBP cascade.
How might these in vitro findings relate to the effects of unsaturated fatty acids on plasma lipoprotein metabolism in vivo? Certainly the effect of fatty acids on SREs does not explain upregulation of the LDL receptor activity in diets rich in polyunsaturated fatty acids. In this regard, recent work has demonstrated major control of LDL receptor gene expression in the LDL receptor promoter region distinct from SREs (11, 12). Thus, it is possible that dietary cholesterol or endogenous cholesterol downregulates the LDL receptor through SREs but that fatty acids have another point of action, still to be defined. In contrast, genes regulating fatty acid and triglyceride synthesis undergo major regulation via SRE expression (3). Our in vitro work suggests that high levels of mono- and polyunsaturated fat would suppress genes important to triglyceride synthesis, and this parallels human in vivo studies in which high polyunsaturated fat diets lead to decreased endogenous triglyceride synthesis. In contrast, diets very low in fat are associated with increased plasma triglyceride levels and can also lead to increased endogenous synthesis of triglycerides endogenously, and associated higher plasma levels of triglyceride-rich lipoproteins (13, 14).
We realize that the interactions between cholesterol, fatty acid, and carbohydrate metabolism are complex, and can occur on multiple levels. Work that will emerge within the next few years will help unravel the molecular mechanisms defining the interactions between sterol, fatty acid, and carbohydrate metabolism. Still, linking the results of our group and other laboratories with the impact of low-fat diets in clinical population studies (14, 15), we suggest that a "minimum" level of mono- and polyunsaturated fat intake may be needed to suppress expression of genes directly linked to triglyceride synthesis. We hypothesize that very low levels of exogenous fat intake are associated with higher levels of SRE-mediated gene expression in genes important to de-novo triglyceride synthesis, and this will contribute to the hypertriglyceridemia associated with very low dietary intake of fat.
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Footnotes |
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References |
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This article has been cited by other articles:
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  R. H. Knopp Introduction: Low-Saturated Fat, High-Carbohydrate Diets: Effects on Triglyceride and LDL Synthesis, the LDL Receptor, and Cardiovascular Disease Risk Experimental Biology and Medicine, December 1, 2000; 225(3): 175 - 177. [Full Text]
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