Feedback Inhibition Of Cholesterol Synthesis Research Articles

Cholesterol-induced conformational changes in the sterol-sensing domain of the Scap protein suggest feedback mechanism to control cholesterol synthesis

Scap is a polytopic protein of endoplasmic reticulum (ER) membranes that transports sterol regulatory element-binding proteins to the Golgi complex for proteolytic activation. Cholesterol accumulation in ER membranes prevents Scap transport and decreases cholesterol synthesis. Previously, we provided evidence that cholesterol inhibition is initiated when cholesterol binds to loop 1 of Scap, which projects into the ER lumen. Within cells, this binding causes loop 1 to dissociate from loop 7, another luminal Scap loop. However, we have been unable to demonstrate this dissociation when we added cholesterol to isolated complexes of loops 1 and 7. We therefore speculated that the dissociation requires a conformational change in the intervening polytopic sequence separating loops 1 and 7. Here we demonstrate such a change using a protease protection assay in sealed membrane vesicles. In the absence of cholesterol, trypsin or proteinase K cleaved cytosolic loop 4, generating a protected fragment that we visualized with a monoclonal antibody against loop 1. When cholesterol was added to these membranes, cleavage in loop 4 was abolished. Because loop 4 is part of the so-called sterol-sensing domain separating loops 1 and 7, these results support the hypothesis that cholesterol binding to loop 1 alters the conformation of the sterol-sensing domain. They also suggest that this conformational change helps transmit the cholesterol signal from loop 1 to loop 7, thereby allowing separation of the loops and facilitating the feedback inhibition of cholesterol synthesis. These insights suggest a new structural model for cholesterol-mediated regulation of Scap activity.

Insig Proteins Mediate Feedback Inhibition of Cholesterol Synthesis in the Intestine

Enterocytes are the only cell type that must balance the de novo synthesis and absorption of cholesterol, although the coordinate regulation of these processes is not well understood. Our previous studies demonstrated that enterocytes respond to the pharmacological blockade of cholesterol absorption by ramping up de novo sterol synthesis through activation of sterol regulatory element-binding protein-2 (SREBP-2). Here, we genetically disrupt both Insig1 and Insig2 in the intestine, two closely related proteins that are required for the feedback inhibition of SREBP and HMG-CoA reductase (HMGR). This double knock-out was achieved by generating mice with an intestine-specific deletion of Insig1 using Villin-Cre in combination with a germ line deletion of Insig2. Deficiency of both Insigs in enterocytes resulted in constitutive activation of SREBP and HMGR, leading to an 11-fold increase in sterol synthesis in the small intestine and producing lipidosis of the intestinal crypts. The intestine-derived cholesterol accumulated in plasma and liver, leading to secondary feedback inhibition of hepatic SREBP2 activity. Pharmacological blockade of cholesterol absorption was unable to further induce the already elevated activities of SREBP-2 or HMGR in Insig-deficient enterocytes. These studies confirm the essential role of Insig proteins in the sterol homeostasis of enterocytes.

Feedback inhibition of cholesterol synthesis and uptake

AbstractCells can obtain the cholesterol they need for membrane function either by synthesizing it or by taking it up from the circulation. Both processes are regulated by feedback inhibition. The capacity for synthesis can be changed by changing the amount of the enzyme HMG‐CoA reductase while the capacity for uptake can be changed by changing the amount of the LDL receptor. Transcription factors called SREBPs regulate synthesis of both proteins. SREBPs are synthesized as inactive precursors which are retained in the endoplasmic reticulum (er) when membrane cholesterol levels are high. However, when membrane cholesterol levels begin to fall SREBPs are released from the er, activated in the Golgi apparatus and stimulate the synthesis of both proteins in the nucleus. The Nobel Prize winners Michael Brown and Joseph Goldstein have established the molecular mechanism for this example of feedback inhibition. This has led to the discovery of cellular proteins such as SCAP which can monitor membrane cholesterol content.

Schoenheimer effect explained - feedback regulation of cholesterol synthesis in mice mediated by Insig proteins

End-product feedback inhibition of cholesterol synthesis was first demonstrated in living animals by Schoenheimer 72 years ago. Current studies define Insig proteins as essential elements of this feedback system in mouse liver. In cultured cells, Insig proteins are required for sterol-mediated inhibition of the processing of sterol regulatory element-binding proteins (SREBPs) to their nuclear forms. We produced mice with germline disruption of the Insig2 gene and Cre-mediated disruption of the Insig1 gene in liver. On a chow diet, these double-knockout mice overaccumulated cholesterol and triglycerides in liver. Despite this accumulation, levels of nuclear SREBPs and mRNAs for SREBP target genes in lipogenic pathways were not reduced. Whereas cholesterol feeding reduced nuclear SREBPs and lipogenic mRNAs in wild-type mice, this feedback response was severely blunted in the double-knockout mice, and synthesis of cholesterol and fatty acids was not repressed. The amount of HMG-CoA reductase protein was elevated out of proportion to the mRNA in the double-knockout mice, apparently owing to the failure of cholesterol to accelerate degradation of the enzyme. These studies indicate that the essential elements of the regulatory pathway for lipid synthesis function in liver as they do in cultured cells.

Reduced binding and removal of chylomicron remnants by ethionine-induced premalignant liver†

The suppression of cholesterol synthesis by dietary cholesterol which occurs in the livers of normal animals is absent in hepatomas. This abnormality has been reported to occur in the livers of animals fed hepatocarcinogens, even before there is any histologic evidence of malignancy (premalignant liver). We have proposed, in an earlier publication, that the deletion of feedback inhibition of cholesterol synthesis in malignancy is due, at least in part, to the loss of receptors which bind chylomicron remnants, the lipoprotein particles that transport dietary cholesterol to the liver. This hypothesis was further tested in the premalignant liver model. Rats were fed a diet containing 0.25% of a known hepatic carcinogen, ethionine. After 3 to 5 weeks on this diet, the liver had no histologic evidence of malignancy; the rate of [14C]acetate incorporation into cholesterol by liver homogenates was elevated as compared to that of controls (5.13 +/- 0.70 vs. 0.65 +/- 0.14 nmoles cholesterol per gm per hr), and in contrast to control animals, this was not reduced by the inclusion of 5% cholesterol in the diet for 48 hr before killing. The serum (44.4 +/- 6.3 vs. 51.4 +/- 3.8 mg per 100 ml) and hepatic (15.8 +/- 0.2 vs. 17.0 +/- 0.4 micrograms per mg protein) cholesterol contents were not substantially different in ethionine-fed as compared to control-fed rats. Hepatic cholesterol content increased when cholesterol was included in the diet (15.8 +/- 0.2 to 25.8 +/- 7.3 micrograms per mg protein and 17.0 +/- 0.4 to 36 +/- 3.7 micrograms per mg protein in ethionine-fed and control-fed animals, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

The Effect of Dried and Fresh Eggs on Plasma Cholesterol and Atherosclerosis in Chickens

Oxidation products of cholesterol, which have been observed to be formed in powdered eggs and other dehydrated cholesterol-containing foods, have been suspected as the probable primary cause of atherosclerotic lesions. The feeding of powdered vs. fresh egg yolks to young male chickens up to 30 and 43 weeks of age, respectively, produced approximately the same increase in plasma cholesterol, liver fat, and liver cholesterol when compared to the feeding of a soybean-based cholesterol-free diet. Aortic atherosclerosis, however, was found to be more severe in a group of birds fed fresh egg yolks than in those fed powdered egg or the cholesterol-free diet. A previously demonstrated, enhanced feedback inhibition of cholesterol synthesis by oxidized cholesterol derivatives may at least partly explain these results.

Effect of lactation on cholesterol synthesis in rats.

Lactation induces a variety of morphological and functional changes in the gastrointestinal tract. In the present study we employed tritiated water as the substrate to demonstrate that in the intact rat lactation results in a twofold increase in cholesterol synthesis in the small intestine. Feeding a high-cholesterol diet did not markedly inhibit small intestinal cholesterol synthesis in either control or lactating animals, and the difference in cholesterol synthesis between the two groups persisted. In the large intestine, cholesterol synthesis is increased threefold in the lactating animals, and feeding a high-cholesterol diet did not affect synthesis in either the control or lactating animals. In the liver, lactation stimulated cholesterol synthesis, and quantitatively this increase in hepatic cholesterol synthesis is much greater than the increase observed in the intestines. Feeding the rats a high-cholesterol diet markedly inhibited hepatic cholesterol synthesis in both control and lactating animals, a finding that demonstrates that the feedback inhibition of cholesterol synthesis in the liver is not impaired by lactation. In the lactating animals, the quantity of labeled cholesterol in 1 ml of serum is 2.4 times greater than observed in controls. Feeding the rats a high-cholesterol diet markedly decreased the quantity of labeled cholesterol in the serum in both groups and obliterated the difference between control and lactating animals. This suggests that the increased hepatic cholesterol synthesis in the lactating animals is responsible for the differences in labeled cholesterol in the serum. Cholesterol feeding also reduced the quantity of labeled cholesterol localized to the mammary glands in lactating animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion of a nonspecific lipid transfer protein by hepatoma cells in culture

Nonspecific lipid transfer protein (sterol carrier protein 2) has previously been proposed to function as (i) a catalyst for intracellular movement of newly synthesized phospholipid, (ii) a cofactor in the biosynthesis and metabolism of cholesterol, and (iii) a cofactor in the feedback inhibition of cholesterol synthesis. Each of these functions is based upon the premise that nonspecific lipid transfer protein (nsLTP) is cytosolic. However, evidence presented in this report suggests that, at least in the case of cultured hepatoma cells, nsLTP is secreted. This conclusion is supported by three observations. First, after culture of hepatoma cells for 10 h, 88% of the nsLTP (as judged by its phosphatidylethanolamine transfer activity) appears in the medium, whereas the cytosolic level of transfer activity remains unchanged. Furthermore, this is accompanied by the appearance in the medium of a polypeptide of M r 12,200–12,500, which corresponds to the known molecular weight of nsLTP. Finally, it was observed that the appearance of both the activity and the polypeptide in the medium are inhibited by monensin, an inhibitor of secretion. Thus their appearance seems to represent secretion and not simply leakage from the cells. Further evidence that nsLTP does not play an important role in the cytosolic transport of phospholipid and sterol is provided by our observation that hepatoma cells containing a level of nsLTP only 10–15% of that found in liver nevertheless possess near-normal membrane phospholipid compositions and retain the ability to feedback-inhibit cholesterol biosynthesis.

Lipoprotein metabolism by rat hepatomas. Studies on the etiology of defective dietary feedback inhibition of cholesterol synthesis.

In contrast to normal liver, it is known that in vivo hepatomas fail to decrease their rate of cholesterol biosynthesis in response to increased dietary cholesterol. From a consideration of the available data it has been hypothesized that the defect might lie in the delivery of cholesterol to the hepatoma cell. To study this further, lipoprotein interactions with rat hepatoma cells in tissue culture (HTC 7288C) and with the same cell line in vivo were investigated. HTC cells grown in a medium containing 10% calf serum exhibited saturable, specific, calcium-dependent binding of rat 125I-chylomicron remnants at 4 degrees C with half maximal saturation at 4.8 micrograms protein/ml and maximum binding of 96 ng protein/10(6) cells. At 4 degrees C, HTC cells also bound human 125I-low density lipoprotein (LDL) specifically, but bound it with a much lower affinity. These cells also exhibited specific binding for rat LDL and rat hypercholesterolemic very low density lipoprotein (VLDL). All these lipoproteins were degraded by HTC cells. Thus, it was concluded that hepatoma cells possess lipoprotein receptors that recognize and process LDL, VLDL, and chylomicron remnants. Overnight incubation of HTC cells in lipid-depleted medium containing 0.5 microM compactin increased binding of rat chylomicron remnants and of hypercholesterolemic VLDL approximately 1.7-fold without a significant change in binding affinity. LDL binding also increased, by approximately 3.5-fold. These changes were also observed when binding and internalization were measured at 37 degrees C. After HTC cells were incubated in lipid-depleted medium, the rate at which [14C]acetate was incorporated into [14C]cholesterol increased 2.5-fold. Inclusion of rat chylomicron remnants at 5-10 micrograms protein/ml prevented this increase in acetate incorporation or, if added after culture in lipid-depleted medium, reduced the increased levels back to control values. However, the rate of acetate incorporation into cholesterol by cells grown in complete medium was not decreased to levels below base line by rat chylomicron remnants. Inclusion of human LDL only partially prevented the rise or only partially reduced the increased levels back to control and did not reduce control levels below base line. Hypercholesterolemic VLDL, which contain more cholesterol per particle than chylomicron remnants, did reduce [14C]acetate incorporation to below control levels. Therefore, the intracellular mechanism for down regulation of cholesterol synthesis by lipoproteins is intact in these cells. Based on these results we hypothesized that a relative lack of lipoprotein receptors expressed by hepatomas in vivo in comparison with those expressed by normal liver would explain the apparent absence of feedback inhibition of cholesterol synthesis. Consistent with this hypothesis, the binding of chylomicron remnants to liver cell membranes was 3-5 times greater than to membranes from tumors grown in vivo subcutaneously or intramuscularly. Membranes from tumor cells grown in vitro bound remnants least well. It is proposed that the relative lack of receptors places the hepatoma at a disadvantage in competing with the liver for lipoproteins of dietary origin and may account for the lack of feedback regulation of cholesterol synthesis in hepatomas.

Relationship of alcoholic hyperlipidemia to the feed-back regulation of hepatic cholesterol synthesis by chylomicron remnants.

Pairfeeding of ethanol liquid diet for 6 weeks to male Wistar rats resulted in defective extrahepatic as well as hepatic catabolism of rat lymph chylomicrons. Based upon the exponential decay curves of the chylomicrons in the blood compartment it was concluded that chronic ethanol feeding caused 30 and 67% decreases in the rate of degradation of the triacylglycerol and cholesterol moieties, respectively. As a consequence, abnormal chylomicron remnants accumulated in chronic ethanol-fed but not in control animals. These abnormal remnants were not as efficient as the normal remnants in causing the feedback inhibition of cholesterol synthesis in hepatocytes from normal meal-fed rats. Furthermore, the hepatocytes from chronic ethanol-fed animals exhibited defective feedback regulation of cholesterol synthesis by normal chylomicron remnants. The net result of all these abnormalities caused by chronic ethanol feeding would be the delayed clearance of triacylglycerol-rich lipoproteins from the blood compartment and enhanced synthesis and secretion of the triacylglycerol-rich lipoproteins by the liver.

Diagnosis of familial hypercholesterolemia by measurement of sterol synthesis in cultured skin fibroblasts

The incorporation of radioactive acetate into the digitonin precipitable fraction (cholesterol) was measured in monolayers of primary cultures of skin fibroblasts. Mean incorporation was increased approximately 20-fold in 4 subjects homozygous for familial hypercholesterolemia (FH) and 4-fold in 6 heterozygotes derived from the immediate family of homozygotes. Incorporation was normal in 4 subjects with Type IV and V hyperlipoproteinemia. In cells that had been preincubated in lipid free medium, incorporation by cells from homozygotes was equal to controls, denoting a derangement in the feedback inhibition of cholesterol synthesis by medium lipids in FH. The activity of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase paralleled the values obtained for sterol synthesis. The assay described could be useful in making an “etiologic” diagnosis of familial hypercholesterolemia and could possibly identify variants of monogenic hyperbetalipoproteinemia.

Feedback regulation of cholesterol biosynthesis: studies with cholestyramine.

Regulation of hepatic cholesterol biosynthesis is thought to occur at the step involving formation of mevalonate from 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA); this reaction is catalyzed by HMG-CoA reductase. Oral administration of cholestyramine, an agent that interferes with cholesterol reabsorption and is associated with a compensatory increase in hepatic cholesterol synthesis, was used to evaluate feedback regulation of cholesterol biosynthesis. Liver slices and cell-free fractions were prepared from control rats and rats fed 3% cholestyramine for 3-7 days. Cholestyramine feeding was associated with a four- to eightfold increase in incorporation of [ 14 C]acetate into cholesterol and mevalonate. However, there was no effect on incorporation of [ 14 C]acetate into CO 2 , fatty acids, or acetoacetate or on incorporation of [ 14 C]mevalonate into cholesterol, indicating stimulation at an early regulatory site prior to mevalonate formation. In fractionated liver homogenates, incorporation of [ 14 C] acetate, [ 14 C]acetyl-CoA, and [ 14 C]HMG-CoA into mevalonate increased markedly following cholestyramine administration. In the absence of microsomes, incorporation of [ 14 C]acetate and [ 14 C]acetyl-CoA into HMG was also increased. Changes in incorporation of [ 14 C]acetate into HMG, mevalonate, and cholesterol corresponded to changes in product synthesis. I concluded that (1) interruption of the enterohepatic circulation of cholesterol causes a release of feedback inhibition of cholesterol synthesis, (2) mevalonate synthesis catalyzed by HMG-CoA condensing enzyme and HMG-CoA reductase, and (3) HMG-CoA condensing enzyme, by limiting the availability of HMG-CoA, may have an important regulatory function in cholesterol biosynthesis.