Feedback Control Of Cholesterol Synthesis Research Articles

Age-related changes in regiospecific expression of Lipolysis Stimulated Receptor (LSR) in mice brain.

The regulation of cholesterol, an essential brain lipid, ensures proper neuronal development and function, as demonstrated by links between perturbations of cholesterol metabolism and neurodegenerative diseases, including Alzheimer’s disease. The central nervous system (CNS) acquires cholesterol via de novo synthesis, where glial cells provide cholesterol to neurons. Both lipoproteins and lipoprotein receptors are key elements in this intercellular transport, where the latter recognize, bind and endocytose cholesterol containing glia-produced lipoproteins. CNS lipoprotein receptors are like those in the periphery, among which include the ApoB, E binding lipolysis stimulated lipoprotein receptor (LSR). LSR is a multimeric protein complex that has multiple isoforms including α and α’, which are seen as a doublet at 68 kDa, and β at 56 kDa. While complete inactivation of murine lsr gene is embryonic lethal, studies on lsr +/- mice revealed altered brain cholesterol distribution and cognitive functions. In the present study, LSR profiling in different CNS regions revealed regiospecific expression of LSR at both RNA and protein levels. At the RNA level, the hippocampus, hypothalamus, cerebellum, and olfactory bulb, all showed high levels of total lsr compared to whole brain tissues, whereas at the protein level, only the hypothalamus, olfactory bulb, and retina showed the highest levels of total LSR. Interestingly, major regional changes in LSR expression were observed in aged mice which suggests changes in cholesterol homeostasis in specific structures in the aging brain. Immunocytostaining of primary cultures of mature murine neurons and glial cells isolated from different CNS regions showed that LSR is expressed in both neurons and glial cells. However, lsr RNA expression in the cerebellum was predominantly higher in glial cells, which was confirmed by the immunocytostaining profile of cerebellar neurons and glia. Based on this observation, we would propose that LSR in glial cells may play a key role in glia-neuron cross talk, particularly in the feedback control of cholesterol synthesis to avoid cholesterol overload in neurons and to maintain proper functioning of the brain throughout life.

Hypoxia-inducible factor 1α activates insulin-induced gene 2 (Insig-2) transcription for degradation of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase in the liver

Cholesterol synthesis is a highly oxygen-consuming process. As such, oxygen deprivation (hypoxia) limits cholesterol synthesis through incompletely understood mechanisms mediated by the oxygen-sensitive transcription factor hypoxia-inducible factor 1α (HIF-1α). We show here that HIF-1α links pathways for oxygen sensing and feedback control of cholesterol synthesis in human fibroblasts by directly activating transcription of the INSIG-2 gene. Insig-2 is one of two endoplasmic reticulum membrane proteins that inhibit cholesterol synthesis by mediating sterol-induced ubiquitination and subsequent endoplasmic reticulum-associated degradation of the rate-limiting enzyme in the pathway, HMG-CoA reductase (HMGCR). Consistent with the results in cultured cells, hepatic levels of Insig-2 mRNA were enhanced in mouse models of hypoxia. Moreover, pharmacologic stabilization of HIF-1α in the liver stimulated HMGCR degradation via a reaction that requires the protein's prior ubiquitination and the presence of the Insig-2 protein. In summary, our results show that HIF-1α activates INSIG-2 transcription, leading to accumulation of Insig-2 protein, which binds to HMGCR and triggers its accelerated ubiquitination and degradation. These results indicate that HIF-mediated induction of Insig-2 and degradation of HMGCR are physiologically relevant events that guard against wasteful oxygen consumption and inappropriate cell growth during hypoxia.

Contribution of Accelerated Degradation to Feedback Regulation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase and Cholesterol Metabolism in the Liver

Accumulation of sterols in endoplasmic reticulum membranes stimulates the ubiquitination of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), which catalyzes a rate-limiting step in synthesis of cholesterol. This ubiquitination marks HMGCR for proteasome-mediated degradation and constitutes one of several mechanisms for feedback control of cholesterol synthesis. Mechanisms for sterol-accelerated ubiquitination and degradation of HMGCR have been elucidated through the study of cultured mammalian cells. However, the extent to which these reactions modulate HMGCR and contribute to control of cholesterol metabolism in whole animals is unknown. Here, we examine transgenic mice expressing in the liver the membrane domain of HMGCR (HMGCR (TM1-8)), a region necessary and sufficient for sterol-accelerated degradation, and knock-in mice in which endogenous HMGCR harbors mutations that prevent sterol-induced ubiquitination. Characterization of transgenic mice revealed that HMGCR (TM1-8) is appropriately regulated in the liver of mice fed a high cholesterol diet or chow diet supplemented with the HMGCR inhibitor lovastatin. Ubiquitination-resistant HMGCR protein accumulates in the liver and other tissues disproportionately to its mRNA, indicating that sterol-accelerated degradation significantly contributes to feedback regulation of HMGCR in vivo Results of these studies demonstrate that HMGCR is subjected to sterol-accelerated degradation in the liver through mechanisms similar to those established in cultured cells. Moreover, these studies designate sterol-accelerated degradation of HMGCR as a potential therapeutic target for prevention of atherosclerosis and associated cardiovascular disease.

The cytoplasmic and extracellular domains of LRP1 promote a convergent mechanism for feedback control of cholesterol synthesis and export, and protect against intracellular cholesterol accumulation

The cytoplasmic and extracellular domains of LRP1 promote a convergent mechanism for feedback control of cholesterol synthesis and export, and protect against intracellular cholesterol accumulation

Ablation of gp78 in Liver Improves Hyperlipidemia and Insulin Resistance by Inhibiting SREBP to Decrease Lipid Biosynthesis

gp78 is a membrane-anchored ubiquitin ligase mediating the degradation of HMG-CoA reductase (HMGCR) and Insig-1. As a rate-limiting enzyme in cholesterol biosynthesis, HMGCR undergoes rapid sterol-promoted degradation. In contrast, destruction of Insig-1 releases its inhibition on SREBP and stimulates the expression of lipogenic genes. Thus, gp78 has opposite effects on lipid biosynthesis. We here generated liver-specific gp78 knockout (L-gp78(-/-)) mice and showed that although the degradation of HMGCR was blunted, SREBP was suppressed due to the elevation of Insig-1/-2, and therefore the lipid biosynthesis was decreased. The L-gp78(-/-) mice were protected from diet-/age-induced obesity and glucose intolerance. The livers of L-gp78(-/-) mice produced more FGF21, which activated thermogenesis in brown adipocytes and enhanced energy expenditure. Together, the major function of gp78 in liver is regulating lipid biosynthesis through SREBP pathway. Ablation of gp78 decreases the lipid levels and increases FGF21, and is beneficial to patients with metabolic diseases.

Joseph Goldstein and Michael Brown

Joseph Goldstein and Michael Brown

The INSIG1 gene, not the INSIG2 gene, associated with coronary heart disease: tagSNPs and haplotype-based association study

Dyslipidemia, especially hypercholesterolemia, is a major risk factor of coronary heart disease (CHD). The insulin induced gene 1 (INSIG1) and insulin induced gene 2 (INSIG2) encode two proteins which mediate feedback control of cholesterol synthesis. We conducted a case-control study to investigate whether the common variants in INSIG genes were associated with CHD in Chinese Han population. Three single nucleotide polymorphisms (SNPs) of the INSIG1 gene and four SNPs of the INSIG2 gene were chosen as haplotype-tagging SNPs (htSNPs) and genotyped in 853 patients with CHD and 948 unrelated control subjects. Haplotype analysis showed that the haplotype Hap4 (TTA) of the INSIG1 gene significantly increased the risk of CHD (adjusted odds ratio [OR]1.59, 95% confidence interval [CI] 1.22-2.06, p = 0.0006), while the haplotype Hap3 (TGA) significantly decreased the risk of CHD (adjusted OR 0.74, 95%CI 0.60-0.92, p = 0.006) compared with the reference haplotype Hap1 (GGA). No significant associations were found between polymorphisms of INSIG2 gene and CHD. In addition, the single polymorphism analysis showed that rs9769826 of the INSIG1 gene was associated with glucose in controls. The G-allele (minor allele) carriers had higher glucose level (5.74 +/- 2.03 mM) than AA genotype carriers (5.45 +/- 1.37 mM, p = 0.015). The present study indicated that the INSIG1 gene, but not the INSIG2 gene, was associated with CHD in the Chinese population.

Juxtamembranous aspartic acid in Insig-1 and Insig-2 is required for cholesterol homeostasis.

Insig-1 and Insig-2 are closely related proteins of the endoplasmic reticulum (ER) that mediate feedback control of cholesterol synthesis by sterol-dependent binding to the following two membrane proteins: the escort protein Scap, thus preventing proteolytic processing of sterol regulatory element-binding proteins; and the cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl CoA reductase, thus inducing the ubiquitination and ER-associated degradation of the enzyme. Here, we report that the conserved Asp-205 in Insig-1, which abuts the fourth transmembrane helix at the cytosolic side of the ER membrane, is essential for its dual function. When Asp-205 was mutated to alanine, the mutant Insig-1 lost the ability to bind to Scap and, thus, was unable to suppress the cleavage of sterol regulatory element-binding proteins. The mutant Insig-1 was ineffective also in accelerating sterol-stimulated degradation of 3-hydroxy-3-methylglutaryl CoA reductase. Alanine substitution of the corresponding aspartic acid in Insig-2 produced the same dual defects. These studies identify a single amino acid residue that is crucial for the function of Insig proteins in regulating cholesterol homeostasis in mammalian cells.

Sterol-regulated ubiquitination and degradation of Insig-1 creates a convergent mechanism for feedback control of cholesterol synthesis and uptake

This paper describes a convergent mechanism for the feedback control of cholesterol synthesis and uptake mediated by SREBPs, membrane bound transcription factors. Endoplasmic reticulum (ER) bound SREBPs form complexes with Scap, a polytopic ER protein. In sterol-overloaded cells, Scap/SREBP binds to Insig-1, which retains the complex in the ER. Upon sterol deprivation, the Scap/SREBP complex dissociates from Insig-1, which is then ubiquitinated on lysines 156 and 158 and degraded in proteasomes. Scap/SREBP moves to the Golgi, where SREBP is processed to liberate a nuclear fragment that activates genes for cholesterol synthesis and uptake and the gene for Insig-1. Ubiquitination is not necessary for release of Scap/SREBP from Insig-1, but it establishes a requirement for synthesis of new Insig-1 for feedback inhibition. When the new Insig-1 and cholesterol converge on Scap, Scap/SREBP binds to Insig-1, preventing ubiquitination. The Insig-1/Scap/SREBP complex accumulates in the ER, ready for liberation when the cell is again sterol deprived.

Isolation of Sterol-resistant Chinese Hamster Ovary Cells with Genetic Deficiencies in Both Insig-1 and Insig-2

Insig-1 and Insig-2, a pair of endoplasmic reticulum (ER) membrane proteins, mediate feedback control of cholesterol synthesis through their sterol-dependent binding to the following two polytopic ER membrane proteins: sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Sterol-induced binding of Insigs to SCAP prevents the proteolytic processing of SREBPs, membrane-bound transcription factors that enhance the synthesis of cholesterol, by retaining complexes between SCAP and SREBP in the ER. Sterol-induced binding of Insigs to reductase leads to the ubiquitination and ER-associated degradation of the enzyme, thereby slowing a rate-controlling step in cholesterol synthesis. Here we report the isolation of a new line of mutant Chinese hamster ovary cells, designated SRD-15, deficient in both Insig-1 and Insig-2. The SRD-15 cells were produced by gamma-irradiation of Insig-1-deficient SRD-14 cells, followed by selection in high levels of the oxysterol, 25-hydroxycholesterol. Sterols neither inhibit SREBP processing nor promote reductase ubiquitination/degradation in SRD-15 cells. Sterol regulation of SREBP processing and reductase ubiquitination/degradation is fully restored in SRD-15 cells when they are transfected with expression plasmids encoding either Insig-1 or Insig-2. These results demonstrate an absolute requirement for Insig proteins in the regulatory system that mediates lipid homeostasis in animal cells.

Insig-mediated degradation of HMG CoA reductase stimulated by lanosterol, an intermediate in the synthesis of cholesterol

Feedback control of cholesterol synthesis is mediated in part by sterol-induced binding of HMG CoA reductase to Insig proteins in the endoplasmic reticulum (ER). Binding leads to ubiquitination and proteasomal degradation of reductase, a rate-controlling enzyme in cholesterol synthesis. Using in vitro and in vivo assays, we show that lanosterol, the first sterol intermediate in cholesterol synthesis, potently stimulates ubiquitination of reductase, whereas cholesterol has no effect at 10-fold higher concentrations. Lanosterol is not effective in mediating the other action of Insigs, namely to promote ER retention of SCAP-SREBP complexes, a reaction that is mediated directly by cholesterol. A pair of methyl groups located in the C4 position of lanosterol confers this differential response. These data indicate that buildup of cholesterol synthesis intermediates represses the pathway selectively at reductase and reveal a previously unappreciated link between feedback inhibition of reductase and carbon flow through the cholesterol synthetic pathway.

Inhibition of protein translocation across the endoplasmic reticulum membrane by sterols.

Cholesterol and related sterols are known to modulate the physical properties of biological membranes and can affect the activities of membrane-bound protein complexes. Here, we report that an early step in protein translocation across the endoplasmic reticulum (ER) membrane is reversibly inhibited by cholesterol levels significantly lower than those found in the plasma membrane. By UV-induced chemical cross-linking we further show that high cholesterol levels prevent cross-linking between ribosome-nascent chain complexes and components of the Sec61 translocon, but have no effect on cross-linking to the signal recognition particle. The inhibiting effect on translocation is different between different sterols. Our data suggest that the protein translocation machinery may be sensitive to changes in cholesterol levels in the ER membrane.

Ultrastructural changes in chemically induced preneoplastic focal lesions in the rat liver: a stereological study

Ultrastructural changes were investigated and quantified, using a stereological approach, in early gamma-glutamyltranspeptidase (GGT)-positive focal lesions, induced in the rat liver by treatment with a single initiating dose of diethylnitrosamine (DENA) followed by promotion with phenobarbitone (PB) for 30 weeks. Within the extra-hepatocyte environment of focal tissue, the mean volume occupied by Ito cells was markedly decreased, whilst that occupied by endothelial and Kupffer cells was increased, when compared to uninvolved tissue from the same rat livers. The bile canaliculi were dilated, but no significant differences in the mean volume occupied by the sinusoidal and Disse spaces were noted. In focal hepatocytes there was a striking overproduction of lipid droplets and proliferation of smooth endoplasmic reticulum (sER). Whorls of concentrically arranged, parallel ER membranes were found only in the hepatocytes of preneoplastic foci, in association with the proliferated sER, and never in the surrounding, uninvolved tissue. The increase in mean volume of the sER, lipid droplet and cytoplasmic matrix compartments, together with the appearance of whorls, were the major contributing factors to the marked hypertrophy seen in focal hepatocytes. The mean volume of the rough endoplasmic reticulum, mitochondrial, lysosomal, peroxisomal and nuclear compartments per hepatocyte also increased, but contributed to a lesser extent to the cellular hypertrophy. It is speculated that whorls may be structural adaptations, resulting from a possible alteration in the normal feedback control of cholesterol synthesis, for the production of sterols and the biogenesis of sER in eosinophilic-type focal cells. The significance of changes observed in focal tissue, and the high biological variation noted between foci, is discussed in relation to the hepatocarcinogenic process.

Feed-back control of cholesterol synthesis in partially hepatectomized rats

Partial hepatectomy caused a marked stimulation of cholesterol and fatty acid syntheses without affecting serum total cholesterol, total phospholipid and triacylglycerol concentrations of rats so far examined 48 h after the operation. Serum free cholesterol level, however, was increased by the treatment and the ratio of lysophosphatidylcholine to phosphatidylcholine was concomitantly decreased, suggesting the impairment of serum lecithin: cholesterol acyltransferase activity. The lipid content in the liver, especially triacylglycerol and ester cholesterol, was increased markedly by the operation. Feeding of a high cholesterol diet which elevated serum cholesterol and phospholipid levels to the partially hepatectomized rats, accelerated the accumulation of hepatic triacylglycerol and ester cholesterol by the partial hepatectomy. The weight of the regenerating liver was not influenced by cholesterol feeding, which suggested that cholesterol feeding did not inhibit the regeneration mechanism of the liver. The increase of cholesterol synthesis after partial hepatectomy was inhibited by cholesterol feeding. Therefore, it is conceivable that the negative feed-back control of cholesterol synthesis is induced by cholesterol feeding under the stimulated cell divisions of the liver after partial hepatectomy. It is suggested from the present data that a large amount of the cholesterol which is necessary for cell growth can be taken up from serum, when serum cholesterol concentration is high.

The intracellular mechanism responsible for dietary feedback control of cholesterol synthesis

The question under discussion is, “By what intracellular means does dietary cholesterol inhibit cholesterol synthesis in the liver?” This inhibition is known to occur primarily at the biosynthetic step catalysed by the microsomal enzyme, HMGR, i.e. β-hydroxy-β-methylglutaryl co-enzyme A reductase [mevalonate:NADP oxidoreductase (acylating CoA);EC.1.1.1.34]. In this review we propose and defend the hypothesis that, “The activity of hepatic HMGR is critically regulated by the fluidity of its supporting microsomal membrane”. Alterations to the activity of membrane-bound enzymes by alterations to membrane fluidity are well known, but only as experimental manipulations. We consider that in the case of HMGR this represents the actual method of physiological control.

Metabolic controls in precancerous liver—VII. Time course of loss of dietary feedback control of cholesterol synthesis during carcinogen treatment

A technique is described, involving continuous low-level feeding of AY 9944 (trans- 1,4-bis(2-chlorobenzyl-aminomethyl) cyclohexane dihydrochloride), whereby the dietary feedback status of hepatic cholesterol synthesis in individual rats can be monitored at frequent intervals. Use of this technique has shown that the hepatocarcinogens ethionine ( 0·25% in feed) and methylazoxymethanol acetate (MAM-acetate) ( 50 mg/kg of body wt. i.p.) cause a rapid and sudden loss of feedback inhibition of hepatic cholesterol synthesis at approximately 7–9 and 11–14 days respectively. Loss of control is not caused by the non-carcinogenic hepatotoxin carbon tetrachloride.

Metabolic controls in precancerous liver—VI. Studies in vivo and in vitro on feedback regulation of Δ-aminolevulinic acid synthetase in ethionine-fed rats

We have studied induction of δ-aminolevulinic acid (ALA) synthetase by the drug allylisopropylacetamide and the ability of exogenous heme to repress such induction in normal rat liver, in the livers of rats fed the hepatocarcinogen, ethionine and in host livers and hepatomas of rats bearing Morris hepatomas 5123C, 7794A and 9618A. We also measured changes in tyrosine aminotransferase. In experiments in vivo, ALA synthetase was shown to be inducible in normal liver, in ethionine-treated liver, in host livers of hepatoma-bearing rats and in hepatoma 5123C. Under our conditions it was non-inducible in hepatomas 9618A and 7794A. In normal and host livers injected heme repressed drug-stimulated induction, but in ethionine-treated livers and in hepatoma 5123C this feedback-type repression by heme was absent. This apparent defect in metabolic control in both cancerous and precancerous liver parallels the similar loss of feedback control of cholesterol synthesis previously reported. In contrast to findings in vivo for ethionine-fed rats, heme repression was found to be fully operative when studied in vitro, i.e. in liver cells isolated from ethionine-treated rats. The possible significance of this difference between findings in vivo and in vitro, in relation to the nature of ethionine-induced changes in precancerous liver, is discussed.

Properties of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Solubilized from Rat Liver and Hepatoma

In hepatomas, the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-controlling enzyme in cholesterol biosynthesis, is not normally suppressed by cholesterol. To examine the biochemical mechanism of this loss of feedback control of cholesterol synthesis, a comparison was made of the properties of 3-hydroxy-3-methylglutaryl coenzyme A reductase after solubilization and partial purification from microsomes of normal rat liver and of a minimal deviation rat hepatoma. The solubilized enzyme from the two sources behaved identically with respect to substrate kinetics, heat inactivation, cold inactivation, and gel filtration. By the use of a new method that permits assay of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity after polyacrylamide gel electrophoresis, it was shown that the solubilized hepatoma enzyme and the normal liver enzyme had similar electrophoretic mobility. It is concluded that the failure of hepatomas to suppress 3-hydroxy-3-methyl-glutaryl coenzyme A reductase activity is not due to the production of an altered form of the enzyme.

Metabolic controls in precancerous liver—IV. Loss of feedback control of cholesterol synthesis and impaired cholesterol uptake in ethionine-fed rats

We have examined several aspects of the control of cholesterol synthesis in vivo (measured as plasma levels of 7-dehydrocholesterol following a single dose of AY- 9944, an inhibitor of cholesterogenesis) in livers of rats fed the hepatocarcinogen, ethionine. Within two weeks of beginning ethionine treatment hepatic cholesterol synthesis was increased and feedback control over this synthesis was lost. This confirms our earlier studies with two other carcinogens, N-2-fluorenylacetamide and aflatoxin. The degree of loss of control of cholesterol synthesis did not correlate with the variable toxicity of the ethionine to the test rats. The hepatic uptake of 14 C-cholesterol, particularly as cholesterol ester, following a single, intragastric dose of 4- 14 C-cholesterol was considerably reduced by ethionine feeding. This suggests that the ethionine-treated liver is defective in its mechanism for removing chylomicron-cholesterol from the plasma, an essential requirement for effective feedback control of cholesterol synthesis. Defective uptake of dietary cholesterol has also been observed in fully-developed hepatomas, all of which similarly lack feedback control of cholesterol synthesis.

Metabolic controls in precancerous liver—II: Loss of feedback control of cholesterol synthesis, measured repeatedly in vivo, during treatment with the carcinogens N-2-fluorenylacetamide and aflatoxin

We have examined feedback control of cholesterogenesis in rat liver during the course of carcinogen treatment, using an assay for cholesterol synthesis which permits repeated tests on the same animal. Rats were treated with AY- 9944 (which inhibits the conversion of 7-dehydrocholesterol to cholesterol), and then 24 hr later blood levels of 7-dehydrocholesterol were measured. This gives an accurate estimation of liver sterol synthesis during that 24 hr period. By feeding 5% cholesterol for two days before each alternate assay, it was possible to measure feedback control of cholesterol synthesis for each individual animal during the carcinogen treatment. Rats injected with a single LD 50 dose of aflatoxin showed a severe loss of control of cholesterogenesis 3 to 5 weeks later. Most animals had recovered normal control 10 weeks after the aflatoxin treatment. N- 2-fluorenylacetamide feeding caused a similar marked loss of control within 1 to 2 weeks, and here also most animals regained normal control later. With both carcinogens the actual degree of loss of control of cholesterol synthesis was extremely variable between individual animals. Only a few animals showed complete loss of control and there was a wide range of varying degrees of partial control. Control of cholesterogenesis, as measured by two entirely independent assay systems, has now been shown to be defective after treatment with two different carcinogens, thus confirming the importance in the carcinogenic process, at least in the liver, of this loss of control previously known to occur in all fully-developed hepatomas.