Insig Proteins Mediate Feedback Inhibition of Cholesterol Synthesis in the Intestine

McFarlane, Matthew; Liang, Guosheng; Engelking, Luke J.

doi:10.1074/jbc.m113.524041

Cited by 34 publications

(42 citation statements)

References 49 publications

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“…To answer these questions, we fi rst disrupted Insig-1 and Insig-2 in intestinal epithelium. We observed signifi cant increases in the amount of intestinal nuclear SREBPs and cholesterol synthesis, such that synthesis in the intestine exceeded that in liver on an organ-by-organ basis ( 14 ). Similar results were found when truncated SREBP-2 was expressed in intestinal epithelia ( 15 ).…”

Section: Quantitative Real-time Rt-pcrsupporting

confidence: 73%

“…The current study does not identify which cell populations in the crypt are most reliant on Scap for growth; we previously found neutral lipid accumulation in the crypt bases of mice with intestine-specifi c Insig disruption ( 14 ), which could suggest a possible role for Scap/Insig sterol regulation in Paneth cells or stem cells that compose the crypt base. Future studies will be necessary to precisely determine which cell population(s) in the crypts are most dependent on SREBPs, potentially unraveling details of the function of the SREBP pathway and more largely of lipid biology in adult stem cells.…”

Section: Discussionmentioning

confidence: 95%

“…Sterols synthesized de novo or absorbed in the intestine are packaged into lipoproteins and ultimately reach the liver via the plasma, where they mediate feedback inhibition of hepatic sterol synthesis. The current study, taken together with our prior studies in the liver and small intestine ( 14,41 ), demonstrates that Scap/Insig-mediated sterol sensing imparts cell-autonomous feedback regulation on SREBPs in hepatocytes and in small IECs to balance cholesterol fl ux between the liver and the small intestine.…”

Section: Discussionmentioning

confidence: 99%

“…Whole-cell extracts, membrane fractions, and nuclear extracts were prepared individually from organoids or IECs of mice, and then samples were pooled as indicated in the fi gure legends and subjected to SDS-PAGE and immunoblot analysis as described ( 9,14 ). Chemiluminescence was imaged with a LI-COR Odyssey ® Fc imager and quantifi ed using LI-COR Image Studio™ software (LI-COR Biotechnology, Lincoln, NE).…”

Section: Immunoblot Analyses Of Iecs and Organoidsmentioning

confidence: 99%

“…Total RNA was prepared from mouse tissues, IECs, or organoids and quantitative real-time RT-PCR (QPCR) was performed as described ( 14 ). Mouse Scap exon 2 primers were 5 ′ -CCGAG-GATGACCCTGACTGA-3 ′ and 5 ′ -AGAGCAGCCCATGGTTG-TAGA-3 ′ .…”

Section: Quantitative Real-time Rt-pcrmentioning

confidence: 99%

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Scap is required for sterol synthesis and crypt growth in intestinal mucosa

McFarlane

Cantoria

Linden

et al. 2015

Journal of Lipid Research

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This article is available online at http://www.jlr.org endoplasmic reticulum (ER) to Golgi, where they are processed proteolytically to yield active nuclear forms. When cells are sterol-replete, Insig , an ER-resident membrane protein, binds Scap and retains the Scap-SREBP complex in the ER, thereby preventing the proteolytic activation of SREBPs ( 2, 3 ).In cells lacking Scap, SREBP transport to the Golgi is abolished, blocking SREBP proteolysis and leading to reduced rates of de novo cholesterol and fatty acid synthesis. As a result, Scap-defi cient cultured cells cannot grow without supplementation with exogenous cholesterol and fatty acids ( 4 ). The in vivo function of Scap has been explored most thoroughly in the liver, which is quantitatively the most important organ for cholesterol synthesis in most mammals ( 5 ). When Scap is ablated through gene knockout in rodent livers, nuclear SREBPs are not detectable, leading to reduced rates of hepatic cholesterol and fatty acid synthesis and reduced levels of cholesterol and triglycerides in the liver and plasma ( 6 ). Mice with defi ciency of Scap in the liver appear phenotypically normal and have grossly normal liver function. These mice are protected from development of fatty liver and carbohydrate-induced hypertriglyceridemia, suggesting that Scap inhibition may be a potential therapeutic strategy for the treatment of nonalcoholic fatty liver disease and hyperlipidemia ( 7 ).Determining the extrahepatic role of Scap is of interest both to elucidate the role of SREBP-mediated lipid homeostasis in extrahepatic tissues and to assess for toxicity arising from Scap inhibition in the context of the whole Grant HL-20948. M.R.M. is an Howard Hughes Medical Institute International Student Research Fellow. L.J.E. was supported by NIH Institutional Training Grant 2T32-DK-007745-16 and by NIH Grant 1K08DK102652-01. Manuscript received 31 March 2015 and in revised form 17 April 2015. Published, JLR Papers in Press, April 20, 2015 DOI 10.1194 Scap is required for sterol synthesis and crypt growth in intestinal mucosa Abbreviations: 4-OHT, 4-hydroxytamoxifen; ChgA, chromogranin A; CREB, cAMP response element binding protein; ER, endoplasmic reticulum; H&E, hematoxylin and eosin; HMGR, HMG-CoA reductase; IEC, intestinal epithelial cell; LGR5, leucine-rich repeat containing G protein-coupled receptor 5; M ␤ CD, methyl-␤ -cyclodextrin; NGS, normal goat serum; NPC1L1, Niemann-Pick C1-like 1 protein; PAS/AB, periodic acid-Schiff-Alcian Blue; QPCR, quantitative real-time RT-PCR; Scap, SREBP cleavage-activating protein; SREBP, sterol regulatory element-binding protein; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling .

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Section: Quantitative Real-time Rt-pcrsupporting

confidence: 73%

Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

Section: Immunoblot Analyses Of Iecs and Organoidsmentioning

confidence: 99%

Section: Quantitative Real-time Rt-pcrmentioning

confidence: 99%

See 3 more Smart Citations

Scap is required for sterol synthesis and crypt growth in intestinal mucosa

McFarlane

Cantoria

Linden

et al. 2015

Journal of Lipid Research

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CAT‐2003: A novel sterol regulatory element‐binding protein inhibitor that reduces steatohepatitis, plasma lipids, and atherosclerosis in apolipoprotein E*3‐Leiden mice

Zimmer

Bista

Benson

et al. 2017

Hepatology Communications

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CAT‐2003 is a novel conjugate of eicosapentaenoic acid (EPA) and niacin designed to be hydrolyzed by fatty acid amide hydrolase to release EPA inside cells at the endoplasmic reticulum. In cultured liver cells, CAT‐2003 blocked the maturation of sterol regulatory element‐binding protein (SREBP)‐1 and SREBP‐2 proteins and decreased the expression of multiple SREBP target genes, including HMGCR and PCSK9. Consistent with proprotein convertase subtilisin/kexin type 9 (PCSK9) reduction, both low‐density lipoprotein receptor protein at the cell surface and low‐density lipoprotein particle uptake were increased. In apolipoprotein E*3‐Leiden mice fed a cholesterol‐containing western diet, CAT‐2003 decreased hepatic inflammation and steatosis as evidenced by fewer inflammatory cell aggregates in histopathologic sections, decreased nuclear factor kappa B activity in liver lysates, reduced inflammatory gene expression, reduced intrahepatic cholesteryl ester and triglyceride levels, and decreased liver mass. Plasma PCSK9 was reduced and hepatic low‐density lipoprotein receptor protein expression was increased; plasma cholesterol and triglyceride levels were lowered. Aortic root segments showed reduction of several atherosclerotic markers, including lesion size, number, and severity. CAT‐2003, when dosed in combination with atorvastatin, further lowered plasma cholesterol levels and decreased hepatic expression of SREBP target genes. Conclusion: SREBP inhibition is a promising new strategy for the prevention and treatment of diseases associated with abnormal lipid metabolism, such as atherosclerosis and nonalcoholic steatohepatitis. (Hepatology Communications 2017;1:311–325)

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Insulin increases cholesterol uptake, lipid droplet content, and apolipoprotein B secretion in CaCo‐2 cells by upregulating SR‐BI via a PI3K, AKT, and mTOR‐dependent pathway

Fuentes

Santander

Cortés

2018

J of Cellular Biochemistry

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The actions of insulin on intestinal cholesterol absorption and lipoprotein secretion are not well understood. Herein, we determined the effects of insulin on the levels of cholesterol transporter scavenger receptor, class B, type I (SR‐BI), cellular cholesterol uptake, intracellular lipid accumulation, and lipoprotein secretion in a cellular model of human intestinal epithelium. Methods: CaCo‐2 cells were cultured to postconfluency in Transwell filters and stimulated with glucose (25 mM) in the presence or absence of insulin (100 nM) at their basolateral surface. SR‐BI mRNA and protein levels were quantified by quantitative reverse transcription‐PCR and immunoblot, respectively. Polarized localization of SR‐BI was determined by cell surface proteins biotinylation and streptavidin precipitation. Activities of PI3K, AKT, mTOR, and SR‐BI were pharmacologically antagonized. Cholesterol uptake was assessed by NBD‐cholesterol incorporation. Apolipoprotein (apo) B concentration was quantified by ELISA. Subcellular localization of neutral lipids (BODIPY) and SR‐BI (immunofluorescence) was determined by confocal microscopy. Results: In polarized CaCo‐2 cells, insulin increased SR‐BI at the mRNA and protein levels. SR‐BI was exclusively present at apical cell surface, as indicated by biotinylation and confocal microscopy analysis. Glucose did not modify SR‐BI abundance or subcellular localization. Effects of insulin on SR‐BI levels were abrogated by PI3K, AKT, or mTOR pharmacological antagonism. Cholesterol uptake, neutral lipid abundance, and apo B secretion were increased by insulin in CaCo‐2 cells, and these effects were prevented by SR‐BI pharmacological antagonism with block lipid transport‐1. Conclusions: insulin promotes cholesterol uptake, intracellular lipid store, and apo B–containing lipoproteins secretion by SR‐BI‐dependent mechanisms in a model of human intestinal epithelium.

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Insig Proteins Mediate Feedback Inhibition of Cholesterol Synthesis in the Intestine

Cited by 34 publications

References 49 publications

Scap is required for sterol synthesis and crypt growth in intestinal mucosa

Scap is required for sterol synthesis and crypt growth in intestinal mucosa

CAT‐2003: A novel sterol regulatory element‐binding protein inhibitor that reduces steatohepatitis, plasma lipids, and atherosclerosis in apolipoprotein E*3‐Leiden mice

Insulin increases cholesterol uptake, lipid droplet content, and apolipoprotein B secretion in CaCo‐2 cells by upregulating SR‐BI via a PI3K, AKT, and mTOR‐dependent pathway

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