Insig proteins mediate feedback inhibition of cholesterol synthesis in the intestine

Abstract

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.

Keywords: Cholesterol Regulation; Dyslipidemia; Ezetimibe; Gene Knockout; HMG-CoA Reductase; Insig; Intestine; Lipid Absorption; Lipid Synthesis; SREBP.

FIGURE 1.

Disruption of Insig1 and Insig2 along the longitudinal axis of intestine. Equal amounts of RNA from isolated enterocytes from intestine segments of control and Vil-Insig⁻ mice (female, 11–15 weeks of age, 4 mice/group) were pooled and subjected to quantitative RT-PCR mice using cyclophilin as the invariant control. Each value represents the amount of mRNA relative to that in the duodenum of the control mice, which is arbitrarily defined as 1.0. Intestines were anatomically divided as follows. Duodenum (D) denotes the segment from the distal end of the pylorus to 1 cm past the insertion of the common bile duct. The remaining small bowel was further divided into four equal sections: jejunum 1 and 2 and ileum 1 and 2 (J1, J2, I1, and I2). The colon (C) was analyzed without further division.

FIGURE 2.

Constitutive SREBP processing in Insig-deficient enterocytes. A, immunoblot analysis. Whole-cell extracts from enterocytes of control and Vil-Insig⁻ mice (male, 14–21 weeks of age, 7 mice/group) were individually prepared and pooled. Aliquots of the pooled protein (60 μg for SREBP-1 and SREBP-2, 30 μg for all other proteins) were subjected to SDS-PAGE and immunoblot analysis. Precursor and nuclear forms of SREBPs are denoted as P and N, respectively. Asterisks, nonspecific bands. Calnexin was used as a loading control. B, relative mRNA levels in enterocytes of control and Vil-Insig⁻ mice used in A. Total RNA from enterocytes was subjected to quantitative RT-PCR using cyclophilin as the invariant control. Each value represents the mean ± S.E. (error bars) of data from seven mice relative to that of control mice, which was arbitrarily defined as 1.0. *, p < 0.01, the level of statistical significance (two-tailed Student's t test) between control and Vil-Insig⁻ mice.

FIGURE 3.

Increased lipid synthesis in intestines of Vil-Insig⁻ mice. Control and Vil-Insig⁻ mice (male, 17–19 weeks of age, 7 mice/group) were injected intraperitoneally with ³H-labeled water (50 mCi in 0.2 ml of saline). One hour later, tissues were removed and processed for isolation of digitonin-precipitable sterols (A and C) and fatty acids (B and D). The small intestine was divided into three equal length segments termed proximal (prox.), middle (mid.), and distal (dist.). Each bar represents mean ± S.E. (error bars) of data from seven mice. The sterol and fatty acid synthetic rates were calculated as μmol of ³H radioactivity incorporated/h per g (A and B) or per organ (C and D). *, p < 0.01, the level of statistical significance (two-tailed Student's t test) between control and Vil-Insig⁻ mice.

FIGURE 4.

Increased tissue and plasma cholesterol in Vil-Insig⁻ mice. A–D, plasma lipids and FPLC profiles from the same mice described in Fig. 2. For plasma cholesterol (A) and triglycerides (B), each bar represents the mean ± S.E. of values from seven mice. Equal aliquots of plasma from 7 mice/group were pooled and subjected to gel filtration by FPLC. Concentrations of cholesterol (C) and triglycerides (D) in each FPLC fraction were determined. Fractions containing VLDL/chylomicron (V-CM), LDL (L), and HDL (H) are indicated. E–H, lipid contents in isolated enterocytes and livers of control and Vil-Insig⁻ mice (female, 17–20 weeks of age, 6 mice/group). Each bar represents mean ± S.E. (error bars) of data from six mice. E and G, tissue cholesterol levels. Total and free cholesterol are designated. F and H, tissue triglyceride levels. *, p < 0.01, the level of statistical significance (two-tailed Student's t test) between control and Vil-Insig⁻ mice.

FIGURE 5.

Lipid accumulation of in jejunal crypts of Vil-Insig⁻ mice. Representative Oil Red O and hematoxylin-stained histologic sections of jejuna from control and Vil-Insig⁻ mice are shown. Magnification, ×20. Inset magnification, ×40.

FIGURE 6.

Compensatory reduction of nuclear SREBP-2 in the livers of Vil-Insig⁻ mice. The mice used here are the same as those described in the legend to Fig. 2. A, immunoblot analysis. Whole-cell extracts from livers of control and Vil-Insig⁻ mice were individually prepared, pooled, and subjected to SDS-PAGE and immunoblot analysis. Precursor and nuclear forms of SREBPs are denoted as P and N, respectively. Asterisks, nonspecific bands. B, relative mRNA levels in livers of control and Vil-Insig⁻ mice. Total RNA from liver was subjected to quantitative RT-PCR with ApoB as the invariant control. Each value represents the mean ± S.E. (error bars) of data from seven mice relative to that of control mice, which was arbitrarily defined as 1.0. *, p < 0.05; **p < 0.01, the level of statistical significance (two-tailed Student's t test) between control and Vil-Insig⁻ mice.

FIGURE 7.

Ezetimibe fails to further activate SREBP-2 processing and increase HMGR protein in Insig-deficient enterocytes. Control and Vil-Insig⁻ mice (female, 22–35 weeks of age, 4 mice/group) were fed ad libitum with a chow diet, a chow diet containing 0.01% ezetimibe (EZE), or a fat-free diet (FF) for 5 days prior to studies. A, immunoblot analysis of isolated enterocytes. Precursor and nuclear forms of SREBP-2 are denoted as P and N, respectively. Asterisks, nonspecific bands. B, relative mRNA levels in isolated enterocytes. Total RNA from enterocytes was subjected to quantitative RT-PCR using cyclophilin as the invariant control. Each bar represents the mean ± S.E. (error bars) of value from four mice relative to that of control mice, which was arbitrarily defined as 1.0.