ChREBP Rather Than SHP Regulates Hepatic VLDL Secretion

Figures

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  Figure 1. The coordinated effects of carbohydrate response element binding protein (ChREBP) and small heterodimer partner (SHP) on Mttp expression. (A) and (B) The effects of SHP on ChREBP-mediated Mttp (A) and Pklr (B) mRNA induction. After 4 h infection with Ad-daChREBP and/or Ad-SHP in hepatocytes, cells were incubated for 20 h. Mttp mRNA levels were detected by real-time Polymerase chain reaction method. Open symbols and closed symbols indicated adenoviral overexpression of daChREBP and SHP, respectively. The x-axis indicates Shp and Chrebp mRNA levels (fold change). Adenoviral overexpression of ChREBP caused a 2.5-fold increase in Chrebp mRNA levels. Adenoviral SHP expression caused an indicated fold increase in Shp expresssion. Pol2 expression was used as an internal control. n = 4 per group. * p < 0.05; (C) Reporter assay using pGL3-Mttp (−211 bp). The indicated amounts of pcDNA-daChREBP and pcDNA-SHP were transfected with pGL3-Mttp (−211 bp) and pRL-TK vectors and Lipofectamine 2000 reagent into primary rat hepatocytes. After 24 h incubation, cells were collected for the dual luciferase assay. Luciferase activity was normalized to Renilla luciferase activity. N.S., not significant.…

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  Figure 2. ChREBP and SHP target mRNA levels in wild-type, Chrebp−/−, Shp−/−, and Chrebp−/− Shp−/− mice. Carbohydrate response element binding protein (Chrebp) (A); small heterodimer partner (Shp) (B); liver type pyruvate kinase (Pklr) (C); fibroblast growth factor 21 (Fgf21) (D); and Cholesterol 7 alpha-hydroxylase (Cyp7a1); (E) mRNA expression analysis in the livers of wild-type, Chrebp−/−, Shp−/−, and Chrebp−/− Shp−/− (DKO) mice. Open squares indicate fasted conditions; closed squares indicate fed conditions. Pol2 expression was used as an internal control. Each mRNA levels were corrected with mouse RNA polymerase 2 mRNA. * p < 0.05 vs. WT (fast), n = 4. ** p < 0.05 vs. WT (fed), n = 4.

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  Figure 3. Shp deletion failed to recover decreased Mttp expression, and thereby, VLDL secretion rates mediated by Chrebp deletion. (A) Hepatic Mttp mRNA levels in the livers of wild-type (WT), Chrebp−/−, Shp−/−, and DKO mice. Mttp mRNA levels were detected by real-time PCR. Pol2 expression was used as an internal control. * p < 0.05 vs. WT (fast), n = 4. ** p < 0.05 vs. WT (fed), n = 4. Data are represented as means ± SD; (B) Hepatic MTTP protein expression in the livers of WT, Chrebp−/−, Shp−/−, and DKO mice. MTTP protein levels were measured by ELISA and corrected for total protein levels. * p < 0.05 vs. WT, n = 5–7. Data are represented as means ± SD; (C) VLDL secretion rates were measured as previously reported [17]. * p < 0.05 vs. WT, n = 5–7. Data are represented as means ± SD; TG contents in lipoprotein fraction (D) and VLDL particle numbers (E) were analyzed using gel-permeation high-performance liquid chromatography. Data are representative of six samples pooled.

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  Figure 4. ChREBP and SHP independently regulate hepatic lipid metabolism. High carbohydrate diets increase de novo lipogenesis and VLDL secretion through ChREBP activation. Therefore, Chrebp deletion prevents high carbohydrate diet induced fatty liver [30,40,42,43]. In contrast, SHP suppresses PPAR alpha and an Shp deletion prevents high fat diet induced fatty liver [15,45]. In this study, we clarified that ChREBP, rather than SHP, regulates VLDL secretion through Mttp expression (bold line). Thus, ChREBP and SHP independently regulate hepatic lipid metabolism.