Activation of hepatic CREBH and Insig signaling in the anti-hypertriglyceridemic mechanism of R-α-lipoic acid

Abstract

The activation of sterol regulatory element binding proteins (SREBPs) is regulated by insulin-induced genes 1 and 2 (Insig-1 and Insig-2) and SCAP. We previously reported that feeding R-α-lipoic acid (LA) to Zucker diabetic fatty (ZDF) rats improves severe hypertriglyceridemia. In this study, we investigated the role of cyclic AMP-responsive element binding protein H (CREBH) in the lipid-lowering mechanism of LA and its involvement in the SREBP-1c and Insig pathway. Incubation of McA cells with LA (0.2 mM) or glucose (6 mM) stimulated activation of CREBH. LA treatment further induced mRNA expression of Insig-1 and Insig-2a, but not Insig-2b, in glucose-treated cells. In vivo, feeding LA to obesity-induced hyperlipidemic ZDF rats activated hepatic CREBH and stimulated transcription and translation of Insig-1 and Insig-2a. Activation of CREBH and Insigs induced by LA suppressed processing of SREBP-1c precursor into nuclear SREBP-1c, which subsequently inhibited expression of genes involved in fatty acid synthesis, including FASN, ACC and SCD-1, and reduced triglyceride (TG) contents in both glucose-treated cells and ZDF rat livers. Additionally, LA treatment also decreased abundances of very low density lipoprotein (VLDL)-associated apolipoproteins, apoB100 and apoE, in glucose-treated cells and livers of ZDF rats, leading to decreased secretion of VLDL and improvement of hypertriglyceridemia. This study unveils a novel molecular mechanism whereby LA lowers TG via activation of hepatic CREBH and increased expression of Insig-1 and Insig-2a to inhibit de novo lipogenesis and VLDL secretion. These findings provide novel insight into the therapeutic potential of LA as an anti-hypertriglyceridemia dietary molecule.

Keywords: Apolipoproteins; Cell signaling; Dyslipidemias; Sterol regulatory element binding proteins; Triglyceride metabolism; Very low density lipoprotein.

Figure 1

LA induces expression of CREBH, Insig-1, and Insig-2a in McA cells. (A) McA cells were treated with PA (0.25 mM) and LA (0.2 mM) for 48 hours, as indicated, and total RNA was extracted to detect mRNA expression of CREBH. Transcript levels were normalized to internal control 18S ribosomal RAN. (B) McA cells were cultured in normal medium (Control, CTL), glucose (6 mM) or glucose plus LA (0.2 mM) for 48 hours. Total RNA was extracted to detect mRNA expression of CREBH. Transcript levels were normalized to internal control 18S ribosomal RAN. (C) McA cells were treated the same as in (B). Protein masses of full-length (F) and nuclear (N) CREBH were detected by immunoblot analysis of cell lysates. Immunoblots were quantified by densitometry and CREBH-F was normalized to β-actin, while CREBH-N was normalized to Histone H3. (D) Total RNA prepared as in (B) was used to determine the mRNA expression of Insig-1, Insig-2a, and Insig-control vector (Mock) or a plasmid expressing WT CREBH (CREBH WT). Forty-eight hours after transfection, cells were collected, total RNA was extracted, and mRNA levels of Insig-1 and Insig-2a were determined by q-PCR. Results are shown as means ± SD for two experiments performed in triplicate. *P<0.05, **P<0.01, ***P<0.001

Figure 2

LA increases hepatic CREBH and Insig-2 expression in ZDF rats. Livers were isolated from 11-wk old ZDF rats fed an LA-supplemented diet for two weeks and from pair-fed controls. Total RNA was extracted from liver tissues to detect mRNA expression of (A) CREBH, as well as (C) Insig-1, Insig-2a, Insig-2b. Transcript levels were normalized to internal control 18S ribosomal RAN. Protein mass of (B) full-length (F) and nuclear (N) CREBH, as well as (D) Insig-1 and Insig-2, were detected by immunoblot analysis of homogenized liver tissues. Immunoblots were quantified by densitometry and normalized to β-actin, except for CREBH-N, which was normalized to Histone H3. Results are shown as means ± SD (n=4/group). *P<0.05, **P<0.01, ***P<0.001.

Figure 3

LA reduces hepatic full-length and nuclear SREBP-1 protein expression in McA cells and ZDF rats. (A) McA cells were cultured in either glucose (6 mM) alone or glucose (6 mM) plus LA (0.2 mM) for 48 hours. Protein masses of precursor (p) and nuclear (n) SREBP-1c and SREBP-2 were detected by immunoblot analysis of cell lysates. (B) Livers were isolated from 11-wk old ZDF rats fed an LA-supplemented diet for two weeks and from pair-fed controls. Protein mass of precursor and nuclear SREBP-1c as well as nuclear SREBP-2 were detected by immunoblot analysis of liver tissues. Immunoblots were quantified by densitometry and precursor proteins were normalized to β-actin, while nuclear proteins were normalized to Histone H3. Results are shown as means ± SD for two experiments performed in triplicate and n of 4 per group for the animal study. *P<0.05.

Figure 4

LA suppresses FA synthesis genes and reduces cellular TG and cholesterol levels in McA cells. McA cells were cultured in high glucose (4.5 g/L) DMEM with the addition of either glucose (6 mM) alone or glucose (6 mM) plus LA (0.2 mM) for 48 hours. (A) Total RNA was extracted to detect mRNA expression of FASN, ACC, and SCD-1 reductase. Transcript levels were normalized to internal control 18S ribosomal RAN. (B) Media was removed and total lipid was extracted from cells to measure TG and cholesterol concentrations. Results are shown as means ± SD for one representative experiment performed in triplicate. Two experiments were performed in triplicate and showed similar results. *P<0.05.

Figure 5

LA reduces intracellular levels and secretion of VLDL-associated apolipoproteins in McA cells and ZDF rats. (A) McA cells were cultured in either glucose (6 mM) alone or glucose (6 mM) plus LA (0.2 mM) for 48 hours. Protein mass of apoB100 and apoE were detected by immunoblot analysis of cell lysates and culture medium. Results are shown as means ± SD for two experiments performed in triplicate. (B, C) Three-hour fasted blood was collected and livers were isolated from 11-wk old ZDF rats fed an LA-supplemented diet for two weeks and from pair-fed controls. Protein mass of apoB100 and apoE were detected by immunoblot analysis of (B) liver tissues and (C) plasma. Results are shown as means ± SD (n=4/group). Immunoblots were quantified by densitometry and normalized to albumin. (D) Total RNA was extracted from liver tissues to detect mRNA expression of LDL-receptor. *P<0.05, **P<0.01.