IRE1α-XBP1s induces PDI expression to increase MTP activity for hepatic VLDL assembly and lipid homeostasis

Abstract

The unfolded protein response (UPR) is a signaling pathway required to maintain endoplasmic reticulum (ER) homeostasis and hepatic lipid metabolism. Here, we identify an essential role for the inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α)-X box binding protein 1 (XBP1) arm of the UPR in regulation of hepatic very low-density lipoprotein (VLDL) assembly and secretion. Hepatocyte-specific deletion of Ire1α reduces lipid partitioning into the ER lumen and impairs the assembly of triglyceride (TG)-rich VLDL but does not affect TG synthesis, de novo lipogenesis, or the synthesis or secretion of apolipoprotein B (apoB). The defect in VLDL assembly is, at least in part, due to decreased microsomal triglyceride-transfer protein (MTP) activity resulting from reduced protein disulfide isomerase (PDI) expression. Collectively, our findings reveal a key role for the IRE1α-XBP1s-PDI axis in linking ER homeostasis with regulation of VLDL production and hepatic lipid homeostasis that may provide a therapeutic target for disorders of lipid metabolism.

Figure 1. Hepatocyte-specific Ire1α-deletion accentuates nutritional stress-induced hepatosteatosis without affecting de novo lipogenesis

(A) and (B) A 16-hr fast increases lipid accumulation in L-Ire1α-KO livers. (A) Liver TG and cholesterol esters were measured from L-Ire1α-Het and L-Ire1α-KO mice, in fed and 16-hr fasted condition n=4. *P< 0.01 vs L-Ire1α-KO. (B) Micrographs of liver sections from mice that were fed or fasted for 16 hrs were stained with Hematoxylin and Eosin (H&E). Scale bars, 40 μm. (C) and (D) A high-fructose diet (HFD) increases TG accumulation in L-Ire1α-KO mice. (C) Liver TG was measured in mice fed with HFD for the indicated times (n ≥ 4). *P< 0.05 vs L-Ire1α-Het. (D) Mice were fed with HFD for indicated time periods and liver sections prepared and stained with H&E. Scale bars, 30μm. (E) and (F) Livers from mice fed a HFD were analyzed for mRNA and proteins levels of lipogenic genes. (E) The abundance of lipogenic gene mRNA was normalized to actin mRNA. Values are relative to mRNA levels of L-Ire1α-Het mice fed with chow. (F) Liver lysates were prepared from mice fed with chow (Co) or HFD (HF) and analyzed by immunoblotting. Calnexin was used as a loading control. (G) Fatty acid synthesis was measured in hepatocytes by labeling with [¹⁴C]-acetic acid for 4 hrs (n=6).

Figure 2. Hepatocyte-specific Ire1α-deleted mice display hypolipidemia and reduced hepatic VLDL secretion

(A) Plasma TG and cholesterol levels are reduced in L-Ire1α-KO mice. Levels of plasma TG and cholesterol were measured in fed and 16-hr fasted mice, n>40. *P< 0.01 vs LIre1α-Het. (B) and (C) Plasma VLDL-TG and HDL-cholesterol levels are reduced in L-Ire1α-KO mice. (D) and (E) VLDL-TG secretion is reduced in L-Ire1α-KO mice. Mice (n≥5) fed with chow or HFD for 2d were fasted 4-hr followed by i.v. injection with tyloxapol. *P< 0.05 vs L-Ire1α-Het. (F) Plasma levels of apoB from the “0” time point to 120 min in Figure 2D were measured.

Figure 3. Ire1α-deletion impairs secretion of TG-rich VLDL particles

(A) and (B) TG secretion is reduced without a change in TG synthesis in L-Ire1α-KO hepatocytes. Hepatocytes were labeled with [³H]-glycerol in the presence of BSA or OA for 3-hr and intracellular (A) and secreted (B) was measured (n=6). *P< 0.05 vs L-Ire1α-Het hepatocytes. (C) ApoB synthesis and secretion are not altered in L-Ire1α-KO hepatocytes. Primary hepatocytes were incubated 48 hrs in vitro and then pulse-labeled with [³⁵S]-Met/Cys for 20 min. Intracellular apoB and secreted apoB were analyzed by immunoprecipitation and SDS-PAGE. (D) Secretion of lipid-rich apoB particles is reduced in L-Ire1α-KO hepatocytes. Secreted apoB-containing lipoproteins from hepatocytes were analyzed by DGUC and immunoblotting (n=3). The abundance of apoB in each fraction was quantified by ImageJ and relative amounts were calculated from the percentage of intensity in each fraction relative to the total intensity across the gel. *P< 0.05 vs L-Ire1α-Het hepatocytes. (E) VLDL assembly is defective in L-Ire1α-KO ER lumen. Lumenal apoB was analyzed by DGUC (n=3). Fractions were either immunoprecipitated with anti-apoB antibody or concentrated and analyzed by immunoblotting for MTP and apoE. The analysis was quantified as described in (D). *P< 0.05 vs L-Ire1α-Het.

Figure 4. Ire1α-deletion reduces TG content in the sER of the liver

(A) and (B) Complementation of IRE1α or spliced XBP1 (XBP1s) restores TG secretion in L-Ire1α-KO hepatocytes. (A) Protein levels of XBP1s and IRE1α were measured by immunoblotting of hepatocytes infected with adenoviruses expressing GFP (Ad-GFP), XBP1s (Ad-XBP1s) and wild-type IRE1α (Ad-IREwt), kinase-dead IRE1α (Ad-IRE599) or RNase-dead IRE1α (Ad-IRE907) (n=6). (B) Rates of TG synthesis and secretion were measured in L-Ire1α-Het hepatocytes and L-Ire1α-KO hepatocytes infected with the above adenoviruses (n=6). *P< 0.05 vs XBP1s infected hepatocytes. (C) Electron micrographs of isolated rER and sER from livers are shown. (D) Immunoblotting analysis of protein markers was performed on isolated cellular organelles. (E) TG accumulation is reduced in L-Ire1α-KO sER. Levels of TGs, cholesterol esters, fatty acids and phospholipids were measured in sER and non-sER fractions (n=4). *P< 0.05 vs L-Ire1α-Het. (F) ER-localized TG is reduced in L-Ire1α-KO hepatocytes. Hepatocytes were incubated with [³H]-glycerol-DMEM media for 3-hr with 0.4mM OA. [³H]-labeled sER was isolated from 5×10⁶ hepatocytes and radioactivity incorporated into TG was measured by TLC of non-sER and sER fractions and presented relative to total protein content (n=4). *P< 0.05 vs L-Ire1α-Het. (G) FAME samples derived from sER-TG and non-sER-TG were analyzed by GC. FAME samples were isolated from 4 livers of each genotype.

Figure 5. Hepatocyte-specific Ire1α-deletion decreases PDI levels and MTP activity in the liver

(A) Proteomic analysis of sER fractions was performed using IEF followed by SDS-PAGE. One spot identified by the circle was subjected to mass spectrum analysis. (B) PDI levels are reduced in L-Ire1α-KO livers. Immunoblot analysis of PDI and MTP in total liver lysates (Total), ER (ER) and ER lumen (ERL) is shown. The intensities of PDI and MTP in the blot were quantified by ImageJ and normalized to the intensity of apoE. The relative abundances are shown relative to PDI levels in each fraction from L-Ire1α-Het samples. The ratios of PDI and MTP in L-Ire1α-KO livers are presented relative to their amount in respective fractions from L-Ire1α-Het samples. (C) MTP activity is reduced in L-Ire1α-KO livers. MTP activity was measured in liver lysates (n=5). *P< 0.01 vs L-Ire1α-Het. (D) Treatment of hepatocytes with 0.3 μM CP-100447 reduces both MTP activity and TG secretion by ~40%. MTP activity, intracellular TG and secreted TG were measured in wild-type hepatocytes treated with MTP inhibitor, CP-100447. (E) Hepatocytes treated with 0.3 μM CP-100447 reduce secretion of apoB in lipid-rich particles. DGUC was repeated twice and the average of the relative intensities were plotted as described in Figure 3D.

Figure 6. Overexpression of PDI, but not MTP, promotes TG secretion in Ire1α-deleted hepatocytes

(A) PDI expression is reduced in L-Ire1α-KO livers. Relative gene expression was normalized to actin mRNA levels and values are compared to mRNA levels from LIre1α-Het mice. *P< 0.05 vs L-Ire1α-Het. (B) and (C) Adenovirus-mediated overexpression of XBP1s increases PDI expression. (B) Values are relative to mRNA levels of L-Ire1α-KO hepatocytes infected with adenovirus expressing GFP. (C) PDI and MTP protein levels were measured at 48-hr after infection of hepatocytes with adenovirus expressing GFP (Ad-GFP), XBP1s (Ad-XBP1s), MTP (Ad-MTP), PDI (Ad-PDI) or mutant PDI (Ad-PDI_Mu). Actin was used as a loading control. (D) and (E) Overexpression of PDI restores MTP activity and promotes hepatic TG secretion in LIre1α-Het hepatocytes. (D) TG synthesis and secretion rates were measured at 48-hr after of infection with the indicated adenoviruses (n=6). *P< 0.05 vs Ad-PDI or mutant PDI Ad-PDI_Mu; **P< 0.05 vs Ad-XBP1s. (E) MTP activity was measured in each sample of (D). *P< 0.05 vs Ad-PDI or Ad-PDI_Mu. (F), (G) and (H) XBP1 knockdown decreases PDI levels, MTP activity and TG secretion in hepatocytes. (F) XBP1 and PDI levels were measured in hepatocytes transfected with control siRNA or XBP1 siRNA. (G) MTP activity was measured in hepatocytes transfected with above siRNAs (n=6). **P<0.05 vs control. (H) TG synthesis and secretion were measured in hepatocytes transfected with above siRNAs (n=6). **P<0.05 vs control.