Regulation of hepatic lipogenesis by the transcription factor XBP1

Abstract

Dietary carbohydrates regulate hepatic lipogenesis by controlling the expression of critical enzymes in glycolytic and lipogenic pathways. We found that the transcription factor XBP1, a key regulator of the unfolded protein response, is required for the unrelated function of normal fatty acid synthesis in the liver. XBP1 protein expression in mice was elevated after feeding carbohydrates and corresponded with the induction of critical genes involved in fatty acid synthesis. Inducible, selective deletion of XBP1 in the liver resulted in marked hypocholesterolemia and hypotriglyceridemia, secondary to a decreased production of lipids from the liver. This phenotype was not accompanied by hepatic steatosis or compromise in protein secretory function. The identification of XBP1 as a regulator of lipogenesis has important implications for human dyslipidemias.

Figure 1. ER stress response in XBP1 deficient mouse liver

(A and B) Hematoxylin and eosin staining of liver sections. (C and D) Transmission electron micrographs of WT and Xbp1Δ liver. (E) Total RNAs were prepared from the liver of WT and Xbp1Δ mice 8 hours after tunicamycin injection. Expression of representative UPR target genes were tested by quantitative real-time PCR analysis. Fold induction represents relative expression level ± SEM compared to that of untreated WT mice. N=4 per group (F) Western blot of nuclear XBP1s and the processed ATF6α(N) proteins. Sp1 expression is a loading control. Levels of IRE1α-mediated XBP1 mRNA (both WT and mutant) splicing were measured by RT-PCR. *non-specific band. G) Total and active JNK protein levels were determined by western blot and c–Jun kinase assay, respectively. (H) IRE1α was detected by immunoprecipitation followed by western blot with IRE1α antibody. One third of the Xbp1Δ immunoprecipitation product was loaded on the last lane for a better comparison of band migration. Phosphorylated IRE1α displayed slowed migration on the gel. (I) IRE1α immunoprecipitation products were treated with λ phosphatase. Western blot analysis shows a band shift upon phosphatase treatment.

Figure 2. Plasma and hepatic lipid profiles of XBP1 deficient mice

Xbp1^f/f and Xbp1^f/f;Mx1-cre mice were injected 3X with poly(I:C). Three weeks after the last injection, plasma TG (A), cholesterol (B), and serum free fatty acid levels (C) were measured. (D) Plasma TG levels were measured over time in mice that received a single injection of 250 µg of poly(I:C). Error bars represent SEM. N=6–7 (E) Distribution of plasma cholesterol was determined by FPLC separation of lipoprotein particles. (F) Fat content in the liver was determined by oil red O staining. (G) Lipid composition in the liver was determined by Lipomics analysis. N=4/group.

Figure 3. Diminished hepatic TG secretion and lipid synthesis, but normal turnover of apoB in the absence of XBP1

(A) Mice were injected with Tyloxapol after a four-hour fast and plasma triglyceride levels measured over time. N=4/group. Primary hepatocytes were labeled with [¹⁴C]-acetate. Radiolabeled fatty acids (B) and sterols (C) extracted from equal numbers of cells were measured by scintillation counting. (D) Primary hepatocytes from WT and Xbp1Δ mice were labeled with ³⁵S-methionine/cysteine for 30 min and then chased for indicated times. Radiolabeled apoB protein species were immunoprecipitated from cells and media and revealed by SDS-PAGE followed by fluorography. (E) The disappearance from cells and the accumulation in media of radiolabeled apoB protein species were determined by plotting relative radioactivity of each band (normalized to initial level in cells or final level in media) versus chase time. (F) Western blot analysis of plasma apoB protein.

Figure 4. XBP1 directly regulates lipogenic genes in the liver

(A) Expression of lipogenic genes in the liver of mice fed a standard chow diet was measured by quantitative real time PCR with actin mRNA as control. Values represent relative amounts of each mRNA compared to WT. Statistical significance of differences between WT and Xbp1Δ were determined. *, p<0.005; **, p<0.0001. (B) Primary hepatocytes were cultured in media containing indicated concentrations of glucose and insulin for 24 h. Nuclear extracts were subjected to western blot analysis. (C) Primary hepatocytes were infected with adenoviruses expressing XBP-1s or GFP control at 2 or 10 pfu per cell. mRNA levels were measured 24 hours after infection by real-time PCR. (D) CHIP assays were performed with liver nuclei of mice untreated or injected with Tm 6 hrs prior to sacrifice. Values represent fold increases of real-time PCR signals compared to the signal for the untreated WT CHIP with control serum.