Mechanisms of liver injury in high fat sugar diet fed mice that lack hepatocyte X-box binding protein 1.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of liver diseases in the United States and can progress to cirrhosis, end-stage liver disease and need for liver transplantation. There are limited therapies for NAFLD, in part, due to incomplete understanding of the disease pathogenesis, which involves different cell populations in the liver. Endoplasmic reticulum stress and its adaptative unfolded protein response (UPR) signaling pathway have been implicated in the progression from simple hepatic steatosis to nonalcoholic steatohepatitis (NASH). We have previously shown that mice lacking the UPR protein X-box binding protein 1 (XBP1) in the liver demonstrated enhanced liver injury and fibrosis in a high fat sugar (HFS) dietary model of NAFLD. In this study, to better understand the role of liver XBP1 in the pathobiology of NAFLD, we fed hepatocyte XBP1 deficient mice a HFS diet or chow and investigated UPR and other cell signaling pathways in hepatocytes, hepatic stellate cells and immune cells. We demonstrate that loss of XBP1 in hepatocytes increased inflammatory pathway expression and altered expression of the UPR signaling in hepatocytes and was associated with enhanced hepatic stellate cell activation after HFS feeding. We believe that a better understanding of liver cell-specific signaling in the pathogenesis of NASH may allow us to identify new therapeutic targets.

Hepatic deletion of X-box binding protein 1 impairs bile acid metabolism in mice.

The unfolded protein response (UPR) is an adaptive response to endoplasmic reticulum stress and the inositol-requiring enzyme 1α/X-box binding protein 1 (IRE1α/XBP1) pathway of the UPR is important in lipid metabolism. However, its role in bile acid metabolism remains unknown. We demonstrate that liver-specific Xbp1 knockout (LS-Xbp1-/-) mice had a 45% reduction in total bile acid pool. LS-Xbp1-/- mice had lower serum 7α-hydroxy-4-cholesten-3-one (C4) levels compared with Xbp1fl/fl mice, indicating reduced cholesterol 7α-hydroxylase (CYP7A1) synthetic activity. This occurred without reductions of hepatic CYP7A1 protein expression. Feeding LS-Xbp1-/- mice cholestyramine increased hepatic CYP7A1 protein expression to levels 2-fold and 8-fold greater than cholestyramine-fed and chow-fed Xbp1fl/fl mice, respectively. However, serum C4 levels remained unchanged and were lower than both groups of Xbp1fl/fl mice. In contrast, although feeding LS-Xbp1-/- mice cholesterol did not increase CYP7A1 expression, serum C4 levels increased significantly up to levels similar to chow-fed Xbp1fl/fl mice and the total bile acid pool normalized. In conclusion, loss of hepatic XBP1 decreased the bile acid pool and CYP7A1 synthetic activity. Cholesterol feeding, but not induction of CYP7A1 with cholestyramine, increased CYP7A1 synthetic activity and corrected the genotype-specific total bile acid pools. These data demonstrate a novel role of IRE1α/XBP1 regulating bile acid metabolism.

Hepatocyte X-box binding protein 1 deficiency increases liver injury in mice fed a high-fat/sugar diet.

Fatty liver is associated with endoplasmic reticulum stress and activation of the hepatic unfolded protein response (UPR). Reduced hepatic expression of the UPR regulator X-box binding protein 1 spliced (XBP1s) is associated with human nonalcoholic steatohepatitis (NASH), and feeding mice a high-fat diet with fructose/sucrose causes progressive, fibrosing steatohepatitis. This study examines the role of XBP1 in nonalcoholic fatty liver injury and fatty acid-induced cell injury. Hepatocyte-specific Xbp1-deficient (Xbp1(-/-)) mice were fed a high-fat/sugar (HFS) diet for up to 16 wk. HFS-fed Xbp1(-/-) mice exhibited higher serum alanine aminotransferase levels compared with Xbp1(fl/fl) controls. RNA sequencing and Gene Ontogeny pathway analysis of hepatic mRNA revealed that apoptotic process, inflammatory response, and extracellular matrix structural constituent pathways had enhanced activation in HFS-fed Xbp1(-/-) mice. Liver histology demonstrated enhanced injury and fibrosis but less steatosis in the HFS-fed Xbp1(-/-) mice. Hepatic Col1a1 and Tgfß1 gene expression, as well as Chop and phosphorylated JNK (p-JNK), were increased in Xbp1(-/-) compared with Xbp1(fl/fl) mice after HFS feeding. In vitro, stable XBP1-knockdown Huh7 cells (Huh7-KD) and scramble control cells (Huh7-SCR) were generated and treated with palmitic acid (PA) for 24 h. PA-treated Huh7-KD cells had increased cytotoxicity measured by lactate dehydrogenase release, apoptotic nuclei, and caspase3/7 activity assays compared with Huh7-SCR cells. CHOP and p-JNK expression was also increased in Huh7-KD cells following PA treatment. In conclusion, loss of XBP1 enhances injury in both in vivo and in vitro models of fatty liver injury. We speculate that hepatic XBP1 plays an important protective role in pathogenesis of NASH.

Mismatch correction modulates mutation frequency and pilus phase and antigenic variation in Neisseria gonorrhoeae.

The mismatch correction (MMC) system repairs DNA mismatches and single nucleotide insertions or deletions postreplication. To test the functions of MMC in the obligate human pathogen Neisseria gonorrhoeae, homologues of the core MMC genes mutS and mutL were inactivated in strain FA1090. No mutH homologue was found in the FA1090 genome, suggesting that gonococcal MMC is not methyl directed. MMC mutants were compared to a mutant in uvrD, the helicase that functions with MMC in Escherichia coli. Inactivation of MMC or uvrD increased spontaneous resistance to rifampin and nalidixic acid, and MMC/uvrD double mutants exhibited higher mutation frequencies than any single mutant. Loss of MMC marginally enhanced the transformation efficiency of DNA carrying a single nucleotide mismatch but not that of DNA with a 1-kb insertion. Unlike the exquisite UV sensitivity of the uvrD mutant, inactivating MMC did not affect survival after UV irradiation. MMC and uvrD mutants exhibited increased PilC-dependent pilus phase variation. mutS-deficient gonococci underwent an increased frequency of pilin antigenic variation, whereas uvrD had no effect. Recombination tracts in the mutS pilin variants were longer than in parental gonococci but utilized the same donor pilS loci. These results show that gonococcal MMC repairs mismatches and small insertion/deletions in DNA and also affects the recombination events underlying pilin antigenic variation. The differential effects of MMC and uvrD in gonococci unexpectedly reveal that MMC can function independently of uvrD in this human-specific pathogen.

Genetic characterization of the nucleotide excision repair system of Neisseria gonorrhoeae.

Nucleotide excision repair (NER) is universally used to recognize and remove many types of DNA damage. In eubacteria, the NER system typically consists of UvrA, UvrB, UvrC, the UvrD helicase, DNA polymerase I, and ligase. In addition, when DNA damage blocks transcription, transcription-repair coupling factor (TRCF), the product of the mfd gene, recruits the Uvr complex to repair the damage. Previous work using selected mutants and assays have indicated that pathogenic Neisseria spp. carry a functional NER system. In order to comprehensively examine the role of NER in Neisseria gonorrhoeae DNA recombination and repair processes, the predicted NER genes (uvrA, uvrB, uvrC, uvrD, and mfd) were each disrupted by a transposon insertion, and the uvrB and uvrD mutants were complemented with a copy of each gene in an ectopic locus. Each uvr mutant strain was highly sensitive to UV irradiation and also showed sensitivity to hydrogen peroxide killing, confirming that all of the NER genes in N. gonorrhoeae are functional. The effect of RecA expression on UV survival was minor in uvr mutants but much larger in the mfd mutant. All of the NER mutants demonstrated wild-type levels of pilin antigenic variation and DNA transformation. However, the uvrD mutant exhibited higher frequencies of PilC-mediated pilus phase variation and spontaneous mutation, a finding consistent with a role for UvrD in mismatch repair. We conclude that NER functions are conserved in N. gonorrhoeae and are important for the DNA repair capabilities of this strict human pathogen.