XBP1 links the 12-hour clock to NAFLD and regulation of membrane fluidity and lipid homeostasis

A distinct 12-hour clock exists in addition to the 24-hour circadian clock to coordinate metabolic and stress rhythms. Here, we show that liver-specific ablation of X-box binding protein 1 (XBP1) disrupts the hepatic 12-hour clock and promotes spontaneous non-alcoholic fatty liver disease (NAFLD). We show that hepatic XBP1 predominantly regulates the 12-hour rhythmicity of gene transcription in the mouse liver and demonstrate that perturbation of the 12-hour clock, but not the core circadian clock, is associated with the onset and progression of this NAFLD phenotype. Mechanistically, we provide evidence that the spliced form of XBP1 (XBP1s) binds to the hepatic 12-hour cistrome to directly regulate the 12-hour clock, with a periodicity paralleling the harmonic activation of the 12-hour oscillatory transcription of many rate-limiting metabolic genes known to have perturbations in human metabolic disease. Functionally, we show that Xbp1 ablation significantly reduces cellular membrane fluidity and impairs lipid homeostasis via rate-limiting metabolic processes in fatty acid monounsaturated and phospholipid remodeling pathways. These findings reveal that genetic disruption of the hepatic 12-hour clock links to the onset and progression of NAFLD development via transcriptional regulator XBP1, and demonstrate a role for XBP1 and the 12-hour clock in the modulation of phospholipid composition and the maintenance of lipid homeostasis.

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Policy information about availability of computer code Data collection Bert W. O'Malley Sep 4, 2020 Metabolite concentrations were obtained using the AbsoluteIDQ kit p180 (Biocrates Life Science AG, Austria) according to manufacturer's instructions on an QTRAP 6500 LC/MS/MS System (AB SCIEX, USA) equipped with an electrospray ionization source, an Agilent G1367B autosampler and the Analyst 1.51 software (AB SCIEX, USA). Mito stress and fuel flex tests were performed with Seahorse kits and the Seahorse XFe96 analyzer. Immunoblot was detected by autoradiography or KwikQuant Imager (Kindle Biosciences).

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October 2018

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Reporting for specific materials, systems and methods GraphPad Prism 8 software was used to analyze the data and to make figures. RNA-seq reads were aligned to the mouse genome (mm10/NCBI38) using HISAT2 (2.1.0). RNA-seq quantification was performed with htseq-count (0.9.1, default parameters) and iGenome annotation (archive-2015-07-17-14-33-26 The genomic datasets generated in this study can be accessed at the GEO public repository using the accession number GSE150890. The array-based mRNA expression profiling of liver samples from NAFLD and NASH patients with healthy obese and controls were obtained from GEO (GSE48325, GSE48452). The data that support the findings of this study are available in the source data files.
Statistical methods were not used to predetermine sample size (n). Number of sample was determined based on experimental approach, availability, feasibility required to obtain definitive results. Sample size was chosen based on our prior studies and published literature using the same types of assays to ensure statistically meaningful results. Both in vitro and in vivo studies were performed with at least three biologically independent samples per group. Statistical tests then performed using GraphPad to provide confidence in the conclusions made.
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Experiments reported are replicated at least twice with similar observations. Animals were assigned to the experimental group based on genotype, and there were no drug treatment groups. Genetic manipulation of cells using adenovirus Cre recombinase. Therefore randomization was not utilized.
For plasma and metabolic profiling and glucose tolerance tests of mice, actual measurements were carried out by service core members of the institute, who did not know which phenotypes were expected. Blinding was performed by removal of identifying information from each sample while primary data was collected during an experiment. Once data had been collected, blinding was not required for statistical or bioinformatic analysis as objective readouts had been used in all experiments and all samples were analyzed using an identical method for each experiment.

October 2018
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Confirm that you have deposited or provided access to graph files (e.g. BED files) for the called peaks. The genotypes of the cell lines were also validated by PCR (Cre) and western blotting (Xbp1 protein expression) All cell lines mentioned above undergo frequently tested for mycoplasma contamination and were verified to be mycoplasma negative before undertaking any experiments with them.
No commonly misidentified cell lines were used.
All animal studies were conducted in accordance with regulations of the Committee on Animal Care and Use at Baylor College of Medicine. Xbp1 flox mice were kindly provided by Dr. Xi Chen at Baylor College of Medicine, and were generated as previously described 27. Xbp1 flox mice were crossed with AlbCre transgenic mice (Jackson Laboratories) to generate AlbCre; Xbp1 flox, as well as Xbp1 flox and AlbCre littermate controls. All offspring from this cross were maintained on a C57BL/6 background. Mice were maintained on a 12h:12h light:dark cycle and allowed free access to regular chow and water under strict temperature control. This study was conducted in male mice, not female mice.
The study did not involve wild animals.
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All animal procedures were completed in accordance with the Guidelines for the Care and Use of Laboratory Animals. All animal studies were conducted in accordance with regulations of the Committee on Animal Care and Use at Baylor College of Medicine.