Dietary Cholesterol‐Induced Gut Microbes Drive Nonalcoholic Fatty Liver Disease Pathogenesis in a Murine Model

Abstract

Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are hepatic manifestations of metabolic syndrome and major indications for liver transplantation. Western diet contributes to disease pathogenesis, partially mediated through the gut microbiome, yet mechanisms remain elusive. Human epidemiological studies identified high dietary cholesterol intake as a NAFLD risk factor and it is essential to drive disease in murine models, yet little is known about its role in reshaping gut microbiota. Using the fast food (FF) diet murine model in germ‐free (GF) mice completely devoid of all microbes and their conventionally‐raised (control) counterparts harboring complex microbiomes, we hypothesized high dietary cholesterol‐induced gut microbiota impact NAFLD onset, progression, and severity.Male C57Bl/6 age‐matched GF and control mice were fed 1 of 4 semi‐purified diets: low‐fat (LF); high fat (HF); FF + 0.2% high cholesterol (FFHC); FF + 2% very high cholesterol (FFVHC) for 8 or 24 weeks. Fecal gut microbiota profiles were tracked over time via Illumina MiSeq 16S rRNA gene amplicon sequencing. Serum alanine transaminase (ALT) and lipopolysaccharide binding protein (LBP, an indicator of gut barrier function) were measured throughout the study. Livers were collected for histology and Illumina NovaSeq RNA‐sequencing.Despite equal caloric intake between GF and controls across diets, significant weight gain and increased liver weight to body weight ratios (P<0.05) were observed only in control mice fed FF diets. GF mice were largely protected from disease, with no elevation in plasma ALT, LBP, or histology‐based NAFLD activity score (NAS) regardless of treatment. Conversely, FFVHC control mice exhibited significantly elevated plasma ALT after 8 weeks on diet, which was exacerbated at 24 weeks relative to LF control and all GF groups. FF diets significantly increased (FFHC: P<0.05; FFVHC: P<0.01) plasma LBP after 24 weeks. Control mice fed FF diets exhibited severe steatosis, where FFVHC significantly increased NAS at 8 (P<0.05) and 24 (P<0.001) weeks relative to LF control and all GF groups. Microbiota profiling revealed no change in α‐diversity regardless of diet in control mice. β‐diversity analysis showed HF and FF diets, particularly FFVHC, rapidly shifted gut microbiota community membership after only 4 weeks, preceding disease onset and was further exacerbated over time. Liver RNA‐seq revealed FFVHC diet in control, but not GF, mice significantly enriched genes involved in the KEGG pathway, “antigen processing and presentation” (Bonferroni P<0.001) relative to HF‐fed counterparts at 24 weeks.Taken together, FF diet‐induced shifts in gut microbes are both a prerequisite for and precede NAFLD/NASH disease onset, which is exacerbated by increased dietary cholesterol, driving liver inflammation. These data provide unique insights into how Western diet components impact host‐microbe interactions in complex liver diseases, which may aid in identifying novel therapeutic interventions.