Non-alcoholic fatty liver disease (NAFLD) is a prevalent chronic liver disease significantly contributing to liver-related mortality. Hepatic steatosis, the initial stage of NAFLD, is influenced by factors such as fatty acid uptake and disposal, de novo lipogenesis, and fatty acid oxidation within the liver. Dietary fatty acid composition plays a crucial role in these processes. NAFLD is also linked to an aberrant gut microbiota; studies in mice have shown that NAFLD-associated gut microbiota contributes to metabolic perturbations. Human studies have identified microbiome signatures differentiating NAFLD patients from healthy individuals, and fecal microbiota transfer from steatosis patients increases hepatic fat content in recipient mice, supporting a causal relationship. The interaction between dietary lipids and the gut microbiota influences host physiology and disease development. Previous research has demonstrated that differences in adiposity and adipose tissue inflammation between mice fed lard or fish oil diets are microbiota-transmitted. Milk fat has been shown to induce a pro-inflammatory response and colitis through a microbiota-mediated mechanism and also induce steatosis. However, the interaction between dietary lipids and the gut microbiota in affecting hepatic steatosis remains largely uncharacterized. This study aims to determine how dietary fatty acid composition affects the gut microbiota profile, microbiota-mediated metabolic regulation, and the development of hepatic steatosis.

Existing literature establishes a strong link between dietary lipids and gut microbiota composition, impacting host metabolism and disease. Studies have shown that different dietary fat sources lead to varying degrees of adiposity and inflammation, effects that are mediated by the gut microbiota. For instance, milk fat has been implicated in inducing pro-inflammatory responses and steatosis. However, the specific mechanisms underlying these interactions and their impact on hepatic steatosis remain largely unclear. This research builds upon previous findings highlighting the role of gut microbiota in metabolic diseases like NAFLD, exploring the interplay between different dietary fatty acids and their subsequent influence on microbiota composition, metabolic processes, and liver health. Prior research has demonstrated that alterations in gut microbiota are associated with NAFLD, and fecal microbiota transplantation experiments further support a causal link. However, a comprehensive understanding of how specific dietary fatty acids modulate this interaction and its consequences for liver steatosis is still lacking. The present study attempts to address this knowledge gap through a combined human and mouse model approach.

The study employed a two-pronged approach: a human cohort study and a mouse model experiment. **Human Study:** 117 subjects (79 women, 38 men) aged 27.2–66.6 years were recruited. Subjects were categorized into tertiles based on saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA) consumption. Fecal samples were collected and analyzed using shotgun metagenomics. Liver fat content was assessed using magnetic resonance imaging (MRI) and the Fatty Liver Index (FLI). Statistical analysis involved principal component analysis (PCA), robust linear regression, and correlation analysis to determine associations between dietary fatty acid intake, gut microbiota composition, and liver fat content. Age, BMI, sex, and dietary fiber intake were controlled for as potential confounders. **Mouse Study:** Male C57BL/6 mice were fed eight isocaloric diets with varying lipid compositions but consistent fiber content for 9 weeks. A milk fat (MF) diet served as a reference. Body weight, glucose tolerance, liver triglyceride levels, and steatosis scores were assessed. Liver transcriptomes were analyzed using microarray technology. Cecal microbiota was profiled using 16S rRNA gene sequencing. Multivariate analysis (DIABLO, rCCA) was performed to study relationships between microbiota, host metabolic features, and hepatic gene expression. Germ-free mice and microbiota transfer experiments were conducted to explore the causal link between microbiota and metabolic phenotypes. Finally, bile acids in vena porta were measured to investigate metabolic mediators. Statistical analyses included Kruskal-Wallis test, Wilcoxon test, ANOSIM, Adonis, and sparse partial least squares regression.

**Human Studies:** Low SFA intake was associated with increased gut microbiota diversity, independent of fiber intake. Several bacteria from phyla Firmicutes and Spirochaetes were negatively associated with SFA. SFA and PUFA intake positively correlated with FLI in obese individuals, while gut microbiota diversity negatively correlated with liver fat. **Mouse Studies:** Diets enriched in stearic acid (diets A and C) led to leaner mice, improved glucose tolerance, and reduced hepatic steatosis compared to the MF diet. Diets enriched in PUFA (diets E and G) also reduced steatosis. Diets A and C increased hepatic cholesterol biosynthesis gene expression, while diets E, F, and G decreased de novo lipogenesis gene expression. Diets A and C significantly altered the cecal microbiota, reducing observed species and phylogenetic diversity, but not bacterial load. A higher proportion of *Akkermansia* and *Bacteroides* and a lower proportion of butyrate-producing bacteria were observed. Multivariate analysis showed strong correlations between these diets, altered microbiota, and host metabolic phenotypes. Cecal levels of long-chain SFAs (C18:0 to C24:0) were negatively correlated with the abundance of many zOTUs. Network analysis showed long-chain SFAs were negatively associated with *Roseburia*, *Anaerotruncus*, and *Intestinimonas* and positively associated with *Akkermansia*, *Bacteroides*, and *Christensenellaceae*. Microbiota transfer from mice fed diet A to mice fed the MF diet improved glucose tolerance and reduced steatosis, along with non-significant increase in *Hmgcr* expression and increased fecal free fatty acid saturation. The diet A microbiota transplantation experiments also resulted in decreased observed species and phylogenetic diversity, but no change in total bacterial counts. *Lactobacillus* and *Acetatifactor* abundance were increased in recipient mice. Bile acid analysis revealed that mice fed diet A had higher total bile acid levels and TBMCA levels, with altered proportions of various bile acids. These alterations were largely transmitted by microbiota transfer. Hepatic *Shp* expression was decreased in mice with diet A microbiota.

This study provides evidence that dietary SFA, particularly stearic acid in poorly absorbed forms, has a strong impact on gut microbiota composition and function, affecting host metabolism and liver steatosis. The findings in human subjects reinforce the relevance of these observations in a clinical setting. The study demonstrates that diets enriched in stearic acid can improve metabolic profiles and reduce liver fat accumulation, possibly through microbiota-mediated mechanisms. The improved metabolic phenotype observed in mice fed stearic acid-rich diets could be attributed to changes in bile acid metabolism, increased fecal free fatty acid saturation (potentially through enhanced biohydrogenation by *Lactobacillus* strains), and alterations in the relative abundances of specific bacterial genera. The successful transfer of these beneficial metabolic effects through microbiota transplantation underscores the crucial role of the gut microbiome in mediating the effects of dietary fatty acids on host health. The study also clarifies that while PUFA-rich diets offer protection against steatosis, their effect on the gut microbiota is less pronounced, suggesting that their metabolic benefits are partly microbiota-independent.

This research highlights the significant interplay between dietary fatty acids and the gut microbiome in influencing host metabolism and liver steatosis. The results demonstrate that poorly absorbed long-chain SFAs, particularly stearic acid, can modulate gut microbiota composition and function, leading to improved metabolic outcomes and reduced liver fat accumulation. The microbiota-mediated effects of these fatty acids involve alterations in bile acid profiles and fecal fatty acid saturation. These findings provide crucial insights into developing dietary strategies targeting gut microbiota modulation for improving metabolic health and managing NAFLD. Further research should focus on elucidating the specific microbial mechanisms involved in mediating the observed effects and exploring the potential of targeted dietary interventions to enhance these beneficial interactions.

The human study had a relatively small sample size, potentially limiting the statistical power and generalizability of the findings. The mouse model, while providing valuable mechanistic insights, may not fully recapitulate the complexity of human physiology and dietary habits. While the study carefully controlled for several factors, the possibility of residual confounding effects cannot be entirely ruled out. Further research with larger human cohorts and more diverse dietary interventions would strengthen the clinical relevance and generalizability of the findings.