SAT-700 - Methylation as A Protective Mechanism Against MASLD Development in Female Mice on HFD

It is widely known that premenopausal females, but not males, are relatively protected against development of metabolic dysfunction-associated steatotic liver disease (MASLD). While some mechanisms of sexually dimorphic gene expression have been explained, how these differences result in protection against MASLD exclusively in females remains unclear.
High fat diet (HFD), associated with the development of MASLD, is known to induce significant metabolic disturbances in the liver. A central metabolic pathway that is disrupted on HFD is One Carbon Metabolism, and in particular the methionine cycle, a significant function of which is facilitating all methylation reactions in the cells. It has been shown that in the liver the methionine cycle exhibits sexual dimorphism.
We hypothesize that differences in methylation patterns between males and females contribute to female-specific protection against MASLD on HFD through methylation-driven epigenetic and epitranscriptomic mechanisms.
All experiments were performed on livers collected from 16-week-old C57BL/6 male and female mice placed on either normal chow diet (NCD) or 60% high fat diet (HFD) for 10 weeks (n=3-4 per condition).
To validate that the methionine cycle is differently affected between males and females on HFD, we measured methionine cycle enzymes at the RNA and protein levels. Bhmt RNA was significantly increased in males on HFD, but not in females, and Mat2a and Ahcy significantly dropped only in females. Furthermore, proteins of MTR, BHMT, MAT1A, MAT2A in males showed an increase in their abundance, while in females only MTR was upregulated.
Next, we validated that these methylation cycle differences carried over to two main mechanisms of methylation-driven gene expression regulation – DNA and RNA methylation. LC/MS analysis of global levels of DNA 5mC and RNA m6A methylations demonstrated that on HFD both global RNA and DNA methylation levels increased in females only.
To understand the specific mechanisms of anti-steatotic protection by methylation, we performed m6A-sequencing using Oxford Nanopore Technology, combined with RNA sequencing and proteomics of the same samples. Proteomics analysis revealed that an anti-steatotic lipid oxidation pathway was significantly more enriched in females vs males on HFD. We discovered that while increase in protein levels on HFD in this pathway could not be explained by increased RNA levels (R^2=0.002), it could be potentially explained by decreased 5’UTR methylation of those transcripts (R^2=0.33). This suggest that selective demethylation of 5’UTRs of lipid oxidation transcripts could contribute to upregulation of this anti-steatotic pathway on HFD in females only.
Our findings for the first time connect the hepatic methylation system to specific effects on sexually dimorphic gene expression on HFD, thereby potentially explaining female protection from MASLD.

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