Sex Differences in Dietary Copper-Fructose Interaction-Induced Alterations of Gut Microbial Activity are Not Correlated to Hepatic Steatosis

Background: Inadequate copper intake and increased fructose consumption represent two important nutritional problems in the US. Diet copper-fructose interactions alter gut microbial activity and contribute to the development of nonalcoholic fatty liver disease (NAFLD). The aim of this study is to determine whether dietary copper-fructose interactions alter gut microbial activity in a sex-differential manner, and whether sex differences in gut microbial activity are associated with sex differences in hepatic steatosis. Methods: Male and female weanling Sprague-Dawley (SD) rats were fed ad libitum with an AIN-93G purified rodent diet with defined copper content for 8 weeks. The copper content is 6mg/kg and 1.5mg/kg in adequate copper diet (CuA) and marginal copper diet (CuM), respectively. Animals had free access to either deionized water or deionized water containing 10% fructose (F) (w/v) as the only drink during the experiment. Body weight, calorie intake, plasma ALT, AST and liver histology were evaluated. Fecal microbial contents were analyzed by 16S rRNA sequencing. Fecal and cecal short chain fatty acids (SCFAs) were determined by gas chromatography-mass spectrometry (GC-MS).Results: Male and female rats exhibit similar trends of changes in the body weight, body weight gain and calorie intake in response to dietary copper and fructose, with a generally higher level in male rats. Several female rats in CuAF group developed mild steatosis, while no obvious steatosis was observed in male rats fed with CuAF or CuMF. Fecal 16S rRNA sequencing analysis revealed distinct alterations of the gut microbiome in male and female rats. Linear discriminant analysis (LDA) effect size (LEfSe) identified sex-specific abundant taxa in different groups. Further, total SCFAs, as well as, butyrate were decreased in a more pronounced manner in female CuMF rats than in male rats. Of note, the decreased SCFAs are concomitant with the reduced SCFA producers, but not correlated to hepatic steatosis. Conclusions: Our data demonstrated sex differences in the alterations of gut microbial activities and hepatic steatosis in response to dietary copper-fructose interaction in rats. Tissue-specific responses to dietary copper and fructose likely contribute to the sex differences in gut microbial activity and metabolic phenotype.