Testosterone deficiency synergistically exacerbates fructose-induced hepatic steatosis through gut microbiota and pyruvate in mice

Hepatic steatosis is the initial stage of metabolic dysfunction-associated steatotic liver disease (MASLD) and is highly prevalent among middle-aged men. Low testosterone levels and dietary fructose intake are independent risk factors for MASLD, although these can occur simultaneously. This study investigated the combined effects of testosterone deficiency and fructose intake on hepatic steatosis, focusing on the involvement of gut microbiota. Male mice were castrated or sham-operated at 8 wk of age and administered fructose water with or without antibiotics for 8 wk after being divided into six groups: Sham/Control, Sham/Fructose, Sham/Fructose + Antibiotics, Castration/Control, Castration/Fructose, and Castration/Fructose + Antibiotics. The castrated groups had lower body weights than the sham-operated groups, whereas castration did not affect portal and circulating fructose concentrations. Although castration alone did not affect hepatic lipid accumulation, it synergistically promoted fructose-induced triglyceride accumulation, which was alleviated by antibiotic treatment. The expression of lipogenesis-related genes (<i>Srebp-1c</i>), fatty acid transporters (<i>Cd36</i>), and fructose metabolism-related genes (<i>Aldob</i>, <i>Khk-A</i>, and <i>Khk-C</i>) was upregulated by the combination of castration and fructose intake, but antibiotic administration did not suppress this effect. Castration, fructose intake, and their combination influenced β-diversity, but not α-diversity of gut microbiota composition. Cecal pyruvate concentrations were increased by the combination of castration and fructose intake and were suppressed by antibiotics. PICRUSt2 and MaAsLin2 analyses supported pyruvate accumulation mediated by alterations in the gut microbiota. Furthermore, pyruvate promoted triglyceride accumulation in primary hepatocytes in the presence of fructose. Our results indicated that testosterone deficiency synergistically exacerbates fructose-induced hepatic steatosis, which is partly mediated by gut-derived pyruvate.<b>NEW & NOTEWORTHY</b> The combined effects of the factors that cause liver dysfunction are unclear. Here, we show that fructose intake and testosterone deficiency synergistically induce intestinal pyruvate accumulation and hepatic steatosis by altering gut microbiota and hepatic gene expression. Our findings provide a notion that pyruvate plays a novel role in hepatic steatosis. Moreover, our results suggest that risk factors for hepatic steatosis can exert a greater impact on disease development through complex mechanisms under certain conditions.