Abstract Objective Metabolic dysfunction-associated fatty liver disease (MAFLD) represents a growing cause of mortality and morbidity and encompasses a spectrum of liver pathologies. Potential therapeutic targets have been identified and are currently being pre-clinically and clinically tested. However, while dozens of preclinical models have been developed to recapitulate various stages of MAFLD, few achieve fibrosis using an experimental design that mimics human pathogenesis. We sought to clarify whether the combination of thermoneutral (T N ) housing and consumption of a classical Western diet (WD) would accelerate the onset of MAFLD and progression in male and female mice. Methods Male and female C57Bl/6J mice were fed a nutrient-matched low-fat control or Western diet (41% Kcal from fat, 43% carbohydrate and 0.2% cholesterol; WD) starting at ∼12 wk of age for a further 16 wk. Mice were divided and housed with littermates at either standard temperature (T S ; 22°C) or thermoneutral conditions (T N ; ∼29°C). Mice underwent tests for glucose tolerance, insulin sensitivity and body composition, as well as intestinal permeability. Following tissue harvest, circulating and liver markers of hepatic disease progression toward steatosis and fibrosis were determined. Results While male mice housed at T N and fed a WD were significantly heavier than T S -housed control animals, no other differences in body weight or composition were observed. WD-fed females housed under T N conditions had higher circulating LDL-cholesterol; however, there were no other significant differences between T N and T S -housing in circulating or hepatic lipid levels. While WD-fed T N males had higher ALT levels, no other differences in markers of liver injury or disease progression were observed. Moreover, females housed at T N conditions and fed a WD remained significantly protected against the induction of fibrosis compared to male counterparts. Interestingly, sex-specific differences were observed in markers of glucose and insulin tolerance, where T N housing and WD feeding resulted in hyperglycemia and impaired insulin responsiveness in both sexes, but glucose intolerance only in male mice. Conclusions While T N housing has been demonstrated to exacerbate high fat-induced hepatic steatosis and inflammation in male and female mice, coupling T N housing with a WD for 16 wk was not sufficient to augment fatty liver progression in male or female mice. Highlights Thermoneutral housing and Western diet feeding does not progress to NASH Female mice are not more susceptible to obesity induced fatty liver under these conditions Temperature and diet had sex-specific effects on glucose tolerance and insulin sensitivity

Thermoneutral housing does not accelerate metabolic dysfunction-associated fatty liver disease in male or female mice fed a Western diet