The present study investigates the longitudinal sex-specific alterations in fecal metabolites and endpoint liver metabolites in HFD exposed C57BL/6J mice. A recent study by Hubbard and colleagues found that a prolonged HFD diet worsens sexually dimorphic obesity (Hubbard et al., 2019). The goal is to compare the sex difference in metabolites and identify specific biomarkers for each sex in both fecal and liver samples in obese mice.
From fecal metabolomic analysis, several biomarkers such as BCAAs, short-chain fatty acids (SCFAs), and glutamate, were shown significant changes impacted by HFD. First, as one of the important BCAAs, isoleucine and its first degradation product 3-methyl-2-oxovalerate were observed to be significantly elevated across all time points (5th, 9th, and 12th months) in the female HFD group compared to its control group. Isoleucine plays an important role in the growth, protein and fatty acid metabolism, immunity, and regulation of key signaling pathways like mTOR (Zhang et al., 2017). Amino acids levels in the body are tightly regulated, and very little is being excreted through the urine or feces. Therefore, elevated fecal isoleucine excretion in our study may indicate increased peripheral isoleucine in the host due to decreased amino acid catabolism or increased gut bacterial metabolism in the female HFD group. Recent studies have shown a strong correlation of BCAA levels with insulin resistance and blood glucose levels in obese human individuals in comparison to non-obese individuals (Allam-Ndoul et al., 2015; Lynch & Adams, 2014). Thus, increased fecal BCAA levels can be used as an effective biomarker for identifying early metabolic alteration associated with obesity in females.
Fecal SCFAs, such as acetate and propionate, were reduced significantly in 9th month female HFD fecal samples compare to its control. The gut bacteria produce SCFAs via fermentation of undigested dietary starch and fibers. SCFAs supplementation is considered a strategy to reduce weight, as it plays an important role in appetite regulation, lipid and glucose metabolism, and integrity of the gastrointestinal barrier. Moreover, propionate regulates lipolysis and lipogenesis rate in fat cells (Kimura et al., 2014). Decreased acetate and propionate indicate a decline in SCFA production by gut bacteria in female mice, which exacerbate the pathophysiology of obesity (Kimura et al., 2013).
Bile acid plays important role in fat digestion, absorption and also serve as signaling molecule keeping up homeostatic state (Agellon, 2002). Bile acid cholate is synthesized in liver and being conjugated before secreting into the small intestine. About 95% of bile acid secreted into the small intestine is reabsorbed and recirculated through enterohepatic circulation. The left over portion is passed on to distal intestine where it is further transformed into secondary metabolites by gut microbes. Thus, little of bile acids is lost in the feces (Chiang, 2013). However, long-term consumption of HFD increased the secretion of bile acid and its secondary products. A study done only in male rats showed the increased bile acids secretion and fats absorption in the development of hepatic steatosis (Hori et al., 2020). Non-alcoholic steatohepatitis (NASH) patients consuming high fat diet showed increased serum and urine bile metabolites in NASH patients compared to healthy individuals (Ferslew et al., 2015). Our study showed significantly increased levels of total bile acid, cholate, and glycocholate in both 9th and 12th month of female HFD fecal samples compare to its control, but not in male HFD mice. Furthermore, the impact of HFD on bile acid catabolism is apparently sex specific demonstrated by our study. All these findings imply that the chronic HFD consumption is evident to hamper the bile acid metabolism and the elevated bile acids secretion may further lead to intestinal hyperpermeability (Murakami et al., 2016; Yoshitsugu et al., 2019, 2020). The HFD induced altered bile acid level maybe also due to increased gut microbial communities which are essential to convert conjugated bile acids into secondary metabolites (Lin et al., 2019).
Glutamate is an important marker in both fecal and liver metabolites. It plays an important role in maintaining intestinal integrity, synthesis of glutathione, enteric bacterial nitrogen metabolism, and brain health (Mazzoli & Pessione, 2016; Newsholme et al., 2003). A cross-sectional study showed lower fecal glutamate level among obese individuals (sex unspecified) compared to lean subjects (Palomo-Buitrago et al., 2019). Intestinal glutamate is interchangeable to glutamine that fuels enterocytes (Burrin & Reeds, 1997). In our study, the female HFD group showed significantly lower level of glutamate in the 9th and 12th month’s fecal samples. The reduced fecal glutamate level in HFD female group may due to the increased intestinal absorption and liver utilization of glutamate (Blachier et al., 2009; Yang et al., 2016, Windmueller & Spaeth, 1975). Moreover, glutamate is also used as substrate by enteric anaerobic bacteria to produce acetate and butyrate (Mardinoglu et al., 2015). Our study showed a trend of decreased acetate and butyrate in both male and female HFD mice compared to their controls (p > 0.05). Our finding in general is supported by the fact that obese individuals have reduced glutamate fermenting enteric bacterial abundance (Liu et al., 2017). Therefore, reduced fecal excretion especially in female HFD group is due to increased intestinal glutamate catabolism and altered bacterial fermentation of glutamate.
In contrast with decreased fecal glutamate level, both the male and female HFD liver extracts showed significant increase of glutamine and glutamate levels compare to their controls. Liver is central in nitrogen metabolism where glutamine is catabolized to produce glutamate and ammonia. Glutamate further enters TCA cycle and act as a substrate for gluconeogenesis (Newsholme et al., 2003), while ammonia generated detoxified through urea cycle. The long-term HFD exposure leads to increased level of glutamine synthetase in the liver of male C57BL/6 mice (Soontornniyomkij et al, 2016). It also leads to fatty liver which further reduces liver ammonia detoxification capacity (Green & Hodson, 2014). In such circumstances, hepatotoxicity is avoided through alternative metabolomics pathway. This pathway synthesize glutamine through glutamate while utilizing the ammonia (Soontornniyomkij et al., 2016; Varani et al., 2022). Increased liver glutamine and glutamate levels in both male and female HFD groups resonates the effect of chronic HFD feeding on liver detoxification capacity and adaptation to the alternative glutamine synthesis(Ruiz et al., 1991). Furthermore, glutamate is a precursor for glutathione which is an important antioxidant. Elevated levels of glutathione found only in female HFD liver samples indicates more active defensive mechanism to overcome liver dysfunction in female HFD group (Ghosh et al., 2011; Yu et al., 1999).
In conclusion, this study implemented a systematic comparison of metabolites between sexes using HFD exposed C57BL/6J mice. In our study, female HFD mice exhibited less HFD induced weight gain compared to male mice over time but also a more discernable perturbation and more robust changes in metabolites. Results from our study of obesity-related changes over time may be overall significant in establishing early fecal and liver biomarkers as they may be first discerned and for their sex-specific characteristics. Future studies will investigate the sex-specific gut microbial composition and gut-liver axis of key metabolites. Such information will likely provide additional insight on molecular mechanisms of HFD-induced specific responses.