A-050 The Role of Gut Microbial Metabolite Trimethylamine N-Oxide in Adrenal Cholesterol Homeostasis

Abstract Background Elevated levels of the gut-microbe-associated metabolite trimethylamine-N-oxide (TMAO) have been associated with increased risk for CVD mortality in many large independent studies. In fact, large laboratory corporations such as Labcorp and Quest Diagnostic now offer TMAO diagnostic tests for the assessment of CVD risk and as a marker for disease-associated dysbiosis. One of the hallmarks of TMAO’s role in CVD development is through alteration of cholesterol homeostasis, contributing to the development and progression of atherosclerosis. Current research investigating TMAO’s effects on cholesterol homeostasis focuses on the gut, liver, and vasculature. However, essentially nothing is known in regard to its impact on cholesterol balance in the adrenal gland, which is responsible for the synthesis of important steroid hormones derived solely from cholesterol, such as glucocorticoids and mineralocorticoids. With TMAO’s known effect on cholesterol homeostasis in other tissues, and the reliance of the adrenal cortex on lipoprotein-derived cholesterol to produce steroid hormones, it is important to further understand whether TMAO may be associated with altered adrenal activity, as conditions of adrenal dysfunction are often associated with increased risk for cardiovascular disease due to the role that glucocorticoids and mineralocorticoids play in the regulation of glucose metabolism and electrolyte homeostasis, respectively. Here, we performed a study on C57BL/6J mice to investigate whether changes in plasma TMAO levels resulted in altered transcriptomic expression of adrenocortical cholesterol transporters and steroidogenic enzymes. Methods Six-week-old female C57BL/6J mice were placed on high fat diets, with half supplemented with gut bacterial choline TMA-lyase inhibitor, fluoromethylcholine (FMC) (n = 6). The mice fed ad libitum for a period of two weeks, after which adrenal glands were harvested. Real time polymerase chain reaction (qPCR) was used to evaluate transcriptomic expression of several adrenal cholesterol transporters and steroidogenesis enzymes. Results were analyzed using GraphPad Prism 10 student unpaired t-test function, where results with p values less than 0.05 were considered statistically significant. Results Three adrenocortical genes were found to be upregulated when mice were treated with FMC. mRNA expression of HDL-c transporter Srb1 trended higher (p = 0.057) in mice fed a diet supplemented with FMC, while Ldlr showed no trends or statistical significance. mRNA expression of cholesterol exporter Abca1 was significantly higher in mice fed a diet supplemented with FMC (p = 0.038), while Abcg1 showed no significance. Additionally, mRNA expression of steroidogenic enzymes Cyp11a1 and Cyp11b1 was significantly higher in mice treated with FMC compared to controls (p values of 0.010 and 0.029, respectively), where Cyp11a1 encodes the cholesterol side-chain cleavage enzyme (P450scc) and Cyp11b1 encodes the corticosterone synthase enzyme, 11B-hydroxylase. Conclusion These results indicate that inhibition of TMAO-production does alter the transcription of critical genes involved in adrenal cholesterol flux and steroidogenesis. It appears that lack of TMAO results in moderate upregulation of an adrenal cholesterol transporter and two steroidogenic enzymes. Collectively, these data support a role for TMAO in adrenocortical cholesterol homeostasis and metabolism and merit further investigation into whether high plasma TMAO can impact adrenal function in a clinically significant way.