Functional metabolite reserves and lipid homeostasis revealed by the MA-10 Leydig cell metabolome

In Leydig cells, intrinsic factors that determine cellular steroidogenic efficiency is of functional interest to decipher and monitor pathophysiology in many contexts. Nevertheless, beyond basic regulation of cholesterol storage and mobilization, systems biology interpretation of the metabolite networks in steroidogenic function is deficient. To reconstruct and describe the different molecular systems regulating steroidogenesis, we profiled the metabolites in resting MA-10 Leydig cells. Our results identified 283-annotated components (82 neutral lipids, 154 membrane lipids, and 47 other metabolites). Neutral lipids were represented by an abundance of triacyglycerols (97.1%), and low levels of cholesterol esters (2.0%). Membrane lipids were represented by an abundance of glycerophospholipids (77.8%), followed by sphingolipids (22.2%). Acylcarnitines, nucleosides, amino acids and their derivatives were the other metabolite classes identified. Among nonlipid metabolites, we recognized substantial reserves of aspartic acid, choline, creatine, betaine, glutamine, homoserine, isoleucine, and pantothenic acid none of which have been previously considered as a requirement in steroidogenic function. Individually limiting use of betaine, choline, or pantothenic acid, during luteinizing hormone-induced steroidogenesis in MA-10 cells resulted in substantial decreases to acute steroidogenic capacity, explained by intermediary metabolite imbalances affecting homeostasis. As such, our dataset represents the current level of baseline characterization and unravels the functional resting state of steroidogenic MA-10 Leydig cells. In identifying metabolite stockpiles and causal mechanisms, these results serve to further comprehend the cellular setup and regulation of steroid biosynthesis.