Melatonin Attenuates Heat Stress-Induced Metabolic Labeling Remodeling in Primary Goat Sertoli Cells

Heat stress impairs male reproductive function, but whether acute heat stress alters glutamine-derived carbon labeling in Sertoli cells and whether melatonin modulates this response remain unclear. This study aimed to determine whether acute heat stress alters glutamine-derived carbon labeling in primary goat Sertoli cells and to evaluate whether melatonin attenuates these heat stress-associated metabolic changes. Primary goat Sertoli cells were assigned to control, heat-stress, or heat-stress plus melatonin groups. Cells were labeled for 24 h with [U-13C5] glutamine. Cells in the heat-stress and heat-stress plus melatonin groups were subsequently exposed to 42 °C for 0.5 h, whereas control cells were maintained at 37 °C. In the heat-stress plus melatonin group, melatonin (0.5 μM) was applied throughout labeling and heat exposure. GC–MS was used to measure mass isotopologue distributions, which were subsequently corrected for natural isotope abundance, and to calculate total 13C-labeled fractions of selected TCA cycle intermediates; extracellular acidification rate (ECAR)-derived parameters were measured as indirect indices of glycolysis-associated acidification. Heat stress increased the total 13C-labeled fraction of cis-aconitate and, as an exploratory combined readout, the unweighted average total 13C-labeled fraction of citrate and cis-aconitate; succinate and malate were unchanged, while fumarate decreased. Melatonin reduced the heat stress-associated total 13C-labeled fractions of citrate and cis-aconitate and decreased the relative abundance of cis-aconitate M + 5. Melatonin also attenuated heat stress-associated increases in ECAR-derived glycolysis, glycolytic capacity, and glycolytic reserve. These findings indicate that acute heat stress is associated with altered citrate/cis-aconitate 13C-labeling patterns that are potentially compatible with reductive carboxylation-related labeling but do not directly demonstrate altered pathway flux; these changes were attenuated by melatonin. Overall, the study objective was achieved by showing that acute heat stress altered citrate/cis-aconitate 13C-labeling patterns and increased ECAR-derived extracellular acidification in primary goat Sertoli cells, and that melatonin attenuated both responses. Further studies incorporating a melatonin-only group, dynamic isotope tracing, mitochondrial respiration measurements, redox analysis, and flux modeling are warranted to define the underlying metabolic routes more directly.