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.