High-Fat Diet Induces Epigenetic and Metabolic Changes in Kisspeptin Neurons in Association with Obesity and Male Secondary Hypogonadism

Abstract Background Obesity and type 2 diabetes mellitus (T2D) are major risk factors for male hypogonadism, a disorder with multi-system impacts on health. However, the mechanisms underlying obesity-associated male secondary hypogonadism remain poorly understood. Here, we aimed to dissect dysfunction of the hypothalamic–pituitary–testicular (HPT) axis and elucidate underlying epigenetic mechanisms, using a high-fat diet mouse model. Methods Male C57BL6 mice were fed standard chow or high-fat diet (HFD, 60% fat) for 16 weeks starting at age 6 weeks. Plasma testosterone levels, sperm counts, and gonadotropin responses to senktide and kisspeptin stimulation were assessed. HFD-induced transcription changes in the hypothalamic arcuate nucleus (ARC) were evaluted using bulk and single-cell RNA-sequencing. Genome-wide changes in 5-hydroxymethylcytosine (5hmC) were analyzed by hydroxymethyl-DNA immunoprecipitation sequencing (hMeDIP-seq). Functional relevance of 5hmC changes was evaluated by ectopic TET expression in an immortalized ARC Kiss1 neuron cell line and RT-qPCR. Results HFD-fed mice developed obesity, hyperglycemia, glucose intolerance, and insulin resistance, indicative of the development of a type 2 diabetes-like metabolic disorder. This was accompanied by low testosterone, reduced sperm counts, and unchanged basal luteinizing hormone (LH), consistent with obesity/T2D-associated male secondary hypogonadism, as observed clinically in humans. Impaired LH responses to senktide, a Kiss1 neuron activator, but not to kisspeptin itself, identified suppressed Kiss1 neuron function as a key mechanism underlying secondary hypogonadism. RNA-seq analysis revealed dysregulation of metabolic and neural pathways in Kiss1 neurons. hMeDIP-seq demonstrated widespread 5hmC alterations in the ARC of HFD-induced obese/diabetic mice, correlated with dysregulation of fatty acid metabolism, neuronal activity, and synapse function pathways. Ectopic TET expression ex vivo in Kiss1 neuronal cell lines restored 5hmC levels and upregulated key metabolic and neuronal genes that were repressed in the ARC of DIO mice Conclusion Our findings demonstrate that Kiss1 neurons are highly sensitive to diet and metabolic changes, and that obesity/diabetes-induced 5hmC modifications play a key role in dysregulating metabolic and neuronal pathways in Kiss1 neurons. These findings reveal a novel mechanism linking metabolic disturbances to reproductive dysfunction, through direct effects on Kiss1 neurons. Graphic Abstract