Obesity is linked to reproductive dysfunction through defects in the hypothalamic-pituitary-gonadal (HPG) axis that arise by unknown mechanisms. Importantly, rodents fed a high-fat diet (HFD) develop similar hypogonadism and reduced ovulatory capacity. During HFD feeding, CNS immune cells (microglia) become activated in the hypothalamus, promoting inflammation, and altering neuronal function indirectly and via cell-cell contacts. Surprisingly, however, genetic ablation of this microglial inflammatory response exacerbates rather than improves HFD-induced HPG axis dysfunction including altering the activity of hypothalamic neurons that express the key reproductive neuropeptide GnRH. Therefore, we hypothesized that increased microglial inflammatory signaling during HFD feeding helps maintain GnRH neuron integrity. To test this hypothesis, we used immunohistochemistry (IHC) to assess microglia-GnRH cell-cell interactions in the hypothalamus of 15-week HFD-fed female mice with microglia-specific deletion of IKKβ (IKKβ-MGKO), a critical regulator of the inflammatory NF-κB pathway. IHC studies using GnRH and Iba1 (microglial marker) revealed fewer cell-cell contacts between GnRH neurons and microglia in the preoptic area of the hypothalamus (POA) of IKKβ-MGKO mice compared with controls. In addition, we found that IKKβ-MGKO mice have reduced levels of Iba1 and total numbers of microglia but no changes in microglial cell morphology as determined by Sholl analysis. These findings suggest that HFD-induced microglial inflammatory signaling promote cell-cell interactions with GnRH neurons that may contribute to maintenance of HPG axis integrity and female reproductive function during diet-induced obesity.

The global pandemic of obesity has increased the prevalence and burden of metabolic diseases, including type 2 diabetes and cardiovascular disease. Obesity and its comorbidities are frequently associated with hypogonadism (low levels of testosterone (T) in men), and both preclinical and clinical evidence support a causative role of hypogonadism in predisposing individuals to metabolic diseases. However, the mechanisms remain unknown. One potential mechanism arises from our recent discovery that in mice, surgical castration (reducing T levels) amplifies the pro-inflammatory response to consumption of a high-fat diet, specifically leading to activation of astrocytes within the hypothalamus, a brain region critical for regulating whole-body metabolism. Concomitantly, there is a striking reduction of the anti-inflammatory neuropeptide neurokinin B (NKB; encoded by the Tac2 gene) in the same brain region. Therefore, we hypothesized that T limits astrocyte inflammation via enhanced NKB-neurokinin-3 receptor (NK3R) signaling. Using primary astrocytes harvested from newborn mice, we found that T and dihydrotestosterone (DHT; a non-aromatizable androgen) increase the expression of tachykinin genes like Tac2. Further, androgen treatment blunted the proinflammatory response of primary astrocytes to lipopolysaccharide (LPS), a sepsis-inducing bacterial cell wall component. To assess the anti-inflammatory capacity of NK3R signaling, we co-incubated astrocytes with the NK3R agonist Senktide and LPS, finding a significant attenuation of proinflammatory cytokine expression. Together, these data suggested that androgen receptor signaling might constrain astrocyte inflammation through induction of NKB-NK3R. However, the ability of DHT to reduce cytokine expression in response to LPS was preserved in the presence of Osanetant, an NK3R antagonist, indicating that the anti-inflammatory actions of androgens are independent of NK3R signaling. These findings form the foundation for future pharmacologic and genetic interventions in obese mouse models to further clarify the role of astrocyte T and NK3R signaling in hypogonadism-associated metabolic diseases.