MON-058 - Exploring the Direct Effects of Adipokines and Other Metabolic Signals on Kndy Neurons using Immortalized Mouse and Sheep Cell Lines

The multiphenotypic hypothalamic KNDy neurons, releasing kisspeptin (KP), neurokinin B (NKB or Tac2/3), and dynorphin (Dyn), play a critical role in the neuroendocrine regulation of reproduction via stimulation of pulsatile GnRH release, and are requisite for fertility. In vivo evidence in mice also identifies this arcuate population in mediating negative feedback effects of gonadal steroids, as well as receiving input from neighboring arcuate populations critical for metabolic regulation.
We previously generated murine immortalized KNDy neuronal cell lines (KTaR-1) to explore molecular mechanisms underlying steroid hormone regulation, and more recently generated and characterized an analogous immortalized line (oKNDy) derived from fetal female sheep brain. These neurons express ovine kiss1, tac3, and pdyn, as well as esr1, esr2, and pgr. Similar to observations in the mouse, low doses (5-50pM) of 17β-estradiol (E2) significantly repress kiss1 and tac3 expression. Since these neurons were derived from short-day seasonally breeding sheep, they can inform how pituitary circannual timing cues may exert gating signals on the reproductive axis as modeled in vivo. Consistent with this, we find high levels of thyroid hormone receptor (thra) expression in oKNDy neurons, and find kiss1/tac3 expression inhibited by exogenous T3.
We used both neuronal cell lines to compare gene expression changes following treatment with multiple adipokines, as well as other metabolic signals such as IGF-1, GLP-1, and ghrelin. We found that both mouse and sheep KNDy neurons express receptors for adipokines adiponectin, leptin, and resistin, while only murine neurons were revealed to express receptors for gut signals GLP-1 and ghrelin. Also, while many of these receptors were expressed constitutively regardless of estradiol exposure in both species, expression of glp1r and ghsr in KTaR-1 cells exhibited significant changes dependent on E2 concentration. In the sheep only, adiponectin was found to increase kiss1 expression in a dose- and E2-dependent fashion, while leptin treatment inhibited its expression, in contrast with what is observed in the less-responsive mouse lines. Use and comparison of these cultured neuronal cell models may provide insight into constructing species-specific molecular models of the direct effects of metabolic inputs to KNDy neurons, in addition to indirect effects via AgRP/POMC afferents.

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