AMPKα alleviates the inhibitory effect of NEFA on the function of bovine follicular granulosa cells cultured in vitro.

High levels of non-esterified fatty acids (NEFA) in cows with subclinical ketosis (SCK) impair postpartum follicular development and disrupt estrus. The precise mechanism through which NEFA impacts the functionality of bovine follicular cells remains elusive. An in vivo experiment was conducted to compare SCK cows without estrus (SCK-E, n = 6) with healthy cows in estrus (C-E, n = 6). In the vitro test, bovine granulosa cells (GCs) were exposed to 0.4 mM NEFA. Notably, the SCK-E group exhibited an elevated ratio of phosphorylated adenosine 5'-monophosphate-activated protein kinase α (AMPKα) to total AMPKα in both liver and ovarian tissues, compared to the C-E group. NEFA treatment of GCs adversely affected steroid hormone synthesis, suppressed the expression of cyclin and proteins crucial for steroid synthesis, and triggered cell apoptosis, thereby inhibiting cell proliferation. Furthermore, it led to a decline in cell mitochondrial membrane potential and an increase in reactive oxygen species production, ultimately causing cellular damage. Subsequently, GCs were co-cultured with adenovirus (ad-AMPKα-siRNA) and NEFA (0.4 mM). Inhibiting AMPKα further exacerbated the detrimental effects of NEFA on steroid hormone synthesis, cell apoptosis, cell proliferation, and mitochondrial function in GCs. Furthermore, upon inhibiting AMPKα, a reduction was observed in both mRNA and protein levels of acetyl-CoA carboxylase 1, accompanied by an elevation in the levels of carnitine palmitoyltransferase-1. These findings suggest that AMPKα becomes activated in SCK cows experiencing elevated NEFA levels, and that AMPKα has the potential to mitigate the detrimental effects of NEFA on GCs function in vitro.