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ABSTRACT: Ketosis is often accompanied by a reduction in milk production in dairy cows, but the molecular mechanism has not been fully elucidated. Ketotic cows possess systemic oxidative stress (OS), which may implicate apoptosis in mammary glands. Sirtuin 3 (SIRT3) is a vital regulator of cellular redox homeostasis and is under the control of AMP-activated protein kinase (AMPK) signaling in nonruminants. Thus, we aimed to investigate (1) the AMPK-SIRT3 and apoptosis status of mammary glands from ketotic cows, (2) the effect of SIRT3 on OS-induced apoptosis in bovine mammary epithelial cells (BMEC), and (3) the role of AMPK signaling on SIRT3-mediated effects on apoptosis. Mammary gland samples were reused from a previous study, which contained healthy and ketotic cows (both n = 15). BMEC were incubated with 0, 0.3, 0.6, or 0.9 mM H2O2 for 6 h with/without a 30 min incubation of an antioxidant MitoQ (1 μM). Then BMEC were incubated with SIRT3 overexpression adenovirus (Ad-SIRT3) for 6 h followed by a 6 h incubation with 0.6 mM H2O2. Finally, BMEC were treated with the AMPK inhibitor Compound C (Cd C,10 μM) for 30 min before the H2O2 challenge, or cells were initially treated with the AMPK agonist MK8722 (10 μM) for 30 min followed by a 30-h culture with/without si-SIRT3 and eventually the H2O2 exposure. Ketotic cows displayed higher levels of Bax, Caspase-3 and Bax/Bcl-2 but lower levels of Bcl-2 in mammary glands. H2O2 incubation displayed similar results, exhibiting a dose-dependent manner between the H2O2 concentration and the apoptosis degree. Mito Q pretreatment reduced cellular reactive oxygen species and rescued cells from apoptosis. Ketotic cows had a lower mammary protein abundance of SIRT3. Similarly, H2O2 incubation downregulated both mRNA and protein levels of SIRT3 in a dose- and time-dependent manner. Ad-SIRT3 infection lowered levels of cellular reactive oxygen species, Bax, Caspase-3 and Bax/Bcl-2 but increased levels of Bcl-2. TUNEL assays confirmed that Ad-SIRT3 infection mitigated H2O2-induced apoptosis. Both ketotic cows and H2O2-induced BMEC had lower levels of p-AMPK and p-AMPK/AMPK. Additionally, Cd C pretreatment decreased SIRT3 and Bcl-2 expression but increased levels of Bax and Caspase-3. Contrary to the inhibitor, MK8722 had opposite effects and reduced the percentage of apoptotic cells. However, these effects of MK8722 were reversed upon SIRT3 silencing. In conclusion, in vivo data confirmed that ketosis is associated with greater apoptosis and restricted AMPK-SIRT3 signaling in mammary glands; in vitro data indicated that SIRT3 mitigates OS-induced apoptosis via AMPK signaling. As such, there may be potential benefits for targeting the AMPK-SIRT3 axis to help counteract the negative effects of mammary glands during ketosis. |
