Oleic acid supports mitochondrial function during lipolytic and pro-oxidant challenges in bovine adipocytes.

Increased energy demand during the transition period in dairy cows leads to excessive lipolysis, oxidative stress, and mitochondrial dysfunction in adipose tissue (AT). Mitochondria play a central role in fatty acid (FA) oxidation and redox regulation during negative energy balance. In humans, palmitic acid (PA) is linked to mitochondrial stress, whereas oleic acid (OA) supports normal function. However, the impact of acute lipolytic or pro-oxidant challenges on mitochondrial function in bovine adipocytes, and whether that impact can be mitigated by FA supplementation, remains unclear. This study aimed to (1) determine how lipolysis and redox imbalance affect mitochondrial respiratory function and (2) evaluate whether OA preserves mitochondrial respiration and function under these conditions. Pre-adipocytes were isolated from subcutaneous AT of nonlactating, nongestating Holstein dairy cows (n = 6) and induced to differentiate. Mature adipocytes were cultured in standard differentiation medium supplemented with PA, OA, or a PA-OA mixture (60% PA, 40% OA) at 300 µM for 7 d. On d 7, lipolysis was stimulated with 1 µM isoproterenol hydrochloride (ISO), and a pro-oxidant challenge was induced with 300 µM H2O2 for 0.5, 2, and 4 h. A basal (BAS) condition with no ISO or H2O2 served as the control. Mitochondrial respiration was assessed in real time by quantifying oxygen consumption rate using an extracellular flux analyzer (Seahorse Pro XF, Agilent Technologies). Isoproterenol reduced basal and ATP-linked respiration at 2 h and decreased maximal respiration and spare respiratory capacity at 0.5 and 2 h, with recovery by 4 h. Hydrogen peroxide progressively impaired basal, maximal, ATP-linked respiration, spare respiratory capacity, and coupling efficiency (%), with peak disruption at 4 h. Hydrogen peroxide increased proton leak at 0.5 and 2 h. Neither ISO nor H2O2 altered nonmitochondrial oxygen consumption. During lipolysis, PA and OA maintained respiration parameters at levels that did not differ from BAS. During redox imbalance, OA improved basal and maximal respiration, as well as spare respiratory capacity, compared with PA; however, none of the FA treatments fully prevented the decline in mitochondrial respiration induced by the pro-oxidant challenge. These findings demonstrate that lipolysis induces a transient suppression of mitochondrial respiration, whereas redox imbalance causes a progressive and sustained decline in mitochondrial function. Although neither FA fully restored mitochondrial function under redox imbalance, OA partially preserved key respiratory parameters. This highlights OA's potential to support mitochondrial bioenergetics in vitro and provides a cellular basis for investigating the bioenergetic mechanisms underlying the metabolic effects of OA-enriched diets in transition dairy cows.