Beta-adrenergic agonists alter mitochondrial metabolite utilization independent of a fiber type shift

Skeletal muscle metabolism and nutrient utilization are dynamic during postnatal growth. While metabolic flexibility of muscle is vital for overall health, the subtleties of metabolite utilization pose a challenge for defining mechanisms that facilitate this adaptability. For example, assessment of metabolic enzymes indicates shifts but may not depict minute fluctuations in metabolite utilization during the transition between glycolytic and oxidative pathways. Therefore, we supplemented pigs with beta-adrenergic agonist (BAA) to investigate changes in myosin heavy chain (MyHC) isoforms and metabolic enzymes as well as mitochondrial utilization of pyruvate, the glycolytic end product. Mature DNA 600 x 241 barrows (DNA Genetics) were fed a standard commercial diet containing 16% crude protein supplemented with 0 ppm (control; n = 8) or 9 ppm ractopamine hydrochloride (BAA; n = 10) for 2 weeks. Samples were collected from the longissimus dorsi (LD, glycolytic muscle), latissimus dorsi (LAT, mixed muscle), semitendinosus (ST, mixed muscle), and masseter (MS, oxidative muscle). Protein abundance of type I MyHC was highest in MS, intermediate in LAT, and lowest in ST and LD ( P < 0.05). Type IIA MyHC was higher in MS than other muscles ( P < 0.05), and type IIX MyHC was lower in MS than other muscles ( P < 0.05). Type IIB MyHC was lowest in MS, intermediate in LAT and LD, and highest in ST ( P < 0.05). However, BAA supplementation did not alter MyHC isoform abundance in muscles evaluated. Activity of phosphofructokinase (PFK) as well as protein abundance of lactate dehydrogenase alpha (LDHα) and succinate dehydrogenase alpha (SDHα) were also assessed. Activity of PFK and LDHα abundance was lowest in MS, intermediate in LAT and LD, and highest in ST ( P < 0.05). However, there were no treatment differences. Further, SDHα abundance was highest in MS, intermediate in LAT and ST, and lowest in LD muscle ( P < 0.05), but there were no treatment differences. To determine if BAA supplementation alters mitochondrial utilization of glucose, isolated mitochondria from LD, LAT, and MS muscles were incubated with [ 13 C 3 ]-pyruvate to assess pyruvate-derived tricarboxylic acid cycle (TCA) intermediate flux. Isotopic enrichment of oxaloacetate (M + 2), citrate (M + 2, M+ 3, and M + 4), and α-ketoglutarate (M + 4 and M + 5) decreased in BAA LAT mitochondria compared to control LAT mitochondria ( P < 0.05). Further, enrichment of α-ketoglutarate (M + 2 and M + 3) and citrate (M + 5 and M + 6) decreased in all BAA mitochondria ( P < 0.05). Collectively, these findings suggest mitochondria with inherent metabolic differences exhibit unique responses to BAA supplementation. Although a lack of BAA mediated changes at a protein level may be attributed to inadequate dosage and duration of feeding, supplementation induced changes in mitochondrial pyruvate utilization. These findings suggest altered metabolite utilization may occur independent of frequently measured indicators of metabolic shifts. Supported by Animal Nutrition, Growth, and Lactation, award no. 2022-67015-36202, from the U.S. Department of Agriculture's National Institute of Food and Agriculture. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.