Carnitine palmitoyltransferase 2 knockout potentiates palmitate-induced insulin resistance in C2C12 myotubes

Authors: BLACKBURN, MICHAEL - Arkansas Children'S Nutrition Research Center (ACNC); ONO-MOORE, KIKUMI - Arkansas Children'S Nutrition Research Center (ACNC); SOBHI, HANY - Coppin State University; Ferruzzi, Mario

Submitted to: American Journal of Physiology - Endocrinology and Metabolism
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/19/2020
Publication Date: 7/22/2020
Citation: Blackburn, M.L., Ono-Moore, K.D., Sobhi, H.F., Adams, S.H. 2020. Carnitine palmitoyltransferase 2 knockout potentiates palmitate-induced insulin resistance in C2C12 myotubes. American Journal of Physiology - Endocrinology and Metabolism. 319(2):E265-E275. https://doi.org/10.1152/ajpendo.00515.2019.
DOI: https://doi.org/10.1152/ajpendo.00515.2019

Interpretive Summary: Saturated fatty acids (SFAs) are implicated in driving whole-body inflammation and contributing to muscle tissue resistance to the blood sugar regulating hormone insulin. The underlying factors that drive the association between SFAs and metabolic health remain to be confirmed. It is also important to acknowledge that fats are important fuels for the muscle, and is is not entirely clear that fats have a negative impact on all aspects of muscle function, including inflammation. SFA derivatives that accumulate with mismatched fatty acids (FA) availability and FA combustion are likely involved in inflammation, cell stress and insulin resistance, and evidence has emerged that select molecules called acylcarnitines should be considered. To understand if excessive long-chain acylcarnitine renders cells more susceptible to lipotoxicity, carnitine palmitoyltransferase 2 knockout muscle cells were generated (CPT2 KO). Since the protein CPT2 is central to fat combustion, limiting its activity increases non-oxidative fates of SFAs including overproduction of acylcarnitines. There was no effect of CPT2 KO on palmitic acid (PA) concentration-dependent increases in media markers of cell stress (IL-6 or adenylate kinase) or other markers inside the cells. In contrast, loss of CPT2 made PA-induced insulin resistance much worse, reducing insulin signaling by as much as ~50-96% compared to control cells. Manipulating the production of acylcarnitines through the use of certain inhibitors or cell culture conditions partly rescued cells from SFA-induced insulin resistance. The results indicate that PA-induced insulin resistance stems in part from palmitoylcarnitine accumulation. The data further support the idea that certain by-products of SFA metabolism (acylcarnitines) participate in muscle cell signaling and when in excess, compromise cell function. This highlights that diet and exercise strategies that promote efficient fat combustion and minimize accumulation of lipid by-products may contribute to more positive health outcomes in muscle.

Technical Abstract: Saturated fatty acids (SFAs) are implicated in muscle inflammation/cell stress and insulin resistance, but the catalog of factors involved is incomplete. SFA derivatives that accumulate with mismatched FA availability and FA oxidation (FAO) are likely involved, and evidence has emerged that select acylcarnitines should be considered. To understand if excessive long-chain acylcarnitine renders cells more susceptible to lipotoxicity, carnitine palmitoyltransferase 2 knockout C2C12 cells were generated (CPT2 KO). CPT2 KO was confirmed by Western blot, increased palmitoylcarnitine accumulation and loss of FAO capacity. There was no effect of CPT2 KO on palmitic acid (PA) concentration-dependent increases in media IL-6 or adenylate kinase. PA at (200, 500 uM) did not trigger other cell stress responses (phosphor-Erk, -JNK, or -p38) above that of vehicle in WT or CPT2 KO cells. In contrast, loss of CPT2 exacerbated PA-induced insulin resistance (acute phospho-Akt; 10 or 100 nM insulin) by as much as ~50-96% compared to WT. The CPT1 inhibitor etomoxir increased p-Akt, and growing cells in carnitine-free media abolished differences between WT and CPT2 KO. The results indicate that PA-induced insulin resistance stems in part from palmitoylcarnitine accumulation, which helps explain increased CPT2 KO susceptibility to PA. The data further support the hypothesis that select acylcarnitines participate in cell signaling and when in excess, compromise cell function. Since etomoxir and carnitine-free conditions could not fully rescue insulin signaling, and CPT2 KO did not alter cell stress responses, the majority of PA-induced "lipotoxicity" in C2C12 myotubes cannot be attributed to palmitoylcarnitine alone.