EXPLORING DIFFERENCES IN INDIVIDUAL SUSCEPTIBILITY TO LIPOTOXICITY AND THE UNDERLYING MECHANISMS IN THE MITOCHONDRIA OF PRIMARY PREADIPOCYTES
Shine, Jared M
Prolonged exposure to fatty acids (FAs), at levels normally seen during fasting, can be cytotoxic in vulnerable cells including preadipocytes. Prior work from our laboratory has shown that FAs cause mitochondrial inner membrane (MIM) permeabilization due to the formation of the permeability transition pore (PTP) and ultimately cell death. Cyclophilin D (CypD) is well known, and the F1F0 ATP synthase has emerged, to be necessary for PTP. However, the role of the Bcl-2 (b-cell lymphoma 2) family members in the formation of the PTP is less clear. The initial hypothesis was that the cytotoxicity of FAs in primary human preadipocytes correlates with the binding of CypD to, and the release of Bcl-xL (b-cell lymphoma extra-large) from, the F1F0 ATP synthase complex. Our results showed a wide range of FA sensitivity in primary human preadipocytes as the number of cells undergoing MIM permeabilization and cell death was 2.3-fold and 3.4-fold higher, respectively, in groups labeled FA-sensitive (FAS) compared to the groups labeled FA-resistant (FAR) quintile. The purpose of selecting these groups was to examine differences in mitochondrial handling of elevated FAs and how this led to differences in preadipocyte cell death. Under thought of the adipose expandability hypothesis, we hypothesize that decreased preadipocyte number (indicative of our FAS group) during times of elevated FAs could play a role in adipose tissue dysfunction and the resulting metabolic abnormalities known as the metabolic syndrome. Furthermore, MIM permeabilization and cell death were prevented by incubating preadipocytes with mitochondrion-selective superoxide and lipid peroxide targeting antioxidants MitoTempo and MitoQ, as well as L-carnitine. Co-immunoprecipitation showed CypD was already present in the F0F1 ATP synthase complex and not further incorporated following FA exposure. Cyclosporine A (CsA), which induced the dissociation of CypD from the F0F1 ATP synthase, prevented FA-induced cytotoxicity. We confirmed that Bcl-xL binds to the F0F1 ATP synthase and that FAs induced the release of Bcl-xL. ABT-737, which inhibits Bcl-xL protein to protein interactions, exacerbated FA-induced ATP depletion and eliminated mitochondrial respiratory reserve capacity in the FAR preadipocytes only. However, this only marginally affected cell viability. Interestingly, FA exposure induced the expression of the carnitine acetyltransferase and sirtuin 3 (SIRT3) genes in the FAR preadipocytes only. We propose that SIRT3 induction decreased acetylation of the F1Fo ATP synthase. In our second study we furthered the aforementioned work by hypothesizing that improving preadipocytes FA sensitivity will correlate with improvements in metabolic health following 12 weeks of exercise training. As observed in our older cohort, the FA sensitivity of preadipocytes ranged several folds. Interestingly, FA sensitivity correlated with lower high-density lipoprotein-cholesterol (HDL-c) levels. Following the training program, the individuals that improved their FA sensitivity also decreased their low-density lipoprotein-cholesterol (LDL-c) levels. One goal of the exercise study was to assess whether fatty acid sensitivity testing prior to exercise could predict the effectiveness of exercise on metabolic health. Though no significance was found, there was a trend for pre-exercise fatty acid sensitivity to correlate with the changes in triglycerides and HDL-c after exercise. The work done in this dissertation provides a deeper understanding of how chronically elevated levels of FA can cause mitochondrial dysfunction and preadipocyte death. The variance in this sensitivity to the toxic effects of FA need further elucidation yet could possibly be at play in determining metabolic health of obese individuals.
Shine, Jared M. (July 2018). EXPLORING DIFFERENCES IN INDIVIDUAL SUSCEPTIBILITY TO LIPOTOXICITY AND THE UNDERLYING MECHANISMS IN THE MITOCHONDRIA OF PRIMARY PREADIPOCYTES (Doctoral Dissertation, East Carolina University). Retrieved from the Scholarship. (http://hdl.handle.net/10342/6976.)
Shine, Jared M. EXPLORING DIFFERENCES IN INDIVIDUAL SUSCEPTIBILITY TO LIPOTOXICITY AND THE UNDERLYING MECHANISMS IN THE MITOCHONDRIA OF PRIMARY PREADIPOCYTES. Doctoral Dissertation. East Carolina University, July 2018. The Scholarship. http://hdl.handle.net/10342/6976. September 19, 2020.
Shine, Jared M, “EXPLORING DIFFERENCES IN INDIVIDUAL SUSCEPTIBILITY TO LIPOTOXICITY AND THE UNDERLYING MECHANISMS IN THE MITOCHONDRIA OF PRIMARY PREADIPOCYTES” (Doctoral Dissertation., East Carolina University, July 2018).
Shine, Jared M. EXPLORING DIFFERENCES IN INDIVIDUAL SUSCEPTIBILITY TO LIPOTOXICITY AND THE UNDERLYING MECHANISMS IN THE MITOCHONDRIA OF PRIMARY PREADIPOCYTES [Doctoral Dissertation]. Greenville, NC: East Carolina University; July 2018.
East Carolina University