1Department of Exercise Science, High Point University, High Point NC, 272682School of Medicine, Medical College of Wisconsin, Milwaukee WI, 53226

Author Contact: johnsm14@highpoint.edu or rvaughan@highpoint.edu

Michele A. Johnson1, Nicholas P. Gannon2, Jamie K. Schnuck1,2, and Roger A. Vaughan, PhD1*

Figure 1. Leucine-induced mitochondrial biogenesis is abolished by concurrent palmitate. Effect of varying leucine concentration with andwithout palmitate at 0.5mM on myotube mRNA expression of (A) peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α), (B) nuclear respiratory factor 1 (NRF1), (C) mitochondrial transcription factor A (TFAM), (D) sirtuin 3 (Sirt3), (E) citrate synthase (CS),and (F) forkhead box protein O1 (Foxo1).

EFFECT OF SATURATED FATTY ACID ON MARKERS OF BCAA-MEDIATED MITOCHONDRIAL BIOGENESIS AND BCAA CATABOLISM IN VITRO

RESULTSABSTRACTPURPOSE: Branched chain amino acids (BCAA) such as leucine,stimulate favorable metabolic processes involved in lean tissuepreservation and skeletal muscle metabolism. However, higher levelsof circulating BCAAs correlate with severity of metabolic disease(including diabetes/insulin resistance), and may result fromderegulated BCAA catabolism. This study investigated therelationship(s) between BCAA concentration and expression ofregulators of metabolism and BCAA catabolism in cultured skeletalmuscle cells. METHODS: C2C12 myotubes were treated with orwithout varying concentrations of either leucine or valine for 24hours, both with and without concurrent palmitate. Data wereanalyzed via Spearman correlation. RESULTS: Increasing leucinetreatment significantly correlated with elevated mRNA expression ofmetabolic targets including peroxisome proliferator-activatedreceptor-gamma coactivator-1alpha (PGC-1a) (rs=0.73, p<0.01),mitochondrial transcription factor A (rs=0.58, p<0.05), sirtuin 3(rs=0.58, p<0.05), citrate synthase (rs=0.56, p<0.05), and forkhead boxprotein O1 (rs=0.66, p<0.05). However, increasing valine treatmentonly correlated with PGC-1a expression. Interestingly, leucine-induced metabolic gene expression was abolished by concurrentpalmitate. Additionally, branched-chain amino transferase 2expression positively correlated with increasing leucine treatment,which was not observed for branched-chain α-ketoicaciddehydrogenase. BCAA catabolic enzyme expression did notsignificantly correlate with leucine-palmitate co-treatments, or valinetreatment concentrations. CONCLUSION: These data suggest leucinepossesses unique metabolic effects compared with other BCAAs.Moreover, the presence of palmitate diminished the metabolic effectsof leucine, suggesting lipids may suppress leucine-mediated celladaptations.

METHODSo C2C12 myotubes were treated with leucine from 0-2mM for 24

hours, both with and without concurrent palmitate at 0.5mM.o Gene expression was measured via qRT PCRo Protein expression was measured via Western Bloto Lipid content was measured using Oil Red O extractiono Cell viability was assessed using WST-1 assayo Data were analyzed via Spearman correlation, 1-way ANOVA, 2-

way ANOVA, or student’s t test.• * or dissimilar letters indicates p<0.05• Bonferonni’s correction was used for ANOVAs

Figure 5. Summary of potential branched-chain aminoacid-mediated mechanisms of stimulating mitochondrialbiogenesis.

Figure 2. Cell viability of cells treated with and withoutleucine at 2mM both with and without palmitate at0.5mM.

REFERENCES• C. Liang, B.J. Curry, P.L. Brown, M.B. Zemel, Leucine Modulates Mitochondrial

Biogenesis and SIRT1-AMPK Signaling in C2C12 Myotubes, J Nutr Metab, (2014)239750.

• J.K. Schnuck, K.L. Sunderland, N.P. Gannon, M.R. Kuennen, R.A. Vaughan, Leucinestimulates PPARbeta/delta-dependent mitochondrial biogenesis and oxidative metabolismwith enhanced GLUT4 content and glucose uptake in myotubes, Biochimie, (2016).

• N.P. Gannon, R.A. Vaughan, Leucine-induced anabolic-catabolism: two sides of the samecoin, Amino Acids, (2015).

• C.B. Newgard, J. An, J.R. Bain, M.J. Muehlbauer, R.D. Stevens, L.F. Lien, A.M. Haqq,S.H. Shah, M. Arlotto, C.A. Slentz, J. Rochon, D. Gallup, O. Ilkayeva, B.R. Wenner, W.S.Yancy, Jr., H. Eisenson, G. Musante, R.S. Surwit, D.S. Millington, M.D. Butler, L.P.Svetkey, A branched-chain amino acid-related metabolic signature that differentiatesobese and lean humans and contributes to insulin resistance, Cell Metab, 9 (2009) 311-326.

• Y. Shimomura, T. Murakami, N. Nakai, M. Nagasaki, R.A. Harris, Exercise promotesBCAA catabolism: effects of BCAA supplementation on skeletal muscle during exercise,J Nutr, 134 (2004) 1583s-1587s.

www.highpoint.edu/healthscienceswww.highpoint.edu/healthsciences

Viability

0 15 30 45 60 75 900

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ControlLeu 2mMPALeu 2mM+PA

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Figure 4. mRNA and Protein Expression of markers ofBCAA Degradation. Effect of varying leucine treatment withand without 0.5mM palmitate on myotube mRNA expressionof (A) BCAT2 and (B) BCKDHa. (C) Protein expression ofBCKDHa (normalized to β-actin).

Figure 3. Palmitate increases lipid content without altering indicators ofmitochondrial biogenesis. (A) Effect of palmitate on mRNA expression ofperoxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A(TFAM), sirtuin 3 (Sirt3), forkhead box protein O1 (Foxo1), and citratesynthase (CS). (B) Lipid content (indicated by oil red O extraction) of cellstreated with and without leucine at 2mM both with and without palmitate at0.5mM for 24 hours.

WORKING HYPOTHESIS

Under conditions of energy homeostasis, BCAAs maypromote improved metabolic phenotypes includingimproved glucose uptake/insulin sensitivity. Underconditions of chronic excess energy, cells (especiallyadipose) appear to lose the ability to degrade BCAAscausing an accumulation of BCAAs and related metabolitesin circulation. As with many metabolites during metabolicdisease (such as lipids and glucose), BCAA accumulationappears to correlate and may have predictive value ofmetabolic disease. It is hypothesized that obesity-relatedsuppression of molecular targets that regulate BCAAmetabolism precedes deregulated BCAA catabolism andaccumulation.