Bioenergetic profile of human coronary artery smooth muscle cells and effect of metabolic intervention

Bioenergetics of artery smooth muscle cells is critical in cardiovascular health and disease. An acute rise in metabolic demand causes vasodilation in systemic circulation while a chronic shift in bioenergetic profile may lead to vascular diseases. A decrease in intracellular ATP level may trigger physiological responses while dedifferentiation of contractile smooth muscle cells to a proliferative and migratory phenotype is often observed during pathological processes. Although it is now possible to dissect multiple building blocks of bioenergetic components quantitatively, detailed cellular bioenergetics of artery smooth muscle cells is still largely unknown. Thus, we profiled cellular bioenergetics of human coronary artery smooth muscle cells and effects of metabolic intervention. Mitochondria and glycolysis stress tests utilizing Seahorse technology revealed that mitochondrial oxidative phosphorylation accounted for 54.5% of ATP production at rest with the remaining 45.5% due to glycolysis. Stress tests also showed that oxidative phosphorylation and glycolysis can increase to a maximum of 3.5 fold and 1.25 fold, respectively, indicating that the former has a high reserve capacity. Analysis of bioenergetic profile indicated that aging cells have lower resting oxidative phosphorylation and reduced reserve capacity. Intracellular ATP level of a single cell was estimated to be over 1.1 mM. Application of metabolic modulators caused significant changes in mitochondria membrane potential, intracellular ATP level and ATP:ADP ratio. The detailed breakdown of cellular bioenergetics showed that proliferating human coronary artery smooth muscle cells rely more or less equally on oxidative phosphorylation and glycolysis at rest. These cells have high respiratory reserve capacity and low glycolysis reserve capacity. Metabolic intervention influences both intracellular ATP concentration and ATP:ADP ratio, where subtler changes may be detected by the latter

Calculation of ATP production rates using the Seahorse XF Analyzer

Oxidative phosphorylation and glycolysis are the dominant ATP-generating pathways in mammalian metabolism. The balance between these two pathways is often shifted to execute cell-specific functions in response to stimuli that promote activation, proliferation, or differentiation. However, measurement of these metabolic switches has remained mostly qualitative, making it difficult to discriminate between healthy, physiological changes in energy transduction or compensatory responses due to metabolic dysfunction. We therefore present a broadly applicable method to calculate ATP production rates from oxidative phosphorylation and glycolysis using Seahorse XF Analyzer data and empirical conversion factors. We quantify the bioenergetic changes observed during macrophage polarization as well as cancer cell adaptation to in vitro culture conditions. Additionally, we detect substantive changes in ATP utilization upon neuronal depolarization and T cell receptor activation that are not evident from steady-state ATP measurements. This method generates a single readout that allows the direct comparison of ATP produced from oxidative phosphorylation and glycolysis in live cells. Additionally, the manuscript provides a framework for tailoring the calculations to specific cell systems or experimental conditions

Differential regulation of mitochondrial pyruvate carrier genes modulates respiratory capacity and stress tolerance in yeast

Mpc proteins are highly conserved from yeast to humans and are necessary for the uptake of pyruvate at the inner mitochondrial membrane, which is used for leucine and valine biosynthesis and as a fuel for respiration. Our analysis of the yeast MPC gene family suggests that amino acid biosynthesis, respiration rate and oxidative stress tolerance are regulated by changes in the Mpc protein composition of the mitochondria. Mpc2 and Mpc3 are highly similar but functionally different: Mpc2 is most abundant under fermentative non stress conditions and important for amino acid biosynthesis, while Mpc3 is the most abundant family member upon salt stress or when high respiration rates are required. Accordingly, expression of the MPC3 gene is highly activated upon NaCl stress or during the transition from fermentation to respiration, both types of regulation depend on the Hog1 MAP kinase. Overexpression experiments show that gain of Mpc2 function leads to a severe respiration defect and ROS accumulation, while Mpc3 stimulates respiration and enhances tolerance to oxidative stress. Our results identify the regulated mitochondrial pyruvate uptake as an important determinant of respiration rate and stress resistance.This work was supported by Ministerio de Economia y Competitividad grant BFU2011-23326 to M.P.; A.T.-G. was supported by a JAE predoctoral grant from Consejo Superior de Investigaciones Cientificas. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Timón Gómez, A.; Proft ., MH.; Pascual-Ahuir Giner, MD. (2013). Differential regulation of mitochondrial pyruvate carrier genes modulates respiratory capacity and stress tolerance in yeast. PLoS ONE. 8(11):1-9. doi:10.1371/journal.pone.0079405S19811Murphy, M. P. (2008). How mitochondria produce reactive oxygen species. Biochemical Journal, 417(1), 1-13. doi:10.1042/bj20081386Pan, Y. (2011). Mitochondria, reactive oxygen species, and chronological aging: A message from yeast. Experimental Gerontology, 46(11), 847-852. doi:10.1016/j.exger.2011.08.007Perrone, G. G., Tan, S.-X., & Dawes, I. W. (2008). Reactive oxygen species and yeast apoptosis. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1783(7), 1354-1368. doi:10.1016/j.bbamcr.2008.01.023Galdieri, L., Mehrotra, S., Yu, S., & Vancura, A. (2010). Transcriptional Regulation in Yeast during Diauxic Shift and Stationary Phase. OMICS: A Journal of Integrative Biology, 14(6), 629-638. doi:10.1089/omi.2010.0069Broach, J. R. (2012). Nutritional Control of Growth and Development in Yeast. Genetics, 192(1), 73-105. doi:10.1534/genetics.111.135731Hedbacker, K. (2008). SNF1/AMPK pathways in yeast. Frontiers in Bioscience, 13(13), 2408. doi:10.2741/2854Martínez-Pastor, M., Proft, M., & Pascual-Ahuir, A. (2010). Adaptive Changes of the Yeast Mitochondrial Proteome in Response to Salt Stress. OMICS: A Journal of Integrative Biology, 14(5), 541-552. doi:10.1089/omi.2010.0020Pastor, M. M., Proft, M., & Pascual-Ahuir, A. (2009). Mitochondrial Function Is an Inducible Determinant of Osmotic Stress Adaptation in Yeast. Journal of Biological Chemistry, 284(44), 30307-30317. doi:10.1074/jbc.m109.050682Saito, H., & Posas, F. (2012). Response to Hyperosmotic Stress. Genetics, 192(2), 289-318. doi:10.1534/genetics.112.140863Ruiz-Roig, C., Noriega, N., Duch, A., Posas, F., & de Nadal, E. (2012). The Hog1 SAPK controls the Rtg1/Rtg3 transcriptional complex activity by multiple regulatory mechanisms. Molecular Biology of the Cell, 23(21), 4286-4296. doi:10.1091/mbc.e12-04-0289Bricker, D. K., Taylor, E. B., Schell, J. C., Orsak, T., Boutron, A., Chen, Y.-C., … Rutter, J. (2012). A Mitochondrial Pyruvate Carrier Required for Pyruvate Uptake in Yeast, Drosophila, and Humans. Science, 337(6090), 96-100. doi:10.1126/science.1218099Herzig, S., Raemy, E., Montessuit, S., Veuthey, J.-L., Zamboni, N., Westermann, B., … Martinou, J.-C. (2012). Identification and Functional Expression of the Mitochondrial Pyruvate Carrier. Science, 337(6090), 93-96. doi:10.1126/science.1218530Winzeler, E. A. (1999). Functional Characterization of the S. cerevisiae Genome by Gene Deletion and Parallel Analysis. Science, 285(5429), 901-906. doi:10.1126/science.285.5429.901Ghaemmaghami, S., Huh, W.-K., Bower, K., Howson, R. W., Belle, A., Dephoure, N., … Weissman, J. S. (2003). Global analysis of protein expression in yeast. Nature, 425(6959), 737-741. doi:10.1038/nature02046Alberti, S., Gitler, A. D., & Lindquist, S. (2007). A suite of Gateway®cloning vectors for high-throughput genetic analysis inSaccharomyces cerevisiae. Yeast, 24(10), 913-919. doi:10.1002/yea.1502Westermann, B., & Neupert, W. (2000). Mitochondria-targeted green fluorescent proteins: convenient tools for the study of organelle biogenesis inSaccharomyces cerevisiae. Yeast, 16(15), 1421-1427. doi:10.1002/1097-0061(200011)16:153.0.co;2-uHong, H.-Y., Yoo, G.-S., & Choi, J.-K. (2000). Direct Blue 71 staining of proteins bound to blotting membranes. Electrophoresis, 21(5), 841-845. doi:10.1002/(sici)1522-2683(20000301)21:53.0.co;2-4Nakai, T., Yasuhara, T., Fujiki, Y., & Ohashi, A. (1995). Multiple genes, including a member of the AAA family, are essential for degradation of unassembled subunit 2 of cytochrome c oxidase in yeast mitochondria. Molecular and Cellular Biology, 15(8), 4441-4452. doi:10.1128/mcb.15.8.4441Boubekeur, S., Bunoust, O., Camougrand, N., Castroviejo, M., Rigoulet, M., & Guérin, B. (1999). A Mitochondrial Pyruvate Dehydrogenase Bypass in the YeastSaccharomyces cerevisiae. Journal of Biological Chemistry, 274(30), 21044-21048. doi:10.1074/jbc.274.30.21044Palmieri, L., Lasorsa, F. M., Iacobazzi, V., Runswick, M. J., Palmieri, F., & Walker, J. E. (1999). Identification of the mitochondrial carnitine carrier in Saccharomyces cerevisiae. FEBS Letters, 462(3), 472-476. doi:10.1016/s0014-5793(99)01555-0Martínez-Montañés, F., Pascual-Ahuir, A., & Proft, M. (2010). Toward a Genomic View of the Gene Expression Program Regulated by Osmostress in Yeast. OMICS: A Journal of Integrative Biology, 14(6), 619-627. doi:10.1089/omi.2010.0046Proft, M., Gibbons, F. D., Copeland, M., Roth, F. P., & Struhl, K. (2005). Genomewide Identification of Sko1 Target Promoters Reveals a Regulatory Network That Operates in Response to Osmotic Stress inSaccharomyces cerevisiae. Eukaryotic Cell, 4(8), 1343-1352. doi:10.1128/ec.4.8.1343-1352.2005Divakaruni, A. S., & Murphy, A. N. (2012). A Mitochondrial Mystery, Solved. Science, 337(6090), 41-43. doi:10.1126/science.1225601Smith, R. A. J., Hartley, R. C., Cochemé, H. M., & Murphy, M. P. (2012). Mitochondrial pharmacology. Trends in Pharmacological Sciences, 33(6), 341-352. doi:10.1016/j.tips.2012.03.010Poteet, E., Choudhury, G. R., Winters, A., Li, W., Ryou, M.-G., Liu, R., … Yang, S.-H. (2013). Reversing the Warburg Effect as a Treatment for Glioblastoma. Journal of Biological Chemistry, 288(13), 9153-9164. doi:10.1074/jbc.m112.440354Soga, T. (2013). Cancer metabolism: Key players in metabolic reprogramming. Cancer Science, 104(3), 275-281. doi:10.1111/cas.1208

Branched-chain amino acid catabolism fuels adipocyte differentiation and lipogenesis

Adipose tissue plays important roles in regulating carbohydrate and lipid homeostasis, but less is known about the regulation of amino acid metabolism in adipocytes. Here we applied isotope tracing to pre-adipocytes and differentiated adipocytes to quantify the contributions of different substrates to tricarboxylic acid (TCA) metabolism and lipogenesis. In contrast to proliferating cells, which use glucose and glutamine for acetyl-coenzyme A (AcCoA) generation, differentiated adipocytes showed increased branched-chain amino acid (BCAA) catabolic flux such that leucine and isoleucine from medium and/or from protein catabolism accounted for as much as 30% of lipogenic AcCoA pools. Medium cobalamin deficiency caused methylmalonic acid accumulation and odd-chain fatty acid synthesis. Vitamin B12 supplementation reduced these metabolites and altered the balance of substrates entering mitochondria. Finally, inhibition of BCAA catabolism compromised adipogenesis. These results quantitatively highlight the contribution of BCAAs to adipocyte metabolism and suggest that BCAA catabolism has a functional role in adipocyte differentiation

Branched-chain amino acid catabolism fuels adipocyte differentiation and lipogenesis

Adipose tissue plays important roles in regulating carbohydrate and lipid homeostasis, but less is known about the regulation of amino acid metabolism in adipocytes. Here we applied isotope tracing to pre-adipocytes and differentiated adipocytes to quantify the contributions of different substrates to tricarboxylic acid (TCA) metabolism and lipogenesis. In contrast to proliferating cells, which use glucose and glutamine for acetyl-coenzyme A (AcCoA) generation, differentiated adipocytes showed increased branched-chain amino acid (BCAA) catabolic flux such that leucine and isoleucine from medium and/or from protein catabolism accounted for as much as 30% of lipogenic AcCoA pools. Medium cobalamin deficiency caused methylmalonic acid accumulation and odd-chain fatty acid synthesis. Vitamin B12 supplementation reduced these metabolites and altered the balance of substrates entering mitochondria. Finally, inhibition of BCAA catabolism compromised adipogenesis. These results quantitatively highlight the contribution of BCAAs to adipocyte metabolism and suggest that BCAA catabolism has a functional role in adipocyte differentiation

Assessing calcium-stimulated mitochondrial bioenergetics using the seahorse XF96 analyzer.

The development of fluorescence-based oxygen sensors coupled with microplate-based assays for quantitative bioenergetics analyses enables screening multiple experimental conditions at once with small biological material and in a timely manner. In this chapter, we outline detailed protocols and practical tips to design and perform controlled measurements of (a) respiratory and glycolytic metabolism of intact cells, (b) substrate-dependent respiration in permeabilized cells and isolated mitochondria, and (c) calcium-dependent regulation of mitochondrial bioenergetics with Seahorse XF Flux Analyzers