The activities of some key enzymes in mitochondria from 135 human omental adipose tissue samples of obese and nonobese patients were analyzed for potential association with the patients’ state of obesity. The activities of respiratory complexes I and II as well as citrate synthase in isolated mitochondria were measured using spectrophotometric enzyme assays. ATP generation of mitochondria was determined with a bioluminescence assay. Protein levels of citrate synthase were quantified by western blot. The rates of ATP generation and the enzymatic activities of complexes I and II did not display associations with age, gender, obesity, or diabetes. By contrast, the enzymatic activities of citrate synthase and its protein levels were significantly reduced in obesity as compared to controls. In diabetic patients, protein levels but not enzymatic activities of citrate synthase were elevated. Thus, this investigation based on enzymatic assay and determination of protein levels revealed that the development of obesity is associated with a significant impact on citrate synthase in mitochondria of human omental adipose tissue. The state of obesity appears to affect mitochondrial function in human omental adipose tissue by limiting this key enzyme of the tricarboxylic acid cycle rather than by limiting the activities of respiratory chain enzymes. 1. Introduction The human adipose tissue is recognized as a multifunctional organ; it stores lipids and with its brown tissue islets also plays an important role in thermogenesis [1, 2]. Furthermore, intra-abdominal and omental adipose tissue compartments exert various endocrine functions [3] by secreting different peptide hormones and adipokines such as adiponectin, plasminogen activator inhibitor-1, interleukin-6, leptin, resistin, or visfatin [4]. The dysregulation of endocrine factors and other functional entities, including mitochondria, are known to be associated with the development of metabolic disorders such as obesity, insulin resistance, metabolic syndrome, and type 2 diabetes [5–10]. Lipid storage and mobilization in adipocytes are tightly linked to the functional state of the mitochondria. Energy expenditure depends on an intact electron transfer chain located in the inner mitochondrial membrane and requires a proton gradient to synthesize ATP from ADP and phosphate [11]. This tightly regulated process can be uncoupled by uncoupling proteins resulting in thermogenesis in brown adipocytes [12] but not in white adipocytes [13]. Different types of cell or tissue contain heterogeneous populations of mitochondria
References
[1]
B. Cannon and J. Nedergaard, “Brown adipose tissue: function and physiological significance,” Physiological Reviews, vol. 84, no. 1, pp. 277–359, 2004.
[2]
J. Nedergaard, T. Bengtsson, and B. Cannon, “Unexpected evidence for active brown adipose tissue in adult humans,” American Journal of Physiology, vol. 293, no. 2, pp. E444–E452, 2007.
[3]
H. Waki and P. Tontonoz, “Endocrine functions of adipose tissue,” Annual Review of Pathology, vol. 2, pp. 31–56, 2007.
[4]
P. Trayhurn, “Adipocyte biology,” Obesity Reviews, vol. 8, supplement 1, pp. 41–44, 2007.
[5]
J. A. Maassen, “Mitochondrial dysfunction in adipocytes: the culprit in type 2 diabetes?” Diabetologia, vol. 49, no. 4, pp. 619–620, 2006.
[6]
H. J. Choo, J. H. Kim, O. B. Kwon et al., “Mitochondria are impaired in the adipocytes of type 2 diabetic mice,” Diabetologia, vol. 49, no. 4, pp. 784–791, 2006.
[7]
A. Guilherme, J. V. Virbasius, V. Puri, and M. P. Czech, “Adipocyte dysfunctions linking obesity to insulin resistance and type 2 diabetes,” Nature Reviews Molecular Cell Biology, vol. 9, no. 5, pp. 367–377, 2008.
[8]
K. F. Petersen, D. Befroy, S. Dufour et al., “Mitochondrial dysfunction in the elderly: possible role in insulin resistance,” Science, vol. 300, no. 5622, pp. 1140–1142, 2003.
[9]
A. E. Civitarese and E. Ravussin, “Minireview: mitochondrial energetics and insulin resistance,” Endocrinology, vol. 149, no. 3, pp. 950–954, 2008.
[10]
B. L. Wajchenberg, “Subcutaneous and visceral adipose tissue: their relation to the metabolic syndrome,” Endocrine Reviews, vol. 21, no. 6, pp. 697–738, 2000.
[11]
L. Ernster and G. Schatz, “Mitochondria: a historical review,” Journal of Cell Biology, vol. 91, no. 3, part 2, pp. 227s–255s, 1981.
[12]
A. Kuroshima, “Brown adipose tissue thermogenesis as physiological strategy for adaptation,” Japanese Journal of Physiology, vol. 43, no. 2, pp. 117–139, 1993.
[13]
R. Justo, J. Oliver, and M. Gianotti, “Brown adipose tissue mitochondrial subpopulations show different morphological and thermogenic characteristics,” Mitochondrion, vol. 5, no. 1, pp. 45–53, 2005.
[14]
T. J. Collins and M. D. Bootman, “Mitochondria are morphologically heterogeneous within cells,” Journal of Experimental Biology, vol. 206, no. 12, pp. 1993–2000, 2003.
[15]
R. A. Menzies and P. H. Gold, “The turnover of mitochondria in a variety of tissues of young adult and aged rats,” Journal of Biological Chemistry, vol. 246, no. 8, pp. 2425–2429, 1971.
[16]
M. Masuyama, R. Iida, H. Takatsuka, T. Yasuda, and T. Matsuki, “Quantitative change in mitochondrial DNA content in various mouse tissues during aging,” Biochimica et Biophysica Acta, vol. 1723, no. 1–3, pp. 302–308, 2005.
[17]
D. P. Kelly and R. C. Scarpulla, “Transcriptional regulatory circuits controlling mitochondrial biogenesis and function,” Genes and Development, vol. 18, no. 4, pp. 357–368, 2004.
[18]
Z. Wu, P. Puigserver, U. Andersson et al., “Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1,” Cell, vol. 98, no. 1, pp. 115–124, 1999.
[19]
A. Lindinger, R. Peterli, T. Peters et al., “Mitochondrial DNA content in human omental adipose tissue,” Obesity Surgery, vol. 20, no. 1, pp. 84–92, 2010.
[20]
D. C. Chan, “Mitochondrial fusion and fission in mammals,” Annual Review of Cell and Developmental Biology, vol. 22, pp. 79–99, 2006.
[21]
S. A. Detmer and D. C. Chan, “Functions and dysfunctions of mitochondrial dynamics,” Nature Reviews Molecular Cell Biology, vol. 8, no. 11, pp. 870–879, 2007.
[22]
G. Mingrone, M. Manco, M. Calvani, M. Castagneto, D. Naon, and A. Zorzano, “Could the low level of expression of the gene encoding skeletal muscle mitofusin-2 account for the metabolic inflexibility of obesity?” Diabetologia, vol. 48, no. 10, pp. 2108–2114, 2005.
[23]
D. Bach, S. Pich, F. X. Soriano et al., “Mitofusin-2 determines mitochondrial network architecture and mitochondrial metabolism,” Journal of Biological Chemistry, vol. 278, no. 19, pp. 17190–17197, 2003.
[24]
J. C. Bournat and C. W. Brown, “Mitochondrial dysfunction in obesity,” Current Opinion in Endocrinology, Diabetes and Obesity, vol. 17, no. 5, pp. 446–452, 2010.
[25]
L. Wilson-Fritch, S. Nicoloro, M. Chouinard et al., “Mitochondrial remodeling in adipose tissue associated with obesity and treatment with rosiglitazone,” Journal of Clinical Investigation, vol. 114, no. 9, pp. 1281–1289, 2004.
[26]
R. Peterli, B. K. W？lnerhanssen, T. Peters, B. Kern, C. Ackermann, and M. Von Flüe, “Prospective study of a two-stage operative concept in the treatment of morbid obesity: primary lap-band followed if needed by sleeve gastrectomy with duodenal switch,” Obesity Surgery, vol. 17, no. 3, pp. 334–340, 2007.
[27]
B. K. W？lnerhanssen, T. Peters, B. Kern et al., “Predictors of outcome in treatment of morbid obesity by laparoscopic adjustable gastric banding: results of a prospective study of 380 patients,” Surgery for Obesity and Related Diseases, vol. 4, no. 4, pp. 500–506, 2008.
[28]
A. Eigentler, A. Draxl, A. Wiethüchter, A. V. Kuznetsov, B. Lassnig, and E. Gnaiger, “Laboratory protocol: citrate synthase, a mitochondrial marker enzyme,” Mitochondrial Physiology Network, vol. 17.04, pp. 1–11, 2012.
[29]
P. A. Srere, “Citrate synthase. [EC 4.1.3.7. Citrate oxaloacetate-lyase (CoA-acetylating)],” Methods in Enzymology C, vol. 13, pp. 3–11, 1969.
[30]
H. U. Bergmeier, Methoden der Enzymatischen Analyse, Chemie, Weinheim, Germany, 3rd edition, 1974.
[31]
A. V. Kuznetsov and E. Gnaiger, “Laboratory protocol: complex I (NADH: ubiquinone oxidoreductase; EC 1.6.5.3) mitochondrial membrane enzyme,” Mitochondrial Physiology Network, vol. 08.15, pp. 1–8, 2010.
[32]
W. Rouslin, “Mitochondrial complexes I, II, III, IV, and V in myocardial ischemia and autolysis,” American Journal of Physiology, vol. 13, no. 6, pp. H743–H748, 1983.
[33]
Y. Hatefi and D. L. Stiggall, “Preparation and properties of NADH: cytochrome c oxidoreductase (complex I–III),” Methods in Enzymology C, vol. 53, pp. 5–10, 1978.
[34]
P. Munujos, J. Coll-Canti, F. Gonzalez-Sastre, and F. J. Gella, “Assay of succinate dehydrogenase activity by a colorimetric-continuous method using iodonitrotetrazolium chloride as electron acceptor,” Analytical Biochemistry, vol. 212, no. 2, pp. 506–509, 1993.
[35]
B. Drew and C. Leeuwenburgh, “Method for measuring ATP production in isolated mitochondria: ATP production in brain and liver mitochondria of Fischer-344 rats with age and caloric restriction,” American Journal of Physiology, vol. 285, no. 5, pp. R1259–R1267, 2003.
[36]
J. C. Pinheiro and D. M. Bates, Mixed-Effects Models in S and S-PLUS; Statistics and Computing, Springer, New York, NY, USA, 2000.
[37]
R. S. Balaban, S. Nemoto, and T. Finkel, “Mitochondria, oxidants, and aging,” Cell, vol. 120, no. 4, pp. 483–495, 2005.
[38]
J. C. Seidell, “Epidemiology of obesity,” Seminars in Vascular Medicine, vol. 5, no. 1, pp. 3–14, 2005.
[39]
J. A. Maassen, “Mitochondrial diabetes: pathophysiology, clinical presentation, and genetic analysis,” American Journal of Medical Genetics, vol. 115, no. 1, pp. 66–70, 2002.
[40]
A. Marcuello, J. González-Alonso, J. A. L. Calbet, R. Damsgaard, M. J. López-Pérez, and C. Díez-Sánchez, “Skeletal muscle mitochondrial DNA content in exercising humans,” Journal of Applied Physiology, vol. 99, no. 4, pp. 1372–1377, 2005.
[41]
M. Jové, J. Salla, A. Planavila et al., “Impaired expression of NADH dehydrogenase subunit 1 and PPARγ coactivator-1 in skeletal muscle of ZDF rats: restoration by troglitazone,” Journal of Lipid Research, vol. 45, no. 1, pp. 113–123, 2004.
[42]
J. He, S. Watkins, and D. E. Kelley, “Skeletal muscle lipid content and oxidative enzyme activity in relation to muscle fiber type in type 2 diabetes and obesity,” Diabetes, vol. 50, no. 4, pp. 817–823, 2001.
[43]
P. A. Kern, R. B. Simsolo, and M. Fournier, “Effect of weight loss on muscle fiber type, fiber size, capillarity, and succinate dehydrogenase activity in humans,” Journal of Clinical Endocrinology and Metabolism, vol. 84, no. 11, pp. 4185–4190, 1999.
[44]
Z. Drahota, M. Milerová, A. Stieglerová, J. Hou？těk, and B. Ostádal, “Developmental changes of cytochrome c oxidase and citrate synthase in rat heart homogenate,” Physiological Research, vol. 53, no. 1, pp. 119–122, 2004.
[45]
J. Marin-Garcia, R. Ananthakrishnan, and M. J. Goldenthal, “Human mitochondrial function during cardiac growth and development,” Molecular and Cellular Biochemistry, vol. 179, no. 1-2, pp. 21–26, 1998.
[46]
V. J. Vieira and R. J. Valentine, “Mitochondrial biogenesis in adipose tissue: can exercise make fat cells ‘fit’?” Journal of Physiology, vol. 587, no. 14, pp. 3427–3428, 2009.
[47]
M. ？apková, J. Hou？těk, H. Hansíková, V. Hainer, M. Kune？ová, and J. Zeman, “Activities of cytochrome c oxidase and citrate synthase in lymphocytes of obese and normal-weight subjects,” International Journal of Obesity, vol. 26, no. 8, pp. 1110–1117, 2002.
