Research article The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 10 October 2006 2791 TNF-α downregulates eNOS expression and mitochondrial biogenesis in fat and muscle of obese rodents Alessandra Valerio, 1 Annalisa Cardile, 1,2 Valeria Cozzi, 1,2 Renata Bracale, 1,2,3 Laura Tedesco, 1,2,4 Addolorata Pisconti, 2,5 Letizia Palomba, 6 Orazio Cantoni, 6 Emilio Clementi, 2,5,7 Salvador Moncada, 8 Michele O. Carruba, 1,4 and Enzo Nisoli 1,4 1 Integrated Laboratories Network, Center for Study and Research on Obesity, Department of Pharmacology, School of Medicine, University of Milan, Milan, Italy. 2 Department of Preclinical Sciences, University of Milan, Milan, Italy. 3 CEINGE Biotecnologie Avanzate, Naples, Italy. 4 Istituto Auxologico Italiano, Milan, Italy. 5 Stem Cell Research Institute, San Raffaele Scientific Institute, Milan, Italy. 6 Istituto di Farmacologia e Farmacognosia, University of Urbino “Carlo Bo,” Urbino, Italy. 7 Eugenio Medea Scientific Institute, Lecco, Italy. 8 Wolfson Institute for Biomedical Research, University College London, London, United Kingdom. Obesity is associated with chronic low-grade inflammation. Thus, at metabolically relevant sites, including adipose tissue and muscle, there is abnormal production of proinflammatory cytokines such as TNF-α. Here we demonstrate that eNOS expression was reduced, with a concomitant reduction of mitochondrial biogenesis and function, in white and brown adipose tissue and in the soleus muscle of 3 different animal models of obesity. The genetic deletion of TNF receptor 1 in obese mice restored eNOS expression and mitochondrial biogenesis in fat and muscle; this was associated with less body weight gain than in obese wild-type controls. Furthermore, TNF-α downregulated eNOS expression and mitochondrial biogenesis in cultured white and brown adipocytes and muscle satellite cells of mice. The NO donors DETA-NO and SNAP prevented the reduction of mitochon- drial biogenesis observed with TNF-α. Our findings demonstrate that TNF-α impairs mitochondrial biogenesis and function in different tissues of obese rodents by downregulating eNOS expression and suggest a novel pathophysiological process that sustains obesity. Introduction Obesity is a complex, chronic disorder that has become a global epidemic (1). It is not just a concern for adults, as the number of overweight and obese children and adolescents has doubled in the past 2–3 decades in the United States. Visceral obesity, with a fat accumulation in abdomen among viscera, is often associated with insulin resistance, dyslipidemia, and hypertension and with an increased risk of accelerated atherosclerosis, i.e., the metabolic syn- drome (1). It results from a positive energy balance in which more calories are consumed than are used up for oxidation (i.e., energy expenditure in skeletal muscle and brown adipose tissue [BAT]) or body building and maintenance, with consequent energy storage as fat (i.e., triglyceride accumulation in white adipose tissue [WAT]). Obesity is associated with a state of chronic inflammation char- acterized by macrophage infiltration of muscle and adipose tis- sue (2) and abnormal production of proinflammatory mediators, including TNF-α (3, 4) and iNOS (5). This inflammatory state is associated with a deficit of energy in the form of ATP (6, 7) and simultaneous overproduction of fat and leptin, which is accompa- nied by leptin resistance in the brain (6, 8). We have previously shown that NO increases mitochondrial biogenesis, oxidative metabolism, and ATP levels in several cell types (9, 10). Consistently, mitochondrial biogenesis and func- tion are markedly decreased in eNOS-null mutant (eNOS –/– ) mice (9, 10), with fat accumulation in the abdomen and body weight increased compared with wild-type animals (9). Here we investi- gated eNOS expression and mitochondrial biogenesis together with the role of TNF-α in different models of obesity. We demon- strate that both eNOS expression and mitochondrial biogenesis are downregulated in fat and muscle tissues of genetically and environmentally obese mice and rats. Moreover, we observed that the genetic deletion of TNF receptor 1 in obese mice restored eNOS expression and mitochondrial biogenesis in the same tis- sues. Thus, our findings indicate that TNF-α impairs mitochon- drial biogenesis and function in metabolically active tissues of obese animals by inhibiting eNOS expression. Results eNOS expression and mitochondrial biogenesis in obese animals. eNOS mRNA and protein levels were measured in WAT from 3 animal models of obesity (ob/ob mice, fa/fa rats, and high-fat diet–induced obese mice [DIO mice]) and were found to be significantly lower than those in the relevant controls (Figure 1). eNOS mRNA and protein levels were also significantly reduced in BAT and soleus muscle from ob/ob mice, fa/fa rats, and DIO mice, although the reduction was less than that in WAT (Supplemental Figure 1; sup- plemental material available online with this article; doi:10.1172/ JCI28570DS1). We next investigated whether eNOS down-regula- tion was accompanied by reduced mitochondrial biogenesis. Lev- els of mitochondrial DNA (mtDNA, a marker of mitochondrial content), peroxisome proliferator–activated receptor γ coactiva- tor 1α (PGC-1α), nuclear respiratory factor-1 (NRF-1), and mito- chondrial transcription factor A (Tfam; all master regulators of Nonstandard abbreviations used: BAT, brown adipose tissue; COX IV, cytochrome c oxidase IV; Ct, threshold cycle; Cyt c, cytochrome c; DETA-NO, (Z)-1-[2-(2-amino- ethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; DIO mice, high-fat diet–induced obese mice; mtDNA, mitochondrial DNA; NRF-1, nuclear respiratory factor-1; PGC-1α, peroxisome proliferator–activated receptor γ coactivator 1α; RNase, ribonuclease; SNAP, S-nitroso-acetyl penicillamine; Tfam, mitochondrial transcrip- tion factor A; WAT, white adipose tissue. Conflict of interest: The authors have declared that no conflict of interest exists. Citation for this article: J. Clin. Invest. 116:2791–2798 (2006). doi:10.1172/JCI28570.