Sustained NFjB inhibition improves insulin sensitivity but is detrimental to muscle health Ning Zhang, 1, * Joseph M. Valentine, 1, * You Zhou, 1 Mengyao E. Li, 1,2 Yiqiang Zhang, 1 Arunabh Bhattacharya, 1 Michael E. Walsh, 1 Katherine E. Fischer, 1,† Steven N. Austad, 1,† Pawel Osmulski, 1 Maria Gaczynska, 1 Steven E. Shoelson, 2 Holly Van Remmen, 1,‡ Hung I. Chen, 3,4 Yidong Chen, 3,4 Hanyu Liang 1 and Nicolas Musi 1,5 1 Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245, USA 2 Joslin Diabetes Center, 1 Joslin Place, Boston, MA 02215, USA 3 Greehey Children’s Cancer Research Institute, 8403 Floyd Curl Dr, San Antonio, TX 78229, USA 4 Department of Epidemiology and Biostatistics, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr, San Antonio, TX 78229, USA 5 San Antonio Geriatric Research, Education and Clinical Center, South Texas Veterans Health Care System, 7400 Merton Minter, San Antonio, TX 78229, USA Summary Older adults universally suffer from sarcopenia and approxi- mately 60–70% are diabetic or prediabetic. Nonetheless, the mechanisms underlying these aging-related metabolic disorders are unknown. NFjB has been implicated in the pathogenesis of several aging-related pathologies including sarcopenia and type 2 diabetes and has been proposed as a target against them. NFjB also is thought to mediate muscle wasting seen with disuse, denervation, and some systemic diseases (e.g., cancer, sepsis). We tested the hypothesis that lifelong inhibition of the classical NFjB pathway would protect against aging-related sarcopenia and insulin resistance. Aged mice with muscle-specific overexpression of a super-repressor IjBa mutant (MISR) were protected from insulin resistance. However, MISR mice were not protected from sarcopenia; to the contrary, these mice had decreases in muscle mass and strength compared to wild-type mice. In MISR mice, NFjB suppression also led to an increase in proteasome activity and alterations in several genes and pathways involved in muscle growth and atrophy (e.g., myostatin). We conclude that the mechanism behind aging-induced sarcopenia is NFjB indepen- dent and differs from muscle wasting due to pathologic condi- tions. Our findings also indicate that, while suppressing NFjB improves insulin sensitivity in aged mice, this transcription factor is important for normal muscle mass maintenance and its sustained inhibition is detrimental to muscle function. Key words: aging; insulin resistance; NFjB; sarcopenia; skeletal muscle. Introduction Aging is characterized by a deterioration in glucose homeostasis and the progressive loss of muscle mass and function. However, the molecular basis for insulin resistance and sarcopenia of aging is unknown. The transcription factor NFjB is a key regulator of inflammatory responses. Accumulating evidence suggests that the NFjB pathway may be involved in the aging process and in the pathogenesis of various aging-related pathologies. For example, upregulation of the NFjB pathway in aged animals and older humans has been demonstrated in multiple tissues such as skeletal muscle, liver, kidney, heart, and gastric mucosa (Helenius et al., 1996; Korhonen et al., 1997; Walter & Sierra, 1998; Xiao & Majumdar, 2000; Ghosh et al., 2015). In line with these findings, a global gene expression microarray analysis of various human and mouse tissues determined that the NFjB motif was the motif most strongly associated with aging (Adler et al., 2007). NFjB is upregulated in muscle of both aged rodents (Phillips & Leeuwenburgh, 2005) and human subjects (Buford et al., 2010; Thalacker-Mercer et al., 2010; Ghosh et al., 2015), and the NFjB pathway has been implicated in the pathogenesis of insulin resistance and type 2 diabetes (Yuan et al., 2001). Therefore, the NFjB pathway may be involved in the glucose metabolism abnormalities that occur during aging. NFjB also is thought to play a key role in the muscle wasting seen with cancer, muscle disuse, and denervation (Cai et al., 2004; Judge et al., 2007; Van Gammeren et al., 2009; Reed et al., 2011). Thus, NFjB may also mediate the muscle loss characteristic of the aging process. Despite the evidence indicating that NFjB may be involved in the glucose metabolism abnormalities and muscle wasting seen with certain pathologic conditions (e.g., type 2 diabetes, cancer, denervation, and disuse), it is not known whether NFjB is a link between the pathogenesis of insulin resistance and the muscle atrophy of aging. In this study, we tested the hypothesis that muscle-specific NFjB inactivation would protect against aging-induced insulin resistance and sarcopenia. If positive, these findings would demonstrate a role for NFjB on the pathophysiology of these metabolic disorders that frequently affect older adults. Such results also would provide proof-of-concept that interventions that target NFjB could be effective in the prevention and treatment of aging-related metabolic conditions. Results Animal model We studied C57BL/6 mice with muscle-specific overexpression of an IjBa super-repressor mutant (MISR) and wild-type (WT) littermates. Genera- tion of these mice and genotyping procedures were described previously (Cai et al., 2004). This is a well-characterized model for studies of suppressed NFjB activity, which carries an IjBa transgene (S32A/S36A) that functions as a potent dominant negative inhibitor of the classical NFjB pathway, because mutating serines 32 and 36 for alanine prevents Correspondence Nicolas Musi, Barshop Institute for Longevity and Aging Studies, 15355 Lambda Drive, San Antonio, TX 78245, USA. Tel.: 210 562 6140; fax: 210 562 6110; e-mail: musi@uthscsa.edu *These authors contributed equally. † Current addreses: University of Alabama at Birmingham Birmingham AL USA. ‡ The Oklahoma Medical Research Foundation Oklahoma OK USA Accepted for publication 16 April 2017 ª 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 847 Aging Cell (2017) 16, pp847–858 Doi: 10.1111/acel.12613 Aging Cell