1 Directing Visceral White Adipocyte Precursors to a Thermogenic 2 Adipocyte Fate Improves Insulin Sensitivity in Obese Mice 3 4 Chelsea Hepler, Mengle Shao, Jonathan Y. Xia, Alexandra L. Ghaben, Mackenzie J. 5 Pearson, Lavanya Vishvanath, Ankit X. Sharma, Thomas S. Morley, William L. Holland, 6 Rana K. Gupta 7 Touchstone Diabetes Center, Department of Internal Medicine, University of Texas 8 Southwestern Medical Center, Dallas, Texas 75390, USA 9 10 Correspondence should be addressed to: 11 Rana K. Gupta 12 Touchstone Diabetes Center 13 Department of Internal Medicine 14 UT Southwestern Medical Center 15 5323 Harry Hines Blvd. 16 K5.240 17 Dallas, TX 75390-8549 18 Phone: 214-648-8721 19 Email: [email protected] 20 Conflicting interests statement. The authors declare that they have no competing 21 financial interests. 22 23 Abstract 24 Visceral adiposity confers significant risk for developing metabolic disease in obesity 25 whereas preferential expansion of subcutaneous white adipose tissue (WAT) appears 26 protective. Unlike subcutaneous WAT, visceral WAT is resistant to adopting a protective 27 thermogenic phenotype characterized by the accumulation of Ucp1+ beige/BRITE 28 adipocytes (termed “browning”). In this study, we investigated the physiological 29 consequences of browning murine visceral WAT by selective genetic ablation of Zfp423, 30 a transcriptional suppressor of the adipocyte thermogenic program. Zfp423 deletion in 31 fetal visceral adipose precursors (Zfp423loxP/loxP; Wt1-Cre), or adult visceral white 32 adipose precursors (PdgfrbrtTA; TRE-Cre; Zfp423loxP/loxP), results in the accumulation of 33 beige-like thermogenic adipocytes within multiple visceral adipose depots. Thermogenic 34 visceral WAT improves cold tolerance and prevents and reverses insulin resistance in 35 obesity. These data indicate that beneficial visceral WAT browning can be engineered 36 by directing visceral white adipocyte precursors to a thermogenic adipocyte fate, and 37 suggest a novel strategy to combat insulin resistance in obesity. 38 39 1 40 Introduction 41 Adipocytes are critical regulators of energy balance and nutrient homeostasis. 42 White adipocytes serve as the principle site for energy storage in mammals. These cells 43 are characterized by a large unilocular lipid droplet and have the capacity to store or 44 release energy depending on metabolic demand. White adipocytes also produce and 45 secrete numerous cytokines and hormones that impact several aspects of physiology 46 (Rosen and Spiegelman, 2014). Eutherian mammals also contain a second major class 47 of adipocytes that function to catabolize stored lipids and produce heat. These 48 thermogenic adipocytes, consisting of brown and beige/BRITE adipocytes, are 49 characterized by their multilocular lipid droplet appearance, high mitochondrial content, 50 and expression of uncoupling protein 1 (Ucp1) (Cohen and Spiegelman, 2015; Harms 51 and Seale, 2013). Brown/beige adipocytes have promising therapeutic potential as their 52 activation in the setting of obesity has a profound impact on metabolic health. 53 White adipose depots are broadly categorized as either subcutaneous or visceral 54 adipose tissue, reflecting their anatomical location. Adipose tissue distribution is a 55 strong predictor of metabolic outcome in obese individuals (Karpe and Pinnick, 2015; 56 Lee et al., 2013). Visceral adiposity strongly correlates with the development of insulin 57 resistance, diabetes, and cardiovascular disease (Kissebah et al., 1982; Krotkiewski et 58 al., 1983; Ohlson et al., 1985; Vague, 1956). Preferential expansion of subcutaneous 59 depots is associated with sustained insulin sensitivity (Manolopoulos et al., 2010). 60 Engineered rodent models highlight the protective role of subcutaneous adipose tissue. 61 Transgenic animals overexpressing adiponectin or mitoNEET develop extreme 62 subcutaneous obesity; however, these animals remain metabolically healthy (Kim et al., 63 2007; Kusminski et al., 2012). 2 64 In humans, the location of visceral adipose tissue itself likely mediates some of 65 its detrimental effects on energy metabolism; lipids, metabolites, and cytokines can 66 drain directly into the portal circulation and affect liver function (Rytka et al., 2011). 67 Transplantation studies, cellular studies, and gene expression analyses, suggest that 68 factors intrinsic to these depots may also determine their effect on nutrient homeostasis 69 (Tran et al., 2008; Yamamoto et al., 2010). Anatomically distinct adipocytes are 70 functionally unique, differing in their ability to undergo lipolysis, lipogenesis, and activate 71 the thermogenic gene program (Lee et al., 2013; Macotela et al., 2012; Morgan-Bathke 72 et al., 2015; Wu et al., 2012). Along these lines, lineage analyses reveal that 73 anatomically distinct white adipocytes can originate from developmentally distinct 74 precursor cells, and emerge at different times during development (Billon and Dani, 75 2012; Chau et al., 2014). As such, it is now widely believed that visceral and 76 subcutaneous adipocytes represent distinct subtypes of fat cells. 77 In mice, visceral and subcutaneous white adipose depots differ remarkably in 78 their ability to remodel under physiological conditions (Hepler and Gupta, 2017). Upon 79 high-fat diet feeding, visceral adipose depots of mice expand by both adipocyte 80 hypertrophy and through the formation of new adipocytes (“adipogenesis”) (Wang et al., 81 2013). Inguinal subcutaneous WAT expands predominantly through adipocyte 82 hypertrophy. The differential capacity for adipogenesis is likely explained by factors 83 present in the local microenvironment (Jeffery et al., 2016). Another notable difference 84 between the inguinal and visceral adipose depots in rodents is the capacity to adopt a 85 thermogenic phenotype. Various stimuli, including β3-adrenergic receptor-agonism and 86 cold exposure, drive the rapid accumulation of beige adipocytes in subcutaneous 3 87 depots (Vitali et al., 2012; Wu et al., 2012). Genetic stimulation of subcutaneous beige 88 adipogenesis renders mice resistant to high-fat diet induced obesity and/or diabetes 89 (Seale et al., 2011; Shao et al., 2016), while inhibition of subcutaneous beiging leads to 90 an earlier onset of insulin resistance during obesity (Cohen et al., 2014). Most visceral 91 depots in mice, particularly the gonadal and mesenteric adipose tissues, are relatively 92 resistant to browning in response to physiological stimuli. With few exceptions (Kiefer et 93 al., 2012), most engineered mouse models of white adipose tissue browning exhibit 94 beige cell accumulation preferentially in subcutaneous WAT depots (Seale et al., 2011; 95 Stine et al., 2016). Visceral WAT depots may harbor mechanisms to suppress 96 thermogenesis in order to ensure its function as a white, energy-storing, depot. 97 We previously established a critical role for the transcription factor, Zfp423, in the 98 establishment and maintenance of the adipocyte lineage. Zfp423 is required for fetal 99 differentiation of subcutaneous white adipocytes (Gupta et al., 2010; Shao et al., 2017). 100 In adult mice, Zfp423 expression defines a subset of committed mural preadipocytes 101 (Gupta et al., 2012; Vishvanath et al., 2016). Upon high-fat diet feeding, these mural 102 cells undergo adipogenesis in visceral depots, contributing to adipocyte hyperplasia 103 (Vishvanath et al., 2016). Zfp423 is also expressed in nearly all mature adipocytes; 104 however, its expression is more abundant in white adipocytes than brown adipocytes 105 (Shao et al., 2016). In the mature adipocyte, Zfp423 acts to maintain the energy-storing 106 status of the white adipocyte through suppression of the thermogenic gene program 107 (Shao et al., 2016). Zfp423 likely exerts this function by serving as a transcriptional co- 108 repressor of the brown/beige lineage determining transcription factor, Ebf2. The loss of 109 Zfp423 in mature white adipocytes triggers a robust conversion of white to beige 4 110 adipocytes in subcutaneous WAT. Amongst the various adipose tissues, visceral WAT 111 expresses the highest levels of Zfp423. Importantly, we observed that visceral 112 adipocytes lacking Zfp423 were also capable of inducing their thermogenic gene 113 program when animals were stimulated pharmacologically with a 3 adrenergic receptor 114 agonist (Shao et al., 2016). This observation affords the possibility of examining 115 whether the thermogenic capacity of visceral white adipose depots can be unlocked 116 under physiological conditions, and whether thermogenic visceral WAT would be 117 ultimately harmful or beneficial to systemic metabolic health. 118 Here, we describe two mouse models of visceral adipose tissue browning 119 derived through selective ablation of Zfp423 in visceral adipose precursors. We reveal 120 that fetal visceral white preadipocytes can be redirected to a beige-like adipocyte fate 121 through the loss of Zfp423, leading to visceral depot mass reduction and fat 122 redistribution towards subcutaneous depots. The browning of visceral depots improves 123 cold tolerance and protects against the development of insulin resistance and 124 hyperlipidemia in obesity. Moreover, we demonstrate that visceral mural preadipocytes 125 in adult mice can also be directed to a thermogenic cell fate; this leads to beige-like, 126 rather than white, adipocyte hyperplasia, in the expanding visceral WAT depots of diet- 127 induced obese animals. Upon activation by β-3 adrenergic receptor