TonEBP in macrophages and adipocytes contributes to obesity and type 2 diabetes

Abstract

The global epidemic of obesity poses serious burden to human health due to its associated risks of chronic diseases including type 2 diabetes and cardiovascular diseases. Adipose tissues have received a lot of attention since as it has become clear that adipocytes and macrophages in them are important integrators of diverse physiological pathways regulating systemic insulin sensitivity and energy homeostasis. It is therefore urgently necessary to identify regulatory convergence points that can be therapeutic targets to improve insulin resistance and impaired energy homeostasis in obesity. Tonicity responsive enhancer-binding protein (TonEBP), also known as NFAT5, is a critical regulator in cellular adaptation to hypertonic stress, macrophage activation, and T-cell development. In this study I found that adipocyte TONEBP expression correlated with body mass index in subcutaneous fat tissues from human subjects. In addition, TONEBP expression in leukocytes correlated with fasting blood glucose in patients with type 2 diabetes. In subcutaneous adipose tissues of mice TonEBP expression increased >50-fold in response to feeding with high fat diet (HFD). In order to understand the role of TonEBP in obesity, I examined a line of mice with TonEBP haplo-deficiency. These animals showed resistance to HFD-induced weight gain and insulin resistance in association with higher energy expenditure and lower adipose tissue inflammation. To delineate TonEBP action mechanisms in adipose tissues, I studied the role of TonEBP in both adipocytes and macrophages further. In order to investigate the role of TonEBP in macrophages, a line of mice with myeloid-specific deletion of TonEBP was generated. When fed with HFD, these animals displayed improved insulin resistance without changes in body weight. Their adipose tissue contained fewer M1 macrophages and more M2 macrophages indicating reduced inflammation. Lipopolysaccharide (LPS)-induced assembly of NFκB enhanceosome was found. In this enhanceosome, TonEBP was required for the recruitment of p300 and RNA polymerase II. TonEBP was rate-limiting in that increased expression of TonEBP enhanced the NFκB activity and reduced TonEBP expression lowered it without affecting NFκB itself. LPS induced the expression of TonEBP leading to elevated assembly of the NFκB enhanceosome. Recombinant TonEBP molecules incapable of recruiting p300 did not stimulate NFκB. A natural small molecule cerulenin was found to specifically disrupt the enhanceosome assembly without altering NFκB. Cerulenin dramatically suppressed the inflammatory activation of macrophages and prevented death by septic shock. Thus, the TonEBP-mediated NFκB enhanceosome offers a promising target for useful anti-inflammatory agents. In addition, TonEBP deficiency promoted M2 macrophage polarization with enhanced PPARγ expression. These data suggest that variations in the level of TonEBP expression determine individual variations in insulin resistance and inflammation regulated by NFκB and PPARγ. Taken together the findings reveal that TonEBP promotes insulin resistance through the stimulation of NFκB in combination with the suppression of PPARγ. To address the role of TonEBP in adipocytes, another line of mice with adipocyte-specific deletion of TonEBP was produced. When fed with HFD these animals displayed improved insulin resistance and lower body weight much like the TonEBP haplo-deficient animals. They also showed elevated energy expenditure and resistance to hypothermia when exposed to cold. In subcutaneous white adipose tissues (WAT) higher levels of beige fat markers and thermogenic genes such as uncoupling protein-1 (UCP-1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) were expressed. This was due to an enhanced expression of the β3-adrenergic receptor (Adrb3). TonEBP recruited DNA methyltransferase 1 (DNMT1) to the Adrb3 promoter and promoted DNA methylation leading to the suppression of the promoter. These data indicate that TonEBP in adipocytes promotes obesity and insulin resistance by the suppression of the Adrb3 expression and blocking the beiging of WAT. In conclusion, TonEBP in adipocytes and macrophages contributes to obesity and insulin resistance due to the suppression of WAT beiging and the promotion of inflammation, respectively. As such, TonEBP offers an attractive therapeutic target for obesity and type 2 diabetes.