Comparative secretome study of brown adipocytes and the role of ITIH4 in adipose biology

Abstract

Adipose tissues in mammals can be broadly classified into two main types: white and brown adipose tissue. Although both are defined as adipose tissues, they differ drastically in their function. The main function of white adipose tissues (WAT) is the storage of fat. Unlike its white counterpart, brown adipose tissue (BAT) specializes in burning fat via thermogenesis and is known to play an important role in non-shivering thermogenesis especially in hibernating animals and newborn babies. Recent evidence of functional BAT in adult humans and its ameliorating effect on metabolic disorders has brought BAT under the spotlight for treatment of metabolic diseases like obesity and type 2 diabetes mellitus. WAT also acts as an endocrine organ by secreting signaling molecules called adipokines such as leptin and adiponectin. Adipokines constitute the secretome of WAT and not only play an important role in WAT function but also affect whole-body energy homeostasis. Various studies have investigated the role of adipose tissue secretome in metabolic disorders like obesity and insulin resistance. The WAT secretome has also been extensively characterized in various settings such as in whole WAT, mature white adipocyte etc. However, the BAT secretome and its adipokines (‘batokines’) have not yet been investigated. Thus, the main aim of this dissertation work was a comparative study of the white and brown adipocyte secretomes using a combination of Click-iT® AHA labeling and pulsed-SILAC (stable isotope labeling by amino-acid in cell culture). In total 1013 proteins were detected and a subset of these proteins was selected based on their secretion with norepinephrine stimulation. An in vitro assay was developed and optimized to test their putative effect on insulin secretion. In addition, one of the secretome candidates, inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) was investigated as a potential batokine and BAT activity marker. Although, the serum levels of ITIH4 did not correlate with BAT activity under cold stimulation, its expression was found to increase with adipogenesis and browning of white adipocytes. Using in vitro knockdown studies, a reduction in differentiation was observed which was characterized by reduction in mature adipocyte functions such as lipolysis, lipid and intracellular triglyceride storage, glucose uptake and lipogenesis. Therefore, rather than being a batokine, ITIH4 was shown to be important for adipogenesis and adipocyte biology. In summary, this dissertation sheds light on BAT secreted proteins and also introduces a new player in field of adipogenesis, both of which might have a significant impact in BAT biology and in the treatment of metabolic disorders like obesity.