There are two types of adipose tissue. White adipose tissue (WAT) accumulates triglycerides (TAG) as the main energy storage form. Obesity from increased WAT mass with increased TAG storage to enlarge cell size, as well as with increased adipocyte number, has become an epidemic. Obesity is associated various diseases, including type 2 diabetes and cardiovascular diseases. In contrast to WAT, brown adipose tissue (BAT) dissipates energy as heat via non-shivering thermogenesis. Moreover, in certain conditions, subcutaneous WAT can become more like BAT by so-called beiging  to perform thermogenesis. Thus, increasing BAT or BAT-like tissues may represent a means for obesity prevention/therapeutics. The broad aims of our research in the Sul Lab are to examine, a) recruitment and differentiation of adipocytes, b) development and function of BAT or BAT-like tissues, and c) regulation of lipid synthesis and breakdown in adipose tissue.

Factors that regulate adipogenesis

       To identify genes that regulate adipogenesis, we cloned and identified Pref-1 (Preadipocyte factor-1), a transmembrane protein with six EGF-repeats at the extracellular domain. Pref-1 is highly expressed in preadipocytes and its expression is extinguished during adipose conversion. When constitutively expressed, Pref-1 blocks, whereas absence of Pref-1 enhances, differentiation. We found that processing of cell-associated Pref-1 by TACE generates a soluble Pref-1 and only the 50 kD soluble form of Pref-1 is active as an inhibitor of adipogenesis. We generated Pref-1 knockout mice as well as transgenic mice ectopically overexpressing Pref-1 in adipose tissue: overexpression of Pref-1 in adipose tissue caused these mice to be lean, but diabetic due to ectopic fat storage in other tissues.  Conversely, ablation of Pref-1 gene caused an increase in adipose mass and insulin resistance. These in vivo experiments unequivocally demonstrate the inhibitory role of Pref-1 in adipogenesis and its effect on glucose/insulin homeostasis. Pref-1 is now widely used as the preadipocyte marker. Recently we found that the soluble Pref-1 interacts with fibronectin to inhibit adipocyte differentiation. Upon binding to fibronectin, Pref-1 increases MEK/ERK phosphorylation in a time- and dose-dependent manner to inhibit adipogenesis. We also found that Sox9 is an important downstream target of Pref-1 to suppress expression of C/EBP and C/EBP, preventing adipocyte differentiation. We found that by regulating Sox9, Pref-1 directs multipotent mesenchymal cells to the chondrogenic lineage and prevents adipogenesis. We generated transgenic mouse models for transient or permanent marking of cells using the Pref-1 promoter, and also to ablate cells in an inducible manner. We found that Pref-1 marked cells are very early adipose precursors and these cells retain proliferative capacity. During embryogenesis, Pref-1 marked cells first appear in the dorsal mesenteric region as early as E10.5, which become lipid-laden adipocytes at E17.5 in the subcutaneous region, whereas visceral WAT develops after birth. Thus, ablation of Pref-1 marked cells prevents not only embryonic WAT development but also later adult adipose expansion upon high fat feeding, demonstrating the requirement of Pref-1 expressing cells for adipogenesis. We used the Pref-1 promoter-rtTA system in mice for labeling Pref-1+ cells and for inducible inactivation of the Pref-1 target, Sox9.  We found that, upon Sox9 inactivation, these Pref-1+ cells become PDGFRα+ cells that express early adipogenic markers and that Pref-1+ cells precede PDGFRα+ cells in the adipogenic pathway. To maintain early adipose precursors, Sox9 activates Meis1, which prevents adipogenic differentiation.

       Recently, we started to examine how adipose tissue mass and adiposity change throughout the lifespan. During aging, while visceral adipose tissue (VAT) tends to increase, peripheral subcutaneous adipose tissue (SAT) decreases significantly. Unlike VAT, which is linked to metabolic diseases, including type 2 diabetes, SAT has beneficial effects. By comparing scRNA-seq of total stromal vascular cells of SAT from young and aging mice, we identified an aging-dependent regulatory cell (ARC) population that emerges only in SAT of aged mice and humans. ARCs express adipose progenitor markers but lack adipogenic capacity; they secrete high levels of pro-inflammatory chemokines, including Ccl6, to inhibit proliferation and differentiation of neighboring adipose precursors. We also found Pu.1 to be a driving factor for ARC development. Thus, we identified an ARC population and its capacity to inhibit differentiation of neighboring adipose precursors, correlating with aging-associated loss of SAT.

Factors that promote thermogenic gene program and thermogenesis

       Uncoupling protein 1 (UCP1) mediates non-shivering thermogenesis in brown fat (BAT) in response to cold exposure. By high-throughput screening using UCP1 promoter, we identify several transcription factors may play critical role in thermogenesis as well as brown adipogenesis. One such gene that we have characterized is Zfp516. Zfp516 can activate UCP1 and other BAT-enriched genes, including PGC1α and Cox8b. Zfp516 is selectively expressed in BAT but not in most other tissues and, through the cAMP-CREB/ATF2 pathway, Zfp516 itself is induced by cold or sympathetic stimulation alone. Zfp516 binds at the proximal promoter regions to activate UCP1 and other BAT genes. Thus, Zfp516 can drive a brown adipogenic program and ablation of Zfp516 prevents BAT development in mice. Ectopic expression of Zfp516 in adipose tissue of mice promotes browning of subcutaneous white fat increasing body temperature and energy expenditure. We also examined the second transcription factor, Zc3h10 that binds far upstream of the UCP1 promoter for activation. Recently, we identified histone modifying enzymes LSD1 and Dot1L, that are recruited by Zfp516 and Zc3h10, respectively, for epigenetic regulation of the BAT gene program during different nutritional conditions.

      In addition to transcription factors and coregulators, we identified Aifm2, a NADH oxidoreductase domain containing flavoprotein, as a lipid droplet (LD)-associated protein, critical for thermogenesis. Aifm2 is induced by cold as well as by diet. Upon cold or β-adrenergic stimulation, Aifm2 associates with the outer side of the mitochondrial inner membrane. As a unique BAT-specific first mammalian NDE (external NADH dehydrogenase)-like enzyme, Aifm2 oxidizes NADH to maintain high cytosolic NAD levels in supporting robust glycolysis and to transfer electrons to the electron transport chain for fueling thermogenesis. Aifm2 in BAT and subcutaneous white adipose tissue promotes oxygen consumption, uncoupled respiration, and heat production during cold- and diet-induced thermogenesis, ameliorating diet-induced obesity and insulin resistance. We continue to search those factors that promote thermogenesis in adipose tissue.

Transcriptional regulation of lipogenic genes in response to feeding/insulin

       To understand how lipogenesis is regulated, we have been investigating the two central enzymes in fat synthesis, fatty acid synthase (FAS) and mitochondrial glycerol-3-phosphate acyltransferase (GPAT).  We have shown that USF1/2 bind to the insulin response sequence for transcriptional activation of lipogenic genes by feeding/insulin. We also found that USF and SREBP directly interacts by binding to the nearby cognate sites, which is a mechanism for the coordinate transcriptional activation of lipogenic genes in response to feeding/insulin. By tandem affinity purification and MS/MS sequencing, we have identified not only the various components of the USF/SREBP complex but also their posttranslational modifications during fasting/feeding. During feeding/insulin treatment, USF-1 recruits and is phosphorylated by DNA-PK, which is first dephosphorylated/activated by PP1.  Phosphorylation of USF-1 allows recruitment and acetylation by P/CAF, resulting in the FAS promoter activation. In fasting, USF-1 is deacetylated by HDAC9 causing the promoter inactivation. Our study demonstrates that DNA-PK mediates the feeding/insulin-dependent lipogenic gene activation, which constitutes a new insulin-signaling pathway. We also found that, by binding to USF-1, Brg1/Brm-associated factor (BAF) 60c functions as a specific chromatin remodeling component for lipogenic gene transcription in liver. In response to insulin, BAF60c is phosphorylated at S247 by atypical PKCζ/λ, which causes translocation of BAF60c to the nucleus and allows a direct interaction of BAF60c with USF-1, which is phosphorylated by DNA-PK and acetylated by P/CAF. Thus, BAF60c is recruited to form the lipoBAF complex to remodel chromatin structure and to activate lipogenic genes in the fed condition. In addition, USF interacts with one of the Mediator subunits, Med17, which is phosphorylated by CK2 for lipogenic gene activation in response to insulin. More recently, we studied a histone demethylase, Jmjd1c which is phosphorylated by mTOR for histone H3K9 methylation at the lipogenic promoter regions. Thus, Jmjd1c ablation in mice prevents diet-induced hepatosteatosis.  We continue to define various transcription factors and coregulators for lipogenic gene activation during feeding and by insulin treatment.   

Novel enzymes in lipid metabolism in adipose tissue

       My laboratory identified several novel enzymes that play critical role in triglyceride (TAG) metabolism. mitochondrial glycerol 3-phosphate acyltransferase (mGPAT) that catalyzes the first-rate limiting step in TAG synthesis, desnutrin/ATGL which is the bona fide adipocyte TAG lipase, and AdPLA (adipocyte specific phospholipase A2) that increases PGE2 levels to suppress lipolysis. GPAT was the first enzyme in glycerophospholipid biosynthesis cloned in mammalian system. mGPAT is induced greatly by feeding/insulin in a similar fashion as fatty acid synthase. In contrast, hydrolysis of TAG (lipolysis) providing fatty acids for use by other tissues as energy source is a unique function of white adipocytes. We identified the bona fide TAG lipase but also a adipose specific phospolipase A2 that regulates lipolysis exquisitely.  We characterized these enzymes by various in vitro biochemical studies and physiological studies by generating tissue specific knockout and overexpressing transgenic mice.  Overall, our research on those proteins that regulate WAT and BAT program, as well as those for adipose lipid metabolism provide better understanding of adipose tissue development and function and also future targets for therapeutics for obesity/diabetes.

For more information, please visit the Sul Lab website.