Hei Sook Sul
Adipocytes are highly specialized cells that play a crucial role in energy balance of most vertebrates by providing the ability to synthesize and deposit fat during times of positive energy balance in preparation for periods of food deprivation. In modern society, however, excess adipose tissue leading to obesity with its associated diseases such as diabetes is a major health problem.
With excess energy intake, there is an increase in lipogenesis and storage of triacylglycerol (TAG) in adipose tissue, that causes enlarged adipocytes (hypertrophy). In addition, precursor cells, preadipocytes, are recruited to become adipocytes, increasing adipocyte number (hyperplasia). Elucidating molecular mechanisms underlying these two processes, hypertrophy and hyperplasia of adipocytes, is critical for understanding obesity and its associated diseases. The long-term goal in our laboratory is to understand (1) adipocyte TAG metabolism that contributes to increased TAG storage and adipocyte size and (2) adipocyte differentiation process that contributes to increased adipocyte number.
Studies on transcriptional regulation of lipogenic enzymes by feeding/insulin
In understanding the process of lipogenesis, we are investigating the two central enzymes in fat synthesis, fatty acid synthase (FAS) and mitochondrial glycerol-3-phosphate acyltransferase (GPAT). These enzymes are under coordinate transcriptional regulation; their transcription is suppressed during fasting but activated upon feeding of a high carbohydrate diet or upon insulin administration. We found that USF1/USF2 binding to the -65 E-box is required for transcriptional activation of the FAS gene. We made transgenic mice containing CAT driven by various 5’-deletions and mutations and found that USF binding to the -65 E-box and SREBP-1c binding to the -150 SRE are necessary for FAS promoter activation by feeing/insulin and that USF binding to the -332 E-box provides maximal induction. Chromatin immunoprecipitation indicated constitutive binding of USF to the -65 and -332 E-boxes and feeding/insulin induced binding of SREBP to the -150 SRE in vivo. We also found that USF occupancy at the -65 E-box is required for SREBP binding to the -150 SRE and that USF and SREBP directly interact to activate transcription of FAS as well as of other lipogenic enzymes such as mitochondrial GPAT. Thus, USF/SREBP binding to their specific sites and their direct interaction is a common mechanism for the coordinate transcriptional activation of lipogenic genes by feeding/insulin treatment.
Recently, by tandem affinity purification and MS/MS sequencing, we have identified not only various components of the USF/SREBP complex but also their posttranslational modifications (phosphorylation and acetylation) during fasting/feeding. We found that 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 promoter inactivation. DNA break/repair components associated with USF also bring about transient DNA breaks during feeding-induced FAS activation. Thus, in DNA-PK deficient SCID mice, feeding induced USF-1 phosphorylation/acetylation, DNA-breaks, and FAS activation leading to lipogenesis are impaired, resulting in decreased liver and circulating triglyceride levels as well as in reduced adiposity. Our study demonstrates that DNA-PK mediates the feeding/insulin-dependent lipogenic gene activation, which constitutes a new insulin signaling pathway.
Identification and structure/function studies of enzymes in lipid metabolism
Lipolysis (hydrolysis of TAG) is a unique pathway that occurs mainly in adipocytes to provide fatty acids for use as an energy source by other tissues during energy shortage. During fasting, levels of catabolic hormones, catacholamines and glucocorticoids increase, increasing lipolysis in adipocytes. Upon feeding, insulin secretion increases, decreasing lipolysis. HSL has been considered to be the rate-limiting step in TAG hydrolysis. HSL is phosphorylated by PKA and is activated during fasting by translocation to lipid droplets in adipocytes. Studies on HSL-null mice that retain 40% of TAG lipase activity and normal WAT mass, however, have challenged such a concept. Distinct but yet to be identified lipase(s) for neutral lipids may exist in adipose tissue. In this regard, HSL is more active in vitro with DAG as a substrate and may be rate-limiting in DAG, but not TAG, hydrolysis. We recently identified adipocyte-specific TAG lipase and we named it desnutrin. Subsequent to our report, two other laboratories also identified this protein as the TAG lipase in adipocytes. We found that desnutrin contains patatin domain with hydrolase fold, that desnutrin expression is induced during fasting, and that glucocorticoids transiently upregulate desnutrin expression. Desnutrin has been found both in the cytoplasm and with lipid droplets. Overexpression of desnutrin in cells causes an increase in TAG hydrolysis and fatty acid release, clearly demonstrating desnutrin function in cultured cells. Transgenic mice overexpressing desnutrin in adipocytes showed increased lipolysis with a lean phenotype.
Phospholipase A2 (PLA2) superfamily of enzymes consists of a broad range of enzymes that catalyze the hydrolysis of the sn-2 ester bond of membrane phospholipids to release unsaturated fatty acids. The fatty acid released by PLA2 such as arachidonic acid serves as the precursor of eicosanoids, which are potent local mediators of signal transduction. Therefore, PLA2 has been regarded as a rate-limiting step in the synthesis of eicosanoids. PLA2 that may be expressed in specific tissues for generation of local lipid mediators in order to regulate tissue-specific function are yet to be identified. In adipocytes, the major prostaglandin produced is PGE2 and its receptor EP3 is present only in mature adipocytes. We identified a novel adipocyte specific phospholipase A2 that we named AdPLA. We found that AdPLA regulates lipolysis via production of PGE2. AdPLA which is expressed mainly in adipose tissue appears to be located in both the cytosol and perinuclear region of adipocytes. We have shown that PGE2 produced upon AdPLA expression plays a dominant role in lipolysis through binding to the Gi-coupled EP3 and inhibits lipolysis by reducing cAMP levels. We characterized AdPLA by generating and purifying the recombinant protein. We also generated AdPLA KO mice that exhibit a drastic decrease in adiposity and are protected from high fat diet induced as well as genetic obesity. We found AdPLA knockout mice had a high rate of lipolysis with decreased PGE2 and increased cAMP levels in adipose tissue. We also found that the impairment of an appropriate TAG storage due to increased lipolysis in adipose tissue leads to an ectopic fat storage in other organs such as liver and muscle and peripheral insulin resistance. Overall, our study showed that AdPLA which is induced upon feeding/insulin, by increasing PGE2 secretion, suppresses lipolysis and this constitutes an important autocrine/paracrine signaling pathway for regulation of TAG metabolism. Our study revealed not only a novel adipocyte-specific PLA2 but a major role of AdPLA in regulating lipolysis and energy balance. AdPLA may constitute a pharmacological therapeutic target for obesity.
Identification/functional studies of inhibitors of adipogenesis
We originally identified an inhibitory molecule for adipogenesis, Pref-1 (Preadipocyte factor-1), a transmembrane protein with six EGF-repeats at the extracellular domain. Pref-1 is highly expressed in 3T3-L1 preadipocytes, its expression is extinguished during adipose conversion, and is not detectable in mature adipocytes. When constitutively expressed, Pref-1 blocks adipocyte differentiation in vitro. There are multiple Pref-1 transcripts generated by alternate splicing with various in-frame deletions of all or part of the sixth EGF repeat. We detected processing of the membrane form of Pref-1 to generate a soluble Pref-1 protein of 50 kD corresponding to the Pref-1 ectodomain. We found that TACE (ADAM17) is responsible for this cleavage. We also found that only the 50 kD large soluble form of Pref-1 is active as an inhibitor of adipocyte differentiation but not an artificial membrane form of Pref-1 that cannot undergo cleavage. While all four of the alternately spliced forms of Pref-1 produce the membrane form of protein, only the two largest alternately spliced forms undergo cleavage to release the 50 kD soluble Pref-1. Thus, alternate splicing may be a mechanism that governs the production of biologically active Pref-1. We generated Pref-1 knockout mice as well as transgenic mice overexpressing Pref-1 in adipose tissue. Overexpression of Pref-1 in adipose tissue caused these mice to be lean but diabetic. In contrast, Pref-1 ablation caused an increase in adipose tissue mass and impaired glucose/insulin homeostasis. These in vivo experiments unequivocally demonstrate the inhibitory role of Pref-1 in adipogenesis and requirement of proper adipocyte differentiation and function in maintaining glucose/insulin homeostasis. Glucocorticoids suppress Pref-1 expression and dexamethasone commonly used for in vitro adipocyte differentiation probably functions partly via suppressing Pref-1. We recently found that Sox9 downregulation is required for adipocyte differentiation and that Pref-1 inhibits adipocyte differentiation through upregulating Sox9 expression. Sox9 directly binds to the promoter regions of C/EBPand C/EBP to suppress their promoter activity, preventing adipocyte differentiation. Furthermore, by inducing Sox9, Pref-1 promotes chondrogenic induction of mesenchymal cells but prevents chondrocyte maturation as well as osteoblast differentiation, which is supported by in vivo evidence in Pref-1 null and Pref-1 transgenic mice. Thus, Sox9 is a Pref-1 target that directs multipotent mesenchymal cells to the chondrogenic lineage but inhibits differentiation into adipocytes as well as osteoblasts and chondrocytes. Pref-1 expression is restricted to certain tissues including preadipocytes and some neuroendocrine type of tissues. However, Pref-1 is widely expressed in the embryo and is a paternally expressed imprinted gene. We are presently studying the role of Pref-1 during embryogenesis and development.
For more information, please visit the Sul Lab page
Selected Recent Publications
Sambeat A, Gulyaeva O, Dempersmier J, Sul HS. Epigenetic Regulation of Brown adipose tissue program and thermogenesis. Trends Endo. Met. 28, 19-31, 2017.
Viscarra J, Wang Y, Kim S-J, Hong I, Sul HS. Transcriptional activation of de novo fatty acid synthesis by insulin requires MED17 phosphorylation by CK2. Science Signaling 10, 467, 2017.
Kim S-J, Tang T, Abbott M, Viscarra JA, Wang Y, Sul HS. AMPK phosphorylates desnutrin/ATGL and HSL to regulate lipolysis and fatty acid oxidation within adipose tissue. Mol. Cell. Biol. 36, 1961-1976, 2016.
Sambeat A, Gulyaeva O, Dempersmier J, Tharp K, Stahl A, Paul SM, Sul HS. LSD1 interacts with Zfp516 to promote UCP1 transcription and Brown Fat Program. Cell Reports 15, 2536-2549 2016.
Perry RJ, Camporez J-PG, Kursawe R, Titchenell PM, Zhang D, Perry CJ, Jurczak MJ, Han MS, Zhang X- M, Ruan H-B, Yang X, Caprio S, Kaech SM, Sul HS, Birnbaum MJ, Davis RJ, Cline GW, Petersen KF, Shulman GI. Hepatic acetyl CoA regulates insulin action and links adipose tissue inflammation with hepatic insulin resistance and type 2 diabetes. Cell 160, 745-758, 2015.
Wang Y, Viscarra JA, Kim, S-J, Sul HS. Transcriptional regulation of lipogenesis. Nat. Rev. Mol. Cell. Biol. 16, 678-689, 2015.
Dempersmier J, Sambeat A, Gulyaeva O, Paul SM, Hudak C, Raposo H, Kwan HY, Kang C, Wong RH, Sul HS. Cold-inducible Zfp516 promotes browning of white Fat and is required for brown fat development. Molecular Cell 57, 235-246, 2015.
Hudak CS, Gulyaeva O, Park SM, Lee L, Kang C, Sul HS. Pref-1 marks early mesenchymal precursors required for adipose tissue development and expansion. Cell Reports 8, 678-687, 2014.
Tang T, Abbott MJ, Ahmadian M, Lopes AB, Wang Y, Sul HS. Desnutrin/ATGL Activates PPARd to Promote Mitochondrial function and Insulin Secretion in Islet b cells. Cell Metabolism 18, 883-895, 2013.
Wang Y, Wong RH, Tang T, Hudak CS, Yang D, Duncan RE, Sul HS. Phosphorylation and recruitment of BAF60c in chromatin remodeling for lipogenesis in response to insulin. Molecular Cell 49, 283-297, 2013.
Ahmadian M, Abbott MJ, Tang T, Hudak CS, Kim Y, Bruss M, Hellerstein MK, Lee H-Y, Samuel VT, Shulman G I, Wang Y, Duncan RE, Kang C, Sul HS. Adipose-specific ablation of desnutrin/ATGL promotes a brown-to-white adipose phenotype; Regulation by AMPK. Cell Metabolism 13,739-748, 2011.
Wang Y, Zhao L Smas CM, Sul HS. Pref-1 interacts with fibronectin to inhibit adipocyte differentiation. Mol. Cell. Biol. 30, 3480-3492, 2010.
Sul HS. Pref-1: role in adipogenesis and mesenchymal cell fate. Mol. Endo. 23. 1717-1725, 2009
Wang Y, Sul HS. Pref-1 regulates mesenchymal cell commitment and differentiation through Sox9. Cell Metabolism 9, 287-302, 2009
Ahmadian M, Duncan RE, Varady KA, Frasson D, Hellerstein MK, Birkenfeld AL, Samuel VT, Shulman GI, Wang Y, Kang C, Sul HS. Adipose overexpression of desnutrin promotes fatty acid use and attenuates diet- induced obesity. Diabetes 58, 855-866, 2009.
Wong RH, Chang I, Hudak CS, Hyun S, Kwan H-Y, Sul HS. A role of DNA-PK for the metabolic gene regulation in response to insulin. Cell 136, 1056-1072, 2009.
Jaworski K, Ahmadian M, Duncan RE, Sarkadi-Nagy E, Varady KA, Hellerstein MK, Lee H-Y, Samuel VT, Shulman GI, Kim K-H, de Val S, Kang C, Sul HS. AdPLA ablation increases lipolysis and prevents obesity induced by high-fat feeding or leptin deficiency. Nature Medicine 15, 159-168, 2009.