Transcriptional regulation of lipogenic genes by feeding/insulin

           

      In elucidating 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).  Originally, we cloned cDNA and genomic sequence for these enzymes and observed coordinate transcriptional induction upon feeding/insulin treatment.  We examined the cis- and trans-factors that bring about activation of these genes by feeding/insulin.  We first defined the insulin response sequence of the FAS gene at -65 that contains a core E-box where USF binds. We then made transgenic mice containing CAT driven by various 5’-deletions and demonstrated -65 E-box where USF binds as well as -150 SRE where SREBP-1c binds are critical for activation of the FAS promoter. Furthermore, generation of transgenic mice containing mutations at the USF or SREBP sites at -65 E-box as well as ChIP clearly showed that binding of USF at -65 E-box is required for SREBP binding to -150SRE during feeding/insulin treatment.  We also found that USF and SREBP directly interact to activate transcription of FAS as well as other lipogenic enzymes including GPAT. USF/SREBP binding to the nearby cognate sites for their direct interaction is a mechanism for the coordinate transcriptional activation of lipogenic genes by feeding/insulin treatment.  Furthermore, by tandem affinity purification and MS/MS sequencing, we have recently identified not only the 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 the 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, circulating triglyceride levels with decreased adiposity. Our study demonstrates that DNA-PK mediates the feeding/insulin-dependent lipogenic gene activation, which constitutes a new insulin-signaling pathway. We 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. Consequently, we found BAF60c promotes lipogenesis in vivo and increases triglyceride levels, demonstrating its role in metabolic adaption to activate lipogenic program in response to feeding and insulin.     

​

Novel enzymes in lipid metabolism in adipose tissue

 

        My laboratory cloned and elucidated several novel enzymes that play critical role in TAG metabolism; mitochondrial glycerol 3-phosphate acyltransferase (GPAT) that catalyzes the first rate limiting step in TAG synthesis, desnutrin which is the bona fide adipocyte TAG lipase, and AdPLA (adipocyte specific phospholipase A2) that increases PGE2 levels to suppress lipolysis.

        First, we originally identified several proteins that are transcriptionally activated during feeding/insulin treatment in a coordinate manner. By overexpression/purification/reconstitution, we identified one such protein as mitochondrial GPAT in early 90s. This was the first enzyme in glycerophospholipid biosynthesis cloned in mammalian system. We further characterized the enzyme by extensive site-directed mutagenesis and identified critical amino acid residues for catalytic activity, as well as substrate binding. Subsequently other enzymes in glycerophospholipid biosynthesis including AGPAT and DGAT were identified by other laboratories due to the presence of homology region, making it possible to elucidate function and regulation of mammalian TAG biosynthesis. 

        Hydrolysis of TAG (lipolysis) providing fatty acids for use by other tissues as energy source is a unique function of white adipocytes. In fasted condition, increase in circulating catabolic hormones, catecholamines and glucocorticoids, stimulate lipolysis, whereas in the fed condition, anabolic hormone, insulin decreases lipolysis. We identified two novel enzymes that play crucial role in lipolysis in adipocytes. We first identified an adipocyte-specific TAG lipase that is induced during fasting/glucocorticoids and we named it desnutrin. Two other laboratories subsequently identified this enzyme as the TAG lipase in adipocytes and thus desnutrin/ATGL/PNPLA2 is now accepted to be the bona fide TAG lipase, whereas previously known HSL functions as DAG lipase. During characterization of desnutrin as a TAG lipase via adenoviral overexpression and mutational analysis, we found that desnutrin is phosphorylated at S406 by AMPK, which activates the TAG lipase activity, representing a new mode of regulation of lipolysis during energy shortage by sensing energy state of the cell. Transgenic mice overexpressing desnutrin in adipose tissue that we generated showed reduced adiposity with increased oxygen consumption and body temperature. Our adipose tissue-specific desnutrin knockout mice showed the unique phenotype of their BAT converting into WAT, greatly impairing thermogenesis. These in vivo mouse models demonstrate the critical role of desnutrin-catalyzed lipolysis in the maintenance of BAT phenotype. We also found that PPARa is activated specifically by fatty acids released by desnutrin acting as PPARa ligand directly or indirectly by conversion to another metabolite. This is a new exciting area since increasing BAT that can dissipate energy in place of the WAT that stores fat would be a future preventive or therapeutic strategy of ever-increasing obesity problem. 

       Phospholipase A2 (PLA2) superfamily of enzymes that catalyze hydrolysis fatty acids from the membrane phospholipids’ sn-2 position enriched with unsaturated fatty acids. Thus, PLA2 can release arachidonic acid and is regarded as the first rate-limiting step in eicosanoid biosynthesis. PLA2 may be expressed in specific tissues for production of local lipid mediators in order to regulate tissue-specific function. We recently identified a novel adipocyte specific phospholipase A2 that we named AdPLA. We identified and characterized AdPLA by overexpression and purification. AdPLA expression is under the nutritional control, very low in the fasted state but induced by feeding/ insulin. We observed AdPLA knockout mice that we generated exhibit a drastic decrease in adiposity and are protected from diet-induced and genetic obesity. In this regard, we detected PGE2 as the major prostaglandin produced in adipocytes and EP3 as the major PGE2 receptor in this cell type. We found that the PGE2 produced upon AdPLA induction in fed state plays a dominant inhibitory role in lipolysis through binding to the Gai-coupled EP3 to reduce cAMP levels, decreasing PKA mediated phosphorylation of HSL. Our study reveals a novel adipocyte-specific PLA2 which, through AdPLA/PGE2/cAMP pathway, plays a major role of in regulating lipolysis in an autocrine/paracrine manner.

 

Novel factors that control adipogenesis

         

        In an attempt to identify genes that regulate adipocyte differentiation, over the years, we have cloned/identified several novel molecules that can control adipogenesis.  We cloned 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 found in mature adipocytes.  When constitutively expressed, Pref-1 blocks adipocyte differentiation in vitro, whereas absence of Pref-1 enhances differentiation. We found that processing of cell-associated Pref-1 by TACE generates a soluble Pref-1 of 50 kD corresponding to its ectodomain. And, only the 50 kD soluble form of Pref-1 is active as an inhibitor of adipocyte differentiation and a membrane form of Pref-1 mutated at the cleavage site was not funtional. We generated Pref-1 knockout mice as well as transgenic mice that ectopically overexpress Pref-1 in adipose tissue: overexpression of Pref-1 in adipose tissue using aP2 promoter in transgenic mice 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. We are currently in pursuing of identifying bone-fie Pref-1 receptor using newly advanced cross-linking techniques.

         In addition to Pref-1, we have identified several other proteins, either intracellular or secretory, that can inhibit adipocyte differentiation. We identified and characterized an actin-binding, kelch-related protein, Enc-1, which is transiently expressed early during adipocyte differentiation and which may play a critical role for the cell shape change that is required for adipose conversion.  We also identified and characterized an adipocyte specific secretory factor that we named ADSF (others identified it as resistin, a factor secreted by adipose tissue that may be responsible for peripheral insulin resistance). Expression of ADSF/resistin that is found only in adipocytes in rodents, increases drastically during in vitro differentiation of adipocytes and ADSF can potently inhibit adipocyte differentiation. ADSF is also under tight hormonal/insulin control and is induced dramatically by feeding/insulin treatment. We generated transgenic overexpressing dominant negative form of ADSF in adipose tissue and found that these mice have higher adipocyte differentiation with improved insulin sensitivity. ADSF/resistin may be an adipose sensor for the nutritional state of the animals and the inhibitory effect on adipocyte differentiation implicate its function as a feedback regulator of adipogenesis.

         Recently, with the recent evidence for the presence of significant functional BAT in human adults, we have initiated a research on brown adipocyte transcription and differentiation. 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 made progress on 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. Zfp516 binds to its response element at the 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. Moreover, ectopic expression of Zfp516 in adipose tissue of mice promotes browning of subcutaneous white fat increasing body temperature and energy expenditure.

 

        Overall, our research on enzymes in TAG metabolism in adipose tissue and those molecules that regulate differentiation of WAT and BAT, provide not only better understanding of adipose tissue function and development, but also future targets for prevention/therapeutics for obesity/diabetes.