NUTRITIONAL AND HORMONAL REGULATION OF LIPOGENESIS AND ADIPOGENESIS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1020689
• Project Start Date: Jan 24, 2020
• Project End Date: Sep 30, 2024
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF CALIFORNIA, BERKELEY
(N/A)
BERKELEY,CA 94720

Performing Department
Nutritional Sciences

Non Technical Summary
White adipose tissue stores fat to increase adiposity, whereas brown adipose tissue dissipates energy as heat for thermogenesis. This research is directed toward understanding how lipid synthesis is regulated and how adipose tissue develops. In addition, identifying proteins and transcriptional regulators that govern white and brown adipose tissue gene expression and development will be studied. Understanding the mechanism and signaling pathway for the action of these proteins will provide us new targets to develop strategies to control obesity and associated diseases, such as diabetes.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
702	7010	1010	100%

Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
7010 - Biological Cell Systems;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

fatty acid and fat synthesis

adipogenesis

thermogenesis and brown adipose tissue

obesity and diabetes

Goals / Objectives
We continue to study our candidate proteins that mayregulateadipose tissue development and function. Specific objectives are:To examine the USF interacting epigenetic factors for lipogenic gene activation by feeding/insulin. 2. To identify and characterize the receptor that mediates Pref-1 inhibition of adipogenesis 3.To study novel transcription factors and coregulators for brown adipogenesis

Project Methods
1. To examine the USF interacting epigenetic factors for lipogenic gene activation by feeding/insulin.Recently, we found a not well studied histone demethylase, JMJD1C, as a USF interacting protein. We will examine histone methylation and chromatin accessibility for FAS transcription upon feeding/insulin treatment, focusing on the role of JMJD1C. JMJD1C is a jmjC domain-containing protein known for H3K9me2 and H3K9me1 demethylation. Indeed, we detected direct interaction between USF1 and JMJD1C via its bHLH domain in USF-GST pulldown assays. We propose that, upon feeding, JMJD1C is recruited to the FAS promoter by USF via direct interaction to bring H3K9 demethylation. We will test this hypothesis by Co-IP of USF (as well as various S262 phosphorylation and K237 acetylation mutants) and JMJD1C. Furthermore, by ChIP, we will examine the effect of shRNA KD or overexpression of JMJD1C on H3K9me3 demethylation to H3K9me2 at the FAS and other lipogenic promoter regions. Results will be compared with expression levels for lipogenic genes. KD and overexpression of JMJD1C will allow us to examine promoter activity of the FAS and other lipogenic genes that can then be compared with H3K9 methylation status of lipogenic genes.In addition, we have detected JMJD1C phosphorylation upon feeding/insulin treatment by western blotting of JMJD1C using pan-phospho-serine and pan-phospho-threonine antibodies. This is exciting since regulation of histone demethylases by phosphorylation has not been well explored. To identify specific phosphorylation site(s), we will purify JMJD1C from livers of mice after feeding/insulin treatment and identify phosphorylation site(s) of JMJD1C by MS analysis. To obtain better evidence of JMJD1C as a downstream target of insulin signaling, we will generate aspartate and alanine mutants of JMJD1C. We will also generate phospho-peptide specific antibodies to examine insulin/feeding dependent phosphorylation. We will then identify specific kinase that phosphorylates JMJD1C. We will determine the effect of phosphorylation on histone H3K9 methylation status and FAS and otherlipogenic gene activation.We will next perform gain- and loss-of function studies for JMJD1C. Similar specific effects on hepatic lipogenic gene expression and TAG levels will be examined upon adenovirus-mediated JMJD1C shRNA KD as well as overexpressing phosphorylation site mutants of JMJD1C in mice. We also have generated liver-specific JMJD1C-KO mice by crossing JMJD1C-floxed mice with albumin-Cre mice. Tissues from these mice will be used for ChIP for occupancy of various proteins at the FAS and other lipogenic promoter regions and for RNA preparation for RT-qPCR. We will also examine at the genome-wide level by ChIP-seq and RNA-seq.Changes in FAS and other lipogenic gene transcription should be reflected in de novo lipogenesis in vivo. We will measure de novo lipogenesis, as well as vivo TAG synthesis and turnover in liver and in adipose tissue using a stable isotope method. Serum lipid metabolite levels, including TAG and FFA, will also be measured. In addition, we will employ CLAMS for whole body O2consumption/CO2production for energy expenditure and RER, which will allow us to calculate whole body fat metabolism. We will perform glucose and insulin tolerance tests, especially since changes in TAG content is known to affect glucose/insulin homeostasis.2. To identify and characterize the receptor mediating Pref-1 inhibition of adipogenesis.Since the cleaved Pref-1 ectodomain is biologically active, we predict that Pref-1 acts as a ligand for a yet to be identified receptorto activate ERK/MEK pathway to inhibit adipocyte differentiation. Thus, identification of the Pref-1 receptor is critical.We have attempted to crosslink Pref-1 with the putative transmembrane Pref-1 receptor, by using a new diazirine photo-reactive linker method that combines robust NHS-ester chemistry with a light-activatable reactive group.We incubated purified HA or His-tagged soluble Pref-1 with a crosslinker and, after quenching and removal of excesscrosslinker, the labeled Pref-1 was incubated with 3T3-L1 preadipocytes in culture, which was then crosslinked by exposure to 320 nm UV. We purified the Pref-1 complex by usingaffinity beads for the tag andsubjecting the samples to SDS-PAGE. The band representing Pref-1-receptor complex was visualized by Pref-1 antibody and was excised for MS analysis. Thus, we identifiedpeptides thatcrosslinked with Pref-1. We propose that, for Pref-1 downstream signaling, Pref-1 binds to the putative Pref-1 receptor that contains the identified peptide sequence.We will examinethe binding characteristics of Pref-1 to this protein, to confirm and characterize the protein as the Pref-1 receptor.We will examine Pref-1 binding to this protein at the plasma membrane by various methods for receptor-ligand interaction. Purified recombinant Pref-1 at varying concentrations will be used for Pref-1 binding to the proteinbyin vitroand in cell-based assays, to determine the binding affinity and sites. Moreover, we will define the Pref-1 binding domain of the protein. Next, we will examine the requirement of this protein for Pref-1signaling in the activation of ERK and for theinhibition of adipocyte differentiation.We will perform gain- and loss-of function studies in examining their roles in Pref-1 mediated ERK activation, Sox9 induction, and the adipocytedifferentiation in 3T3-L1 cells and primary preadipocytes of SVF cells of WAT, as well as MEFs.Next, we will test the function ofthis protein as the putative Pref-1 receptor in mice by treating them with the neutralizing antibody for this putative receptor. We will also employ the KO mice to test the requirement of this protein for Pref-1 action. Moreover, we will generate conditional PreASKO mice that lack the putative Pref-1 receptor specifically in preadipocytes, which will allow us to test thePref-1 function in adipogenic differentiation from preadipocytes. We will examine adipose depot weights, morphology, and gene expression patterns for known adipogenic markers, as well as Pref-1downstream targets, includingSox9, C/EBPband C/EBPd. Finally, we will examine glucose/insulin homeostasis of these mouse models as affected by altered adipogenesis through impaired Pref-1 signaling.3. To study novel transcriptional regulators for brown adipogenesisTo identify novel transcription factors that activate UCP1 transcription, we have screened a library of known and putative transcription factors by co-transfecting them with the -5.5 kb UCP1 promoter-GFP reporter. Zc3h10 is one such factor that we have identified. Zc3h10 contains three C3H-type zinc fingers as well as a ZIP domain. Zc3h10 function in a biological/physiological setting is unknown. We will define Zc3h10 binding and activation of the UCP1 promoter by promoter-reporter assay and EMSA as well as by SELEX. By ChIP, we will document occupancy of Zc3h10 at the promoter regions of UCP1 and other thermogenic genes. Moreover, we will test the requirement of Zc3h10 for expression of UCP1 and other thermogenic genes by using Zc3h10 knockout BAT cells we generated by using the CRISPR-Cas9 system. RNA-Seq and ChIP-Seq will be performed to identify direct targets of Zc3h10 and Zc3h10 binding sites in brown adipocytes at the genome-wide level. We will also identify the coregulators that interact with Zc3h10 in activating BAT genes during thermogenesis. Moreover,we will examinein vivothe function of Zc3h10 by documenting morphology of thermogenic tissues and thermogenesis in our transgenic mice that overexpress Zc3h10 in adipose tissue as well as in our conditional Zc3h10-KO mice. Effects of altered thermogenesis on adiposity and glucose/insulin homeostasis in our mouse models will be studied also.

Progress 01/24/20 to 09/30/20

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The postdoctoral fellows and graduate students have had training in scientific research by performing the proposed research. How have the results been disseminated to communities of interest?The scientists, postdoctoral fellows and graduat students have participated various scientific meetings to give seminar and posters. What do you plan to do during the next reporting period to accomplish the goals?1. Genome-wide screening for human beiging factors using CRISPR-Cas9 activation system 2. Studying a microprotein critical for thermogenesis 3. A brown adipocyte transcription factor used as a marker for BAT but not beige fat. 4. New epigenetic regulators for lipogenesis

Impacts
What was accomplished under these goals? A novel lipid droplet associated protein that inhibits lipolysis in adipose tissue We recently identified a previously uncharacterized 38 kD protein, which contains an apolipoprotein like domain and is specifically expressed in adipose tissue. Due to lack of signal sequence, however, this apoprotein-like protein is not secreted. Rather, it is localized to lipid droplets (LD), making this protein a newly discovered adipose-specific LD-associated protein. We also found that this new LD-associated protein directly interacts with two other well-known LD proteins, Perilipin (Plin1) and Fsp27 (CideC), but not with lipases, Desnutrin/ATGL and HSL. Furthermore, expression of the gene coding for this apolipoprotein-like protein is very low in adipose tissue of fasted mice, which is increased upon feeding, especially when fed a high fat diet. We also found it to be overexpressed in both genetic and diet induced obesity, suggesting its contribution to adiposity. We have generated transgenic mice for overexpression in adipose tissue, as well as global knockout mice by using CRISPR-Cas9 system. Our transgenic mice showed a greatly increased white adipose tissue (WAT) mass with enlarged adipocytes. Conversely, our global knockout mice showed a substantially diminished adipose tissue mass with smaller adipocyte size, protected from diet induced obesity. We also found that lipolysis and fatty acid oxidation are decreased, with no change in lipogenesis, upon overexpression in adipose tissue. Conversely, lipolysis is increased upon ApoL6 ablation in mice. Our long-term goal is to understand the molecular details and physiological significance of the function of this protein as a LD-associated protein to suppress lipolysis in WAT for promotion of TAG storage and adiposity. This research may not only help to fully understand LD biology in adipocytes but also provide future therapeutic targets for obesity/diabetes. 2. Aifm2, a novel NADH oxidase, supports robust glycoysis for cold- and diet-induced thermogenesis Brown adipose tissue (BAT) is highly metabolically active tissue that dissipates energy via UCP1 as heat, and BAT mass is correlated negatively with obesity. The presence of BAT/BAT-like tissue in humans renders BAT as an attractive target against obesity and insulin resistance. Here, we identify Aifm2, a NADH oxidoreductase domain containing flavoprotein, as a lipid droplet (LD)-associated protein highly enriched in BAT. 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 (ETC) for fueling thermogenesis. Aifm2 in BAT and subcutaneous white adipose tissue (WAT) promotes oxygen consumption, uncoupled respiration, and heat production during cold- and diet-induced thermogenesis. Aifm2, thus, can ameliorate diet-induced obesity and insulin resistance. 3. Identification of an epigenetic factor, Dot1L, required for thermogenic program Brown adipose tissue is a metabolically beneficial organ capable of dissipating chemical energy into heat, thereby increasing energy expenditure. Here, we identify Dot1l, the only known H3K79 methyltransferase, as an interacting partner of Zc3h10 that transcriptionally activates theUcp1promoter and other BAT genes. Through a direct interaction, Dot1l is recruited by Zc3h10 to the promoter regions of thermogenic genes to function as a coactivator by methylating H3K79. We also show that Dot1l is induced during brown fat cell differentiation and by cold exposure and that Dot1l and its H3K79 methyltransferase activity is required for thermogenic gene program. Furthermore, we demonstrate that Dot1l ablation in mice usingUcp1-Cre prevents activation ofUcp1and other target genes to reduce thermogenic capacity and energy expenditure, promoting adiposity. Hence, Dot1l plays a critical role in the thermogenic program and may present as a future target for obesity therapeutics.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Viscarra J, Sul HS Epigenetic regulation of hepatic lipogenesis: Role in hepatosteatosis and diabetes. Diabetes. 2020 Apr;69(4):525-531.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Yi D, Nguyen HP, Sul HS. Epigenetic dynamics of the thermogenic gene program of adipocytes. Biochem J. 2020 Mar 27;477(6):1137-1148.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Nguyen HP, Yi D, Lin F, Viscarra JA, Tabuchi C, Ngo K, Shin G, Lee AY, Wang Y, Sul HS. Aifm2, a NADH oxidase, supports robudt glycolysis and is required for cold- and diet-induced thermogenesis. Mol Cell. 2020 Feb 6;77(3):600-617.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Yi D, Nguyen HP, Dinh J, Viscarra JA, Xie Y, Lin F, Zhu M, Dempersmier JM, Wang Y, Sul HS. Dot1l interacts with Zc3h10 to activate UCP1 and other thermogenic genes. Elife. 2020 Oct 27;9:e59990.