IDH1-dependent α-KG regulates brown fat differentiation and function by modulating histone methylation

Journal Pre-proof IDH1-dependent α-KG regulates brown fat differentiation and function by modulating histone methylation

Hyun Sup Kang, Jae Ho Lee, Kyoung-Jin Oh, Eun Woo Lee, Baek Soo Han, Kun-Young Park, Jae Myoung Suh, Jeong-Ki Min, Seung-Wook Chi, Sang Chul Lee, Kwang-Hee Bae, Won Kon Kim PII:

S0026-0495(20)30037-8

DOI:

https://doi.org/10.1016/j.metabol.2020.154173

Reference:

YMETA 154173

To appear in:

Metabolism

Received date:

14 November 2019

Accepted date:

4 February 2020

Please cite this article as: H.S. Kang, J.H. Lee, K.-J. Oh, et al., IDH1-dependent αKG regulates brown fat differentiation and function by modulating histone methylation, Metabolism(2020), https://doi.org/10.1016/j.metabol.2020.154173

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© 2020 Published by Elsevier.

Journal Pre-proof

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IDH1-dependent α-KG regulates brown fat differentiation and function

3

by modulating histone methylation

4 5

Hyun Sup Kang

6

Kun-Young Park c, Jae Myoung Suh c,d, Jeong-Ki Min a,b, Seung-Wook Chi a,b, Sang Chul Lee

7

a,b

a,b,1

, Jae Ho Lee

a,1

, Kyoung-Jin Oh

, Eun Woo Lee a, Baek Soo Han

a,b

,

oo

f

, Kwang-Hee Bae a,b,*, Won Kon Kim a,b,*

a

pr

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9

a,b

Metabolic Regulation Research Center, Korea Research Institute of Bioscience and

Biotechnology (KRIBB), Daejeon 34141, Republic of Korea

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b

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Science and Technology (UST), Daejeon 34141, Republic of Korea

13

c

14

Republic of Korea

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d

e-

10

Pr

Department of Functional Genomics, KRIBB School of Bioscience, Korea University of

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Biomedical Science and Engineering Interdisciplinary Program, KAIST, Daejeon 34141,

rn

Graduate School of Medical Science and Engineering, KAIST, Daejeon, Republic of Korea

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*Corresponding authors.

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E-mail addresses: [email protected] (K.-H. Bae), [email protected] (W.K. Kim).

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1

These two authors contributed equally to this work.

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Abbreviations

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IDH1, isocitrate dehydrogenase 1; α-KG, α-ketoglutarate; UCP1, uncoupling protein 1;

23

PPARγ, peroxisome proliferator-activated receptor-γ; PGC1α, PPARγ -coactivator protein 1-

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alpha; PRDM16, PR-domain-containing 16; C/EBP, CCAAT-enhancer-binding protein. 1

Journal Pre-proof ABSTRACT

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Objective. Brown adipocytes play important roles in the regulation of energy homeostasis by

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uncoupling protein 1-mediated non-shivering thermogenesis. Recent studies suggest that

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brown adipocytes as novel therapeutic targets for combating obesity and associated diseases,

29

such as type II diabetes. However, the molecular mechanisms underlying brown adipocyte

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differentiation and function are not fully understood.

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Methods. We employed previous findings obtained through proteomic studies performed to

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assess proteins displaying altered levels during brown adipocyte differentiation. Here, we

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performed assays to determine the functional significance of their altered levels during brown

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adipogenesis and development.

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Results. We identified isocitrate dehydrogenase 1 (IDH1) as upregulated during brown

36

adipocyte differentiation, with subsequent investigations revealing that ectopic expression of

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IDH1 inhibited brown adipogenesis, whereas suppression of IDH1 levels promoted

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differentiation of brown adipocytes. Additionally, Idh1 overexpression resulted in increased

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levels of intracellular α-ketoglutarate (α-KG) and inhibited the expression of genes involved

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in brown adipogenesis. Exogenous treatment with α-KG reduced brown adipogenesis during

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the early phase of differentiation, and ChIP analysis revealed that IDH1-mediated α-KG

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reduced trimethylation of histone H3 lysine 4 in the promoters of genes associated with

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brown adipogenesis. Furthermore, administration of α-KG decreased adipogenic gene

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expression by modulating histone methylation in brown adipose tissues of mice.

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Conclusion. These results suggested that the IDH1–α-KG axis plays an important role in

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regulating brown adipocyte differentiation and might represent a therapeutic target for

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treating metabolic diseases.

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Keywords: brown adipocyte differentiation, isocitrate dehydrogenase 1 (IDH1), α-

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ketoglutarate (α-KG), histone modification

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1. Introduction

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Obesity is a phenomenon primarily caused by sustained energy imbalance and represents a

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major causal factor of various metabolic disorders [1, 2]. Due to the rapid increase in the

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obese population and associated metabolic diseases, research into strategies for treating

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obesity has become increasingly important. Adipose tissues are mainly composed of adipocytes that play critical roles in regulating

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energy homeostasis. Mammals harbor two types of adipocytes, including white and brown

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adipocytes. White adipocytes comprise a single, large lipid droplet located in the

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subcutaneous and abdominal areas of the body where they store excess energy in the form of

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triglycerides. Brown adipocytes are enriched with numerous mitochondria and mainly located

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in the interscapular region in rodents and human infants. In contrast to white adipocytes,

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brown adipocytes primarily consume energy to generate heat, with their thermogenic capacity

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largely due to induction of the brown adipose tissue-specific protein uncoupling protein 1

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(UCP1) located in the mitochondrial inner membrane [3]. UCP1 dissipates the mitochondrial

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electrochemical gradient through proton leakage to induce uncoupled respiration [4, 5].

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Therefore, the UCP1-mediated thermogenic functions of brown adipocytes are now regarded

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as a therapeutic target for treating obesity [6].

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Brown adipocytes are derived from mesenchymal stem cells (MSCs), which are

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multipotent stromal cells capable of differentiating into various cell types, such as adipocytes,

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myocytes, chondrocytes, and osteoblasts, under specific stimuli [7]. Brown adipocyte

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differentiation from MSCs involves two steps: 1) commitment to preadipocytes from MSCs

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and 2) adipocyte differentiation into mature adipocytes [8, 9]. In particular, MSC

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commitment to a brown adipocyte lineage is controlled by a variety of factors, including PR-

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domain-containing 16 (PRDM16), bone morphogenetic protein 7 (BMP7), and early B cell

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factor 2 (EBF2) [10-12]. In particular, PRDM16 coupled with CCAAT-enhancer-binding 3

Journal Pre-proof protein-β (C/EBPβ) is essential for the determination of brown fat from muscle-precursor

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cells [13]. After lineage determination to brown preadipocytes, these cells sequentially

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differentiate into mature brown adipocytes accompanied by the activities of multiple

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transcriptional regulators, including peroxisome proliferator-activated receptor-γ (PPARγ),

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C/EBPs, and PPARγ coactivator 1-alpha (PGC1α) [14, 15]. Specifically, PPARγ is a key

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transcription factor involved in regulating brown adipocyte differentiation and the browning

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of white adipose tissues [16]. Therefore, several transcriptional regulators are directly

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involved in lineage determination and differentiation; however, the molecular mechanisms

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that coordinate the commitment and differentiation of brown adipocytes remain incompletely

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understood.

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Histone modification plays a key role in the regulation of gene expression and

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numerous cellular functions [17]. Among the various types of histone modification,

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acetylation and methylation on lysine or arginine residues of histones H3 and H4 play

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important biological roles, including transcriptional regulation [18, 19]. Gene expression is

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differentially controlled depending on the site of histone methylation, with methylation of the

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4th and 36th lysine residues of histone H3 responsible for general activation of transcription,

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whereas methylation of the 9th and 27th lysine residues of histone H3 mediate gene

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inactivation [19]. Moreover, methylation of these lysine residues can occur as mono-, di- and

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trimethylation, which regulates genes in different ways [20]. Additionally, histone

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methylation and demethylation play important roles in adipocyte differentiation [21] and

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reportedly control adipocyte differentiation by regulating PPARγ expression [22].

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Furthermore, a recent study reported the involvement of H3K27 methylation and acetylation

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in the thermogenic program of brown adipocytes by regulating the expression of brown

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adipocyte-specific genes, including Ucp1 and Pgc1a [23].

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Journal Pre-proof We previously performed proteomic analysis to screen changes in protein levels

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during brown adipocyte differentiation [24] and identified isocitrate dehydrogenase 1 (IDH1)

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as upregulated during differentiation of primary brown preadipocytes. IDH1 is an NADP+-

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dependent metabolic enzyme responsible for converting isocitrate to α-ketoglutarate (α-KG)

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[25, 26]. Although IDH2 localizes to the mitochondrial matrix, IDH1 localizes to the cytosol;

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however, both IDH1/2 play essential roles in cellular metabolism, including the TCA cycle

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[27]. In addition to its metabolic functions, IDH1 regulates gene expression through

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epigenetic modification of histone proteins [28]. In this study, we investigated the functional

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roles of IDH1 and its regulatory mechanisms associated with brown adipocyte differentiation.

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Journal Pre-proof 2. Materials and methods

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2.1. Cell culture, brown adipocyte differentiation, and Oil-Red-O staining

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An immortalized brown preadipocyte cell line was kindly provided by Dr. Shingo Kajimura

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(UCSF, San Francisco, CA, USA). A brown preadipocyte cell line was obtained from the

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interscapular brown adipose tissue of C57BL/6 mice at postnatal days 1–2 and isolated by

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collagenase dispersion, as described previously [29]. These cells were grown in Dulbecco's

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modified Eagle medium (DMEM) containing 1% antibiotics and 10% fetal bovine serum

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(FBS) at 37C in a humidified atmosphere with 5% CO2. Primary brown preadipocytes were

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isolated from the interscapular brown adipose tissues of 1- to 3-day old mice, as described

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previously [30], and cultured in DMEM containing 1% antibiotics and 20% FBS (Gibco;

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Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified atmosphere with 5% CO2. For

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brown adipocyte differentiation, cells were induced, as previously described [31]. The

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C3H10T1/2 MSC line was provided by Dr. Jae Bum Kim (Seoul National University, Seoul,

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South Korea) and grown in DMEM containing 1% antibiotics and 10% FBS at 37C. The

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conditions used for adipogenic differentiation of C3H10T1/2 cells were previously described

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[32].

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Lipid droplets of differentiated brown adipocytes were subjected to Oil-Red-O staining,

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as described in our previous study [33]. Briefly, cultured cells were washed twice with

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phosphate-buffered saline (PBS) and fixed for 30 min with 10% formaldehyde at room

127

temperature. Fixed cells were then washed with distilled water and stained for 30 min at room

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temperature with 0.3% filtered Oil-Red-O solution in 60% isopropanol (Sigma-Aldrich, St.

129

Louis, MO, USA). The cells were then washed twice with distilled water, and micrographs

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were obtained. For quantification analysis, Oil-Red-O staining dye was eluted, as previously

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described [31].

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Journal Pre-proof 2.2. Transduction using retroviral expression or knockdown vectors

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To construct immortalized brown preadipocytes stably expressing FLAG-tagged mouse Idh1,

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a retroviral infection system was used. For Idh1 expression, DNA encoding the FLAG-tagged

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IDH1 was inserted into the pRetroX-IRES-ZsGreen1 vector (Clontech Laboratories,

137

Mountain View, CA, USA), as described previously [34]. For virus production, GP2-293 cells

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were transfected using Lipofectamine 2000 (Gibco; Invitrogen), and infected cells were

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selected using a FACSAria cell sorter (BD Biosciences, Franklin Lakes, NJ, USA) and

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maintained in growth medium. The ectopic expression of IDH1 was determined by western

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blot analysis and real-time polymerase chain reaction (PCR).

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To knockdown endogenous Idh1 expression, we used a retrovirus-mediated infection

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system. Short-hairpin RNAs (shRNAs) were designed by selecting a target sequence for the

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mouse Idh1 gene according to a previous report [35] and an RNA-interference target-

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sequence selector (Clontech). The gene encoding shRNA against Idh1 was inserted into the

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multi-cloning site of the pSIREN-RetroQ-DsRed vector (Clontech). The following gene-

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specific sequences were used to successfully inhibit Idh1 expression: shIDH1-1, 5′-

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GATCCGCTGCAGAGGCTTTAAAGATTCAAGAGATCTTTAAAGCCTCTGCAGCTTTTTTACGCGTG-3′ and 5′-

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AATTCACGCGTAAAAAAGCTGCAGAGGCTTTAAAGATCTCTTGAATCTTTAAAGCCTCTGCAGCG-3′;shIDH1-2,

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5′-GATCCGCACCATCCGAAACATTCTTTCAAGAGAAGAATGTTTCGGATGGTGCTTTTTTACGCGTG-3′ and 5′-

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AATTCACGCGTAAAAAAGCACCATCCGAAACATTCTTCTCTTGAAAGAATGTTTCGGATGGTGCG;

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shIDH1-3, 5′-GATCCGCATGCATATGGGGACCAATTTCAAGAGAATTGGTCCCCATATGCATGTTTTTTACGCGTG-

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3′ and 5′-AATTCACGCGTAAAAAACATGCATATGGGGACCAATTC TCTTGAAATTGGTCCCCATATGCATGCG-3′.

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Non-targeting control shRNA (scrambled; SCR) was provided by Clontech.

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2.3. Quantitative reverse transcription (qRT)-PCR

7

and

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Total RNAs were extracted from cultured cells using TRIzol reagent (Invitrogen) according

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to manufacturer instructions, and cDNA was synthesized from total RNA using the reverse

159

transcriptase M-MLV and a random primer (Promega, Madison, WI, USA) according to

160

manufacturer instructions [36]. Amplified cDNA was analyzed by qRT-PCR using a SYBR

161

green PCR kit and each primer (Table S1). Gene-expression levels were normalized to that of

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the TATA-binding protein (TBP) gene.

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2.4. Chromatin immunoprecipitation (ChIP) assay

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ChIP assays were performed, as previously described [23, 37]. For each immunoprecipitation

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reaction, proteins in supernatants were immunoprecipitated with antibodies against histone

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H3 trimethylated at lysine 4 (H3K4me3) and lysine 36 (H3K36me3; Abcam, Cambridge,

168

UK), respectively, with anti-rabbit IgG used as a negative control (GE Healthcare, Pittsburgh,

169

PA, USA). Precipitated DNA fragments were analyzed by real-time PCR using primers

170

against

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CCCACTAGCAGCTCTTTGGA-3′

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cAMP-CRE

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AAAAGTAGGCTGGGCTGTCA-3′;

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ATACACTGCCCTGTGTAAGG-3′ and 5′-CTGCTAGGTTGGCAAGGAAT-3′; pyruvate

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dehydrogenase

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GGGAGGTCTAGAGCCCCTAA-3′; and Wntless (Wls) 5′-CTGGCTGTGGCTTGTGTAAA-

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3′ and 5′-GGACAAGAGGCAAAAGCAAC-3′.

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kinase

as

and

follows:

Ucp1

proximal

3

(Pdk3),

promoter,

5′-CTGTGGAGCAGCTCAAAGGT-3′;

5′-CAAAGCTGGCTTCAGTCACA-3′

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promoters,

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mouse

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relevant

Pparg

proximal

5′-

Pgc1a

and

5′-

promoter,

5′-

5′-TTCCTTAAAGCCCCGGTAAC-3′

and

5′-

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2.5. Western blot

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Cells were washed three times with ice-cold PBS and harvested in ice-cold NP-40 lysis buffer

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(137 mM NaCl, 20 mM Tris-Cl, 1 mM EDTA, 10% glycerol, and 1% NP-40) containing a 8

Journal Pre-proof protease inhibitor and a phosphatase-inhibitor cocktail (Roche, Basel, Switzerland). Protein

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concentrations were measured using a Bradford assay (Bio-Rad, Hercules, CA, USA).

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Sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot analyses were

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performed using standard protocols, as described previously [38]. The following primary

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antibodies were used: IDH1 (Cell Signaling, Danvers, MA, USA; #3997), IDH2 (Cell

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Signaling; #56439), UCP1 (Abcam; #ab10983), PGC1α (Invitrogen; #PA5-38021), PRDM16

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(R&D Systems, Minneapolis, MN, USA; #AF6295), PPARγ (Cell Signaling; #2435), heat-

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shock protein 90 (HSP90; Santa Cruz Biotechnology, Dallas, TX, USA; #sc-13119),

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H3K4me3 (Abcam; #ab8580), H3K9me3 (Abcam; #ab8898), H3K27me3 (Abcam; #ab6002),

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H3K36me3 (Abcam; #ab9050), Histone H3 (Abcam; #ab1791), Anti-5′ AMP-activated

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protein kinase-alpha (AMPKα, Cell Signaling; #2532), and anti-phospho-AMPKα (Cell

193

Signaling; #2535). The specific signals were amplified by horseradish peroxidase-conjugated

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secondary anti-rabbit, anti-mouse, or anti-sheep IgG antibody (Santa Cruz Biotechnology),

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and were visualized using an enhanced chemiluminescence system (Fusion Solo S; Vilber

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Lourmat, France). Relative amounts of each protein were quantified using ImageJ software.

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2.6. α-KG assay

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The cell-membrane-permeable α-KG precursor dimethyl-2-ketoglutarate (dm-α-KG) was

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purchased from Sigma-Aldrich [39]. Briefly, 1 mM dm-α-KG was used to treat immortalized

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brown preadipocytes during differentiation, and intracellular α-KG concentrations were

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analyzed using an α-KG assay kit (K677; BioVision, Milpitas, CA, USA) according to

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manufacturer instructions. Similar to in vitro analysis, intracellular α-KG concentrations in

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mouse tissues were measured using identical methods.

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2.7. Animal experiments 9

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All animal housing was in compliance and experiments were conducted in accordance with

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the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Institutional Animal

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Care and Use Committee Guidelines. Mice were housed in a temperature- and humidity-

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controlled, specific pathogen-free animal facility at 22℃ under a 12:12 hour light:dark cycle.

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For studies in brown fat development, interscapular brown adipose tissues of mice from

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embryonic day 18 and after birth from postnatal day 1 to 6 were carefully dissected and

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analyzed by qRT-PCR and Western blotting (n = 2). For administration of α-KG in vivo, 8-

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week-old

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intraperitoneally with saline or 0.5, 1, and 2 g/kg of dm-α-KG (Sigma-Aldrich) for 3 hours (n

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= 4). In addition, we sacrificed mice and dissected brown adipose tissues after 3 days of daily

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intraperitoneal injections of 1 g/kg of dm-α-KG (n = 5). For cold exposure experiments, 11-

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week-old C57BL/6 male mice were randomly divided into four groups: room temperature

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(RT), cold exposure for 1, 3, and 7 days (n = 3). Mice as control group were all placed at RT

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(22°C) for 7 days, while the cold groups were placed at 5°C for the above mentioned times.

mice

(KOATECH,

Pyeongtaek,

Korea)

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were

injected

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C57BL/6

2.8. Statistical analysis

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All experiments were performed at least in triplicate. All data are presented as the mean ±

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standard deviation (SD). Statistical significance of differences between two groups was

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measured using the two-tailed Student's t test. For assessment between more than three

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groups, we used one-way analysis of variance (ANOVA) with multiple comparisons. To

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assess the relationship between two independent variables, two-way ANOVA with multiple

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comparisons was used. One-way ANOVA was followed by Tukey's post hoc test, and two-

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way ANOVA was followed by Sidak's multiple comparison test. Statistical analyses were

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performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA), and

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differences were considered significant at p < 0.05.

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Journal Pre-proof 3. Results

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3.1. IDH1 is upregulated during brown adipogenesis and brown adipose tissue

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development

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We previously performed proteomic analysis to identify differential levels of proteins during

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brown adipocyte differentiation [24], eventually choosing IDH1 for further analysis. In the

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present study, we found that IDH1 levels were clearly elevated in both immortalized brown

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preadipocytes and primary brown preadipocytes during differentiation (Fig. 1A), whereas

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IDH2 levels were unchanged during brown adipogenesis (Fig. 1A). Accordingly, Idh1 mRNA

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levels were upregulated during brown adipogenesis along with upregulated expression of

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brown adipogenic genes such as Ucp1 and Pgc1a (Fig. 1B). Next, we analyzed the

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expression levels of IDH1 protein during brown fat development through late embryogenesis,

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postnatal periods and adulthood. The levels of IDH1 and brown adipogenic proteins, UCP1

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and PGC1α, increased from embryonic day 18 to postnatal day 6 when brown adipose tissue

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became fully mature (Fig. 1C and D). These data show that IDH1 expression is upregulated

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during brown fat adipogenesis both in vitro and in vivo.

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3.2. Ectopic expression of IDH1 inhibits brown adipocyte differentiation

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To investigate the functional role of IDH1 in brown adipocyte differentiation, we established

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immortalized brown preadipocytes stably expressing Idh1 following retroviral infection.

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After flow cytometric sorting of infected cells, stable expression was verified by fluorescence

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microscopy (Fig. 2A), with ectopic expression of IDH1 determined by western blot (Fig. 2B).

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The infected brown preadipocytes were then induced to differentiate into mature brown

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adipocytes by culture in brown adipogenic induction medium for 6 days, followed by

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examination of cellular lipid accumulation using Oil-Red-O staining on day 6 after

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differentiation. The results showed that Idh1 overexpression decreased differentiation of 11

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brown preadipocytes (Fig. 2C and D). Additionally, transcript levels of key genes involved in

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brown adipogenesis, including Ucp1, Pgc1a, Prdm16, and Pparg, were significantly

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suppressed upon IDH1 ectopic expression (Fig. 2E). Moreover, we consistently observed

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attenuated UCP1 levels following differentiation in brown adipocytes ectopically expressing

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IDH1 (Fig. 2B). These results strongly implied that IDH1 negatively regulated brown

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adipocyte differentiation by suppressing the expression of genes involved in brown

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adipogenesis.

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3.3. Idh1 knockdown promotes brown adipogenesis

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To clarify the roles of IDH1 in regulating brown adipocyte differentiation, we generated

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stable Idh1-knockdown brown preadipocytes using the pSIREN-RetroQ-DsRed vector

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system (Fig. 3A). We verified the highly efficient knockdown of endogenous Idh1 expression

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by shRNA IDH1-II according to qRT-PCR and western blot analyses (Fig. 3B) and observed

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continuous suppression of Idh1 expression into the late phases of differentiation (Fig. 3C and

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E). We then differentiated Idh1-knockdown immortalized brown preadipocytes into mature

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brown adipocytes using a standard protocol for 6 days, followed by Oil-Red-O staining to

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visually assess lipid accumulation. In contrast to Idh1 overexpression, Idh1 knockdown

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promoted brown adipocyte differentiation relative to that observed in cells infected with

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scrambled shRNA (Fig. 3D). Additionally, levels of UCP1, a key brown adipocyte protein,

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were enhanced by Idh1 knockdown (Fig. 3E), with transcript levels of Ucp1, Pgc1a, Prdm16,

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and Pparg also significantly upregulated in the presence of attenuated IDH1 levels (Fig. 3F).

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3.4. α-KG inhibits brown adipocyte differentiation by acting at an early phase of

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differentiation

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Because IDH1 localizes to the cytosol and catalyzes the conversion of isocitrate to α-KG [25-

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27], we investigated whether IDH1 regulates brown adipocyte differentiation through α-KG.

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Measurement of intracellular α-KG levels during brown adipogenesis in cells ectopically

284

expressing IDH1 or Idh1-knockdown cells revealed fluctuations in α-KG levels according to

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those of IDH1 during brown adipocyte differentiation (Fig. 4A), with elevated α-KG levels in

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the presence of IDH1 and attenuated α-KG levels in Idh1-knockdown cells. These data

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suggest that increases in α-KG levels might affect brown adipocyte differentiation. To examine the effects of α-KG on brown adipocyte differentiation, brown

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preadipocytes were treated with cell-permeable dm-α-KG during differentiation, and its effect

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on intracellular α-KG levels was determined. As shown in Fig. 4B, intracellular α-KG levels

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were rapidly elevated following administration of dm-α-KG. We then added dm-α-KG to

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culture medium at different time points during brown adipocyte differentiation, finding that

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administration during the early phase (days 0–2) inhibited brown adipocyte differentiation,

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whereas this activity during the middle (days 2–4) or terminal (days 4–6) stages did not affect

295

brown adipocyte differentiation or the expression of genes associated with brown

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adipogenesis (Figs. 4C; S1A and B). Furthermore, we confirmed that protein and mRNA

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levels of brown adipogenic markers, such as Ucp1, Pgc1a, Prdm16, and Pparg, were

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consistently downregulated in mature brown adipocytes treated with α-KG for either 2 days

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(days 0–2) or 6 days (days 0–6) (Fig. 4D and E). These results clearly implied that α-KG

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affected the early phase of brown adipocyte differentiation by suppressing the expression of

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key genes involved in brown adipogenesis, thereby inhibiting brown adipocyte differentiation.

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3.5. IDH1 negatively regulates brown adipocyte differentiation through α-KG

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To clarify the α-KG-dependent effect of IDH1 on brown adipocyte differentiation, we

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investigated whether supplementation of dm-α-KG of IDH1-depleted cells could rescue the 13

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effects of Idh1 knockdown on brown adipocyte differentiation. Interestingly, the enhanced

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brown adipocyte differentiation observed following Idh1 knockdown was potently

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suppressed by administration of dm-α-KG (Fig. 5A). Additionally, qRT-PCR analysis

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revealed that the elevated expression of genes associated with brown adipogenesis in Idh1-

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knockdown cells was downregulated to control levels following dm-α-KG administration

311

(Fig. 5B). These results suggested that IDH1-mediated upregulation of α-KG levels is crucial

312

for regulating brown adipogenic genes and brown adipocyte differentiation. We then examined the effect of treatment with α-KG during brown adipocyte

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differentiation in Idh1-overexpressing cells. Compared with the significant repression of

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brown adipocyte differentiation observed following dm-α-KG administration or IDH1 ectopic

316

expression, Idh1-overexpressing cells administered dm-α-KG showed no additive effect on

317

inhibiting brown adipocyte differentiation (Fig. 5C), with similarly attenuated levels of

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brown adipogenic transcripts also observed according to cell-treatment status (Fig. 5D).

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These data suggested that IDH1-mediated α-KG level or exogenous dm-α-KG alone might be

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sufficient to inhibit the expression of brown adipogenic genes and brown adipocyte

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differentiation.

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3.6. Brown adipocyte differentiation is regulated by α-KG-mediated alteration of

324

histone methylation

325

Previous studies report that IDH1 not only plays roles in metabolic pathways but also

326

promotes epigenetic reprogramming through α-KG [28, 39], which regulates gene expression

327

by activating several histone demethylases [40]. Therefore, we hypothesized that IDH1-

328

mediated α-KG affects brown adipocyte differentiation by modulating epigenetic regulation

329

of gene expression. First, we monitored histone-methylation patterns during brown adipocyte

330

differentiation by western blot using specific antibodies, finding that H3K4me3 and 14

Journal Pre-proof H3K36me3 (but not H3K9me3 and H3K27me3 levels) increased during the first 2 days of

332

brown adipogenesis (Fig. 6A). Since H3K4me3 and H3K36me3 represent active chromatin

333

complexes, we speculated that this might be related to the induction of adipogenic gene

334

expression [41, 42]. As expected, mRNA levels of brown adipogenic genes were elevated at

335

48-h post-induction of differentiation and attenuated in the presence of α-KG (Fig. 6B). We

336

then examined whether IDH1 and/or α-KG affect levels of histone methylation, finding that

337

H3K4me3 and H3K36me3 levels were reduced by treatment with α-KG or IDH1 ectopic

338

expression (Fig. 6C), with further decreases in these levels observed in α-KG-treated Idh1-

339

overexpressing cells (Fig. 6C). By contrast, attenuated IDH1 levels resulted in elevated

340

H3K4me3 and H3K36me3 levels presumably by reducing α-KG levels (Figs. 4A and 6D).

341

Additionally, replenishment of the α-KG level to that present in IDH1-depleted cells rescued

342

H3K4me3 and H3K36me3 levels (Fig. 6D). These results suggested that IDH1 increased

343

intracellular α-KG levels and subsequently suppressed specific histone methylation

344

(H3K4me3 and H3K36me3) to inhibit brown adipocyte differentiation.

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Because histone methylation in the promoters of genes associated with brown adipocyte

346

differentiation is associated with transcriptional regulation of each gene [23], we evaluated

347

the effect of IDH1 and/or α-KG on the pattern of histone methylation in the promoter regions

348

of Ucp1, Pgc1a, and Pparg using ChIP analysis. We found that H3K4me3 levels at these

349

promoters were significantly reduced in cells exhibiting IDH1 ectopic expression or

350

undergoing dm-α-KG treatment (Fig. 6E), whereas they were suppressed in Idh1-knockdown

351

and further attenuated to control levels by additional dm-α-KG treatment (Fig. 6F).

352

Interestingly, we did not observed changes in H3K36me3 levels in these promoters,

353

regardless of dm-α-KG treatment or IDH1 status, indicating that H3K36me3 is not involved

354

in the regulation of brown adipogenic genes during brown adipocyte differentiation (Fig. 6E

355

and F). Additionally, dm-α-KG treatment did not change levels of H3K4me3 and H3K36me3

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in Prdm16-promoter regions (Fig. S2). These results suggested that IDH1-induced α-KG

357

specifically suppressed H3K4me3 in the promoters of brown adipogenic genes, thereby

358

preventing brown adipocyte differentiation. We then investigated whether IDH1 and α-KG also modulate histone methylation in the

360

promoters of white adipocyte-specific genes. ChIP analysis of the promoter regions of two

361

white adipocyte-specific genes, Wls and Pdk3 [37], indicated no significant changes to

362

histone modifications in these promoter regions or their transcript levels according to IDH1

363

status or α-KG treatment (Fig. S3A and B). These data strongly suggested that IDH1- and α-

364

KG-mediated fine-tuning of H3K4me3 levels in the promoter regions of brown adipogenic

365

genes during the early phase of differentiation is critical for brown adipocyte differentiation.

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3.7. α-KG administration decreased brown adipogenic gene expression in vivo

368

It has been demonstrated that brown adipogenic factors, such as UCP1 and PGC1α, are

369

required for the maintenance as well as the differentiation of brown adipocytes [43]. To

370

further investigate the effects of α-KG on maintenance and function of brown fat in vivo,

371

mice were injected intraperitoneally with α-KG. The increase of α-KG level in brown fat was

372

dependent on the concentration of administered α-KG, indicating the efficient delivery of

373

exogenous α-KG to brown adipose tissues (Fig. 7A). Since intracellular α-KG concentrations

374

were saturated by injection of 1 g/kg α-KG, we examined adipogenic gene expression after

375

the injection of 1 g/kg α-KG. Interestingly, exogenous α-KG decreased the mRNA levels of

376

the brown adipocyte-specific genes, such as Ucp1 and Pgc1a, in brown adipose tissues (Fig.

377

7B). In addition, α-KG suppressed the protein levels of brown adipogenic markers in brown

378

fat tissues (Fig. 7C). Moreover, we examined whether α-KG was accompanied by a change in

379

histone methylation in vivo. Similar to in vitro data, α-KG administration altered histone

380

methylation patterns, particularly H3K4me3 levels, in brown adipose tissues (Fig. 7D). In

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accordance with the α-KG-mediated reduction of brown adipocyte-specific gene expression,

382

H3K4me3 levels at the promoters of these genes, including Ucp1 and Pgc1a, were decreased

383

in brown adipose tissues of α-KG-administered mice (Fig. 7E). On the other hand,

384

H3K36me3 occupancy at these promoters was not altered by α-KG supplementation in brown

385

adipose tissues (Fig. 7E). These data imply that α-KG may not only regulate brown

386

adipogenesis but also mature brown fat physiology and function.

387

3.8. AMPK activation prevents differentiation of brown preadipocytes

389

A previous study suggests that activation of the AMPK–α-KG axis increases C3H10T1/2

390

MSC differentiation into brown adipocytes [44]. To evaluate the role(s) of AMPK in brown

391

adipogenesis, we examined the differentiation capacity of brown preadipocytes following

392

treatment with the AMPK activator metformin. As shown in Fig. 8A, metformin efficiently

393

activated AMPK signaling based on elevated levels of phosphorylated AMPK in brown

394

preadipocytes. Consistent with a previous report [44], activation of AMPK enhanced

395

intracellular α-KG levels during brown adipocyte differentiation (Fig. 8B). Additionally,

396

metformin suppressed brown adipogenesis, lipid accumulation, as well as the expression of

397

brown adipogenic genes, including Ucp1, Pgc1a, and Pparg (Figs. 8C–E and S4A). These

398

results suggested that metformin inhibited brown adipogenesis via AMPK-mediated

399

alteration of α-KG level. Subsequent investigation of the effect of α-KG level on C3H10T1/2

400

MSC differentiation to brown adipocytes indicated that no change in differentiation or the

401

expression of brown adipogenic genes during this process (Figs. 8F–G and S4B).

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3.9. IDH1 and α-KG is regulated by cold-induced thermogenic activation in brown

404

adipose tissues

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Journal Pre-proof Brown adipose tissue is a thermogenic organ that protects the body from cold exposure via

406

dissipating energy as heat [45]. Moreover, it has been demonstrated that TCA cycle

407

intermediates, especially succinate, regulate the activation of brown fat thermogenesis [46].

408

To elucidate whether IDH1 and α-KG might be involved in brown fat thermogenesis, we

409

analyzed the levels of IDH1 and α-KG in brown adipose tissues upon cold exposure. In

410

brown adipose tissues, the mRNA levels of Idh1 and thermogenic genes, Ucp1 and Pgc1a,

411

were increased in a manner dependent on the duration of cold exposure (Fig. 9A). In addition,

412

the level of IDH1 protein was upregulated under conditions of thermogenic activation by cold

413

challenge (Fig. 9B). Consistent with cold-induced IDH1 expression, the intracellular α-KG

414

levels in brown adipose tissues were elevated by cold exposure (Fig. 9C). Furthermore, cold

415

challenge modulated histone methylation patterns, especially induction of H3K4me3 levels

416

(Fig. 9D). These data suggested that the IDH1–α-KG axis may regulate in vivo brown fat

417

thermogenesis under cold exposure conditions.

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Journal Pre-proof 4. Discussion

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Brown adipocytes are widely accepted as an optimal target for creating strategies to

420

overcome obesity and its related diseases; therefore, investigating brown adipocyte formation

421

represents an active research area. To identify novel factors involved in brown adipocyte

422

differentiation, we previously screened proteins exhibiting differential levels during brown

423

adipocyte differentiation using two-dimensional gel electrophoresis proteomics [24], which

424

identified IDH1 in primary brown adipocytes, as well as in immortalized brown adipocytes.

425

Moreover, the expression of IDH1 was increased during the development of brown adipose

426

tissues. On the other hand, the level of IDH1 in brown fat in 12-week-old mice was lower

427

compared to the perinatal periods. This expression pattern may reflect the requirement of

428

IDH1 when brown fat thermogenic demand is highest, such as during early postnatal stages

429

of life. IDH1 plays an important role in maintaining metabolic energy homeostasis by

430

converting isocitrate to α-KG [25-27]. Previous studies focused on the metabolic function of

431

IDH1 and its critical role of mutated IDH1 in cancer [47]; however, little is known about the

432

role of IDH1 in adipocyte differentiation and function. Therefore, we investigated the

433

functional roles and regulatory mechanisms of IDH1 in brown adipocyte metabolism.

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We found that ectopic expression of IDH1 in brown preadipocytes significantly

435

inhibited brown adipocyte differentiation, whereas knockdown of endogenous Idh1

436

expression increased brown adipocyte differentiation. Moreover, the presence of IDH1

437

increased α-KG levels, and the addition of exogenous α-KG suppressed brown adipocyte

438

differentiation at the early stage (days 0–2). These results suggested IDH1 as a potent

439

negative regulator of brown adipocyte differentiation at the early stage of differentiation by

440

modulating α-KG level.

441

Interestingly, the expression of IDH1, a negative regulator of brown adipogenesis,

442

increases during brown adipocyte differentiation indicating that IDH1 may play a role in fine19

Journal Pre-proof tuning brown adipocyte differentiation and function. In this regard, a previous study

444

demonstrated that Twist-1, which is induced upon PPARδ activation during brown

445

adipogenesis, inhibits Pgc1α expression in a negative-feedback regulatory loop to tightly

446

modulate PGC1α-controlled brown fat metabolism [48]. In addition, orphan nuclear receptor

447

NR4A, whose expression is upregulated during 3T3-L1 adipogenesis, serves as a potent

448

negative regulator of differentiation by inhibiting mitotic clonal expansion of 3T3-L1

449

preadipocytes [49]. In this study, multiple lines of in vitro and in vivo data support the idea

450

that the IDH1–α-KG axis functions as a negative-feedback regulator to fine-tune brown fat

451

differentiation and function.

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Since IDH1 catalyzes the transformation of isocitrate to α-KG, we focused on α-KG in

453

order to identify the mechanism by which IDH1 regulates brown adipogenic gene expression.

454

α-KG is an important cofactor of ten-eleven translocation dioxygenase, which regulates

455

histone methylation [50], and induces histone demethylation through activation of lysine

456

demethylases (KDMs) [40, 51]. Accordingly, decreased α-KG levels via reduced IDH1

457

activity increases histone-methylation levels [28]. Among various histone proteins and their

458

residues, H3K4 and H3K36 are critical for gene-promoter activation, with methylation of

459

H3K4 and H3K36 activating the transcription of certain gene subsets [21]. Moreover,

460

previous reports indicate that methylation of H3K4 and H3K36 regulates adipogenic gene

461

activity and expression [52, 53]. In particular, promoter methylation at H3K4 by mixed-

462

lineage leukemia 3 (also known as lysine N-methyltransferase 2C) activates adipocyte

463

differentiation [54]. Furthermore, demethylation of H3K4 by lysine-specific histone

464

demethylase 1A differentially controls brown adipose-specific versus white adipose-specific

465

gene expression [37]. Based on these reports, we propose that IDH1-dependent α-KG

466

regulates brown adipocyte-related gene expression through modulation of the histone-

467

methylation pattern in their respective promoters. Interestingly, in the present study, we

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Journal Pre-proof 468

observed elevated H3K4me3 and H3K36me3 levels during the early stage of brown

469

adipocyte differentiation, and IDH1-mediated α-KG levels suppressed H3K4me3 levels in the

470

promoters of brown adipogenic genes. In agreement with in vitro data, in vivo administration

471

of α-KG reduced the expression of brown adipogenic genes in brown adipose tissues, which

472

was accompanied by decreased H3K4me3 levels at brown adipogenic gene promoters. These

473

results suggested that the IDH1–α-KG axis is important for brown adipocyte differentiation

474

and function dependent upon brown adipogenic gene expression. Brown adipocytes play an essential role in thermogenesis via β-adrenergic-dependent

476

and -independent pathways under cold conditions [45]. Recently, it has been demonstrated

477

that succinate, a TCA intermediate, is required for brown fat thermogenesis upon cold

478

challenge [46]. In this study, we observed an increase in the levels of IDH1 and α-KG, along

479

with those of UCP1 and PGC1α in brown adipose tissues by cold exposure. In addition, cold

480

exposure was also accompanied by changes in histone methylation, especially H3K4me3

481

levels, in brown adipose tissues. Unexpectedly, administration of CL-316,243, a β-adrenergic

482

agonist, did not change the level of IDH1 (Fig. S5). It is possible that cold exposure could

483

regulate the levels of IDH1 and α-KG by various factors other than β-adrenergic signaling.

484

These results are consistent with the notion that the IDH1–α-KG axis may play a key role in

485

thermogenesis of brown adipose tissues. Nevertheless, further studies are needed to elucidate

486

the effects of IDH1 and α-KG on the regulation of brown fat thermogenesis.

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By contrast, IDH1 reportedly promotes white adipocyte differentiation and lipid

488

synthesis via NADPH production [55]. In agreement with this report, we found that IDH1

489

was upregulated during differentiation of 3T3-L1 white adipocytes (Fig. S6A). Additionally,

490

Idh1 overexpression in 3T3-L1 cells promoted adipocyte differentiation, whereas Idh1

491

knockdown suppressed adipogenesis (Fig. S6B and C). In contrast to brown adipocytes,

492

H3K4me3 occupancy in the Pparg promoter was not altered by Idh1 overexpression and 21

Journal Pre-proof reduced by Idh1 knockdown in 3T3-L1 adipocytes (Fig. S6B and C). Notably, only

494

enrichment of H3K4me3 at brown adipocyte-specific promoters, but not at white adipocyte-

495

specific promoters, was attenuated by Idh1 overexpression or α-KG treatment (Fig. S3B).

496

Furthermore, differentiation of 3T3-L1 adipocytes was increased by treatment with α-KG and

497

accompanied by upregulated expression of adipogenic genes, including Pparg, aP2, and

498

adiponectin (Fig. S7A and B). These data suggest that IDH1 might differentially regulate

499

adipogenesis of brown and white adipocytes by modulating histone methylation in a cell-

500

type-selective manner. However, it remains unclear why IDH1/α-KG plays an opposing role

501

between white and brown adipocyte differentiation; therefore, extensive studies are warranted

502

to elucidate the specific role and mechanism of IDH1 in white and brown fat development

503

using brown adipocyte-specific Idh1-knockout animal models.

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A recent report indicated that AMPK-mediated α-KG promotes brown adipogenesis in

505

MSCs [44], demonstrating that ablation of AMPKα1 reduces α-KG levels and impairs

506

development of brown adipose tissues. Unexpectedly, our study revealed that the IDH1–α-

507

KG axis inhibits brown adipocyte differentiation; therefore, we suggest that α-KG might be

508

required for committing progenitor cells to the brown adipocyte lineage while possibly

509

inhibiting the differentiation of brown preadipocytes, which are already adipocyte

510

determinant. Generally, brown adipocyte differentiation comprises commitment to the brown

511

adipocyte lineage by MSCs and differentiation from preadipocytes to mature adipocytes [8,

512

9]. MSCs can differentiate into various cell types, including adipocytes, myocytes,

513

chondrocytes, and osteoblasts [7], with adipocytes requiring regulators, such as PRDM16,

514

BMP7, and EBF2, for commitment to the brown adipocyte lineage [10-12]. Moreover, α-KG

515

maintains the pluripotency of embryonic stem cells (ESCs) and inhibits the differentiation of

516

naive-state ESCs via epigenetic regulation [56], whereas α-KG also promotes early stage

517

neuroectodermal differentiation of pluripotent stem cells [57]. Therefore, α-KG is potentially

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Journal Pre-proof involved in self-renewal and/or differentiation depending on the stage of cellular maturity,

519

suggesting that α-KG might control the brown adipogenic process in a stage-selective manner.

520

A previous study suggested stage-specific functions for pre-B-cell leukemia transcription

521

factor-1 in the control of adipocyte development by promoting the adipocyte-progenitor step

522

of ESCs while preventing adipocyte differentiation [58]. Additionally, dexamethasone is an

523

essential inducer of the early stage of adipocyte differentiation but displays anti-adipogenic

524

effects at terminal stages of adipogenesis, indicating time-dependent roles for hormones or

525

metabolites in adipogenesis [59]. Therefore, it is possible that α-KG in MSCs can drive

526

differentiation into the brown adipocyte lineage through DNA demethylation in the Prdm16

527

promoter, whereas α-KG in brown preadipocytes suppresses adipogenesis by inhibiting

528

adipogenic gene expression through histone modification.

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AMPK suppresses the differentiation of 3T3-L1 white adipocytes by modulating the

530

Akt–mammalian target of rapamycin, WNT/β-catenin, and extracellular signal-regulated

531

kinase pathways [60, 61]; however, little is known about the role of AMPK in brown

532

adipogenesis. A previous report showed that daily treatment with the AMPK activator 5-

533

aminoimidazole-4-carboxamide ribonucleotide (AICAR) is toxic to brown adipocytes by

534

decreasing intracellular pH [62]. To clarify the roles of AMPK in brown adipogenesis, we

535

examined the adipogenic capacity of the AMPK activator metformin following administration

536

to brown preadipocytes during differentiation. We found that metformin increased

537

intracellular α-KG levels and inhibited brown adipocyte differentiation, suggesting that

538

AMPK might inhibit differentiation of brown preadipocytes through elevated α-KG

539

production. Additionally, metformin also plays an AMPK-independent role in inhibiting

540

respiratory chain complex I [63]; therefore, it is possible that metformin suppresses brown

541

adipogenesis by regulating the mitochondrial respiratory chain.

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Journal Pre-proof Mitochondrial respiration and lipogenesis are important metabolic pathways not only to

543

adipocyte function but also for cancer-cell survival [35]. PPARγ is a master regulator of

544

adipocyte differentiation and also controls glucose and lipid metabolism in cancer cells [64].

545

Additionally, PPARγ agonists can either promote or suppress tumorigenesis, tumor

546

progression, and metastasis in various tumor types [65]. Moreover, PGC1α plays an

547

important role in mitochondrial biogenesis and oxidative phosphorylation in cancer cells to

548

promote rapid proliferation and metastasis [66]. Therefore, further investigation is needed to

549

determine the roles of IDH1-mediated PPARγ–PGC1α regulatory pathways for cancer

550

metabolism. In the previous decade, gain-of-function mutations in IDH1/2 in cancers, and

551

especially glioma, were extensively studied [28]. Mutation of IDH1 at R132 results in the

552

production of 2-hydroxyglutaric acid (2-HG) rather than α-KG, which rewires cellular

553

metabolism to support tumorigenesis and cancer progression [67]. Furthermore, 2-HG

554

produced by mutant IDH1/2 inhibits the H3K9 demethylase KDM4C, thereby preventing the

555

differentiation of adipocytes, as well as astrocytes [28]. Therefore, IDH1, as well as mutant

556

IDH1/2, might be involved in brown adipocyte differentiation via α-KG- or 2-HG-mediated

557

histone modification, and this pathway might represent a therapeutic target for metabolic

558

diseases, including obesity and cancer.

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Taken together, we demonstrated that the IDH1–α-KG axis inhibits brown adipocyte

560

differentiation through histone modification-mediated transcriptional control of brown

561

adipogenic genes. Our results identified the molecular pathway associated with stage-

562

dependent IDH1-mediated α-KG regulation of brown adipocyte differentiation. Moreover, the

563

data suggest that IDH1–α-KG might inversely modulate adipogenesis of brown and white

564

adipocytes. Further studies are required to understand the underlying mechanisms involved in

565

the cell-type-selective opposite effects of IDH1 on adipogenesis. These findings suggest that

24

Journal Pre-proof IDH1 might represent a therapeutic target for treating obesity and metabolic diseases by

567

controlling adipocyte differentiation.

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Journal Pre-proof 5. Conclusions

569

In summary, we demonstrated that IDH1-mediated α-KG negatively regulated brown

570

adipocyte differentiation. Mechanistically, the IDH1–α-KG axis reduced H3K4me3 levels in

571

the promoters of brown adipogenic genes, which was accompanied by their decreased

572

expression. Moreover, administration of α-KG decreased adipogenic gene expression in

573

brown adipose tissues of mice, which were concomitant with alterations in histone

574

methylation. These findings suggest IDH1 is a key regulator of brown fat differentiation and

575

function. Taken together, the IDH1–α-KG axis might represent a potential therapeutic target

576

for ameliorating metabolic syndrome.

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Journal Pre-proof 577

Author contributions

578

HSK, JHL, K-JO, EWL, SCL, K-HB, and WKK conceived the study design, and data

579

interpretation. HSK, JHL, K-JO, EWL, K-YP, and JMS performed experiments. HSK, JHL,

580

JMS, K-HB, and WKK contributed to writing the manuscript. K-JO, EWL, BSH, J-KM, S-

581

WC, and SCL critically reviewed the study and gave the necessary suggestions. All authors

582

read and approved the final version of the manuscript.

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Acknowledgments

585

We thank Professor Jae Bum Kim at the Seoul National University for providing the

586

C3H10T1/2 cells. Additionally, we thank Dr. Hee Jun Cho at the Korea Research Institute of

587

Bioscience and Biotechnology for sharing the AMPK activator metformin. We also thank

588

Min Wook Kim for proofreading the manuscript. This work was supported by grants from the

589

KRIBB and the Research Program (grants 2017M3A9C4065954, 2015M3A9D7029882,

590

2017R1E1A1A01074745, and 2016R1C1B2013430) through the National Research Foundation

591

of Korea.

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Conflict of interest

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The authors have no conflicts of interest and declare no competing financial interests.

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Journal Pre-proof Figure Legends

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Fig. 1. IDH1 is upregulated during brown fat differentiation and development. (A)

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Protein levels of IDH1 and IDH2 during adipogenesis of immortalized brown preadipocytes

752

and primary brown preadipocytes. Numbers below the immunoblots indicate band intensity

753

(normalized to HSP90) quantified by using ImageJ software. (B) mRNA levels of Idh1 and

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brown adipogenic genes during differentiation of immortalized brown preadipocytes (n = 3).

755

Data represent the mean ± SD. Statistical analysis was performed using one-way ANOVA. *p

756

< 0.05 and *p < 0.005 vs. brown preadipocytes (Day 0). (C) Time course of IDH1 and UCP1

757

protein expression during brown fat development from embryonic day 18 (E18), postnatal

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days 1–6 (P1–6), and 12-week-old (12w) mice. IDH1 and UCP1 levels were measured by

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western blot, with HSP90 used as a loading control. (D) mRNA levels of Idh1 and brown

760

adipogenic genes during various time course of brown fat development as determined by

761

qRT-PCR (n =2). Data represent the mean ± SD. Statistical analysis was performed using

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one-way ANOVA. *p < 0.05 vs. E18. All experiments were repeated independently at least

763

three times and representative results are shown.

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Fig. 2. Ectopic IDH1 expression inhibits differentiation of brown preadipocytes. (A) The

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efficiency of retroviral transduction and monitoring of IDH1 levels by fluorescence

767

microscopy. Retrovirus-transduced cells were selected by GFP-positive cell sorting. (B)

768

IDH1 and UCP1 levels were measured by western blot, with HSP90 used as a loading control.

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(C) Oil-Red-O staining of lipid droplets after differentiation of Idh1-overexpressing brown

770

adipocytes. (D) For quantitation of lipid content, Oil-red-O stain was eluted from cells with

771

isopropanol, and measurements were taken at 490 nm (n = 3). Data represent the mean ± SD.

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(E) mRNA levels of brown adipogenic genes in mature brown adipocytes as determined by

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qRT-PCR and normalized to TBP (n = 3). Data represent the mean ± SD. Statistical analysis 33

Journal Pre-proof 774

was performed using a two-tailed Student's t test. *p < 0.05, **p < 0.005, and ***p < 0.0005

775

vs. Vector. The data shown are representative results of at least three independent

776

experiments.

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Fig. 3. IDH1 suppression inhibits brown adipocyte differentiation. (A) RFP levels were

779

monitored by fluorescence microscopy. Brown preadipocytes were transduced with retroviral

780

vectors expressing pSIREN-RetroQ-DsRed. (B) Efficiency of Idh1 knockdown in IDH1-

781

suppressed brown preadipocytes according to qRT-PCR (n = 2). Data represent the mean ±

782

SD. Statistical analysis was performed using one-way ANOVA. (C) IDH1 levels according to

783

western blot. Relative amount of IDH1 protein was calculated using ImageJ software and

784

normalized to HSP90. (D) Oil-Red-O staining of lipid droplets after differentiation of IDH1-

785

suppressed brown adipocytes and quantification of lipid accumulation (n = 3). Data represent

786

the mean ± SD. Statistical analysis was performed using a two-tailed Student's t test. (E)

787

IDH1 and UCP1 levels according to western blot. Numbers below the immunoblots indicate

788

band intensity (normalized to HSP90) quantified by using ImageJ software. n.d., not detected.

789

(F) mRNA levels of brown adipogenic genes according to qRT-PCR and normalized to TBP

790

(n = 3). Data represent the mean ± SD. Statistical analysis was performed using a two-tailed

791

Student's t test. *p < 0.05 and **p < 0.005 vs. SCR. The data shown are representative of

792

three independent experiments.

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Fig. 4. α-KG negatively regulates brown adipogenesis at the early stage of

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differentiation. (A) α-KG levels following Idh1 overexpression or knockdown monitored

796

during brown adipocyte differentiation (n = 3). Data represent the mean ± SD. Statistical

797

analysis was performed using a two-tailed Student's t test. *p < 0.05, **p < 0.005, and ***p <

798

0.0005 vs. control (Vector or SCR). (B) Cell-permeable α-KG (dm-α-KG, 1 mM) was 34

Journal Pre-proof administered to brown preadipocytes, and α-KG levels were measured according to a time

800

course (n = 3). Data represent the mean ± SD. Statistical analysis was performed using one-

801

way ANOVA. n.s., not significant. (C) Oil-Red-O staining of lipid droplets after

802

differentiation of brown preadipocytes treated with dm-α-KG for 2 days (days 0–2, 2–4, and

803

4–6) and for 6 days (days 0–6) every 24 h. (D) mRNA levels of brown adipogenic genes in

804

dm-α-KG-treated mature brown adipocytes according to qRT-PCR and normalized to TBP (n

805

= 3). Statistical analysis was performed using one-way ANOVA. *p < 0.05, **p < 0.005, and

806

***p < 0.0005 vs. Vehicle. (E) Protein levels of UCP1, PGC1α, PRDM16, and PPARγ were

807

measured by western blot. Numbers below the immunoblots indicate band intensity

808

(normalized to HSP90) quantified by using ImageJ software. n.d., not detected. Similar

809

results were obtained in at least three independent experiments.

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Fig. 5. The effects of α-KG and Idh1 overexpression or knockdown on brown adipocyte

812

differentiation. Brown preadipocytes were treated with dm-α-KG (1 mM) during the early

813

stage of differentiation (days 0–2) or daily (days 0–6). (A, C) Oil-Red-O staining of lipid

814

droplets after differentiation of α-KG-treated Idh1-modulated brown adipocytes. (B, D)

815

mRNA levels of brown adipogenic genes according to qRT-PCR (n = 3). Data represent the

816

mean ± SD. Statistical analysis was performed using two-way ANOVA. *p < 0.05, **p <

817

0.005, and ***p < 0.0005 vs. control (Vehicle-treated control cells). ###p < 0.0005. All

818

experiments were repeated independently at least three times and representative results are

819

shown.

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Fig. 6. α-KG controls epigenetic modification via histone methylation during brown

822

adipocyte differentiation. (A) Levels of H3K4me3, H3K9me3, H3K27me3, and H3K36me3

823

measured during brown adipocyte differentiation for 12 h, 24 h, and 48 h. Numbers below the 35

Journal Pre-proof immunoblots indicate band intensity (normalized to Histone H3) quantified by using ImageJ

825

software. (B) mRNA levels of brown adipogenic genes were monitored during brown

826

adipocyte differentiation in the presence of dm-α-KG (n = 3). Data represent the mean ± SD.

827

Statistical analysis was performed using two-way ANOVA. **p < 0.005 and ***p < 0.0005

828

vs. control (Vehicle-treated cells on day 0). #p < 0.05 and ##p < 0.005. n.s., not significant.

829

(C, D) Levels of histone H3 lysine methylation on the second day were examined during

830

differentiation of dm-α-KG-treated Idh1-overexpressing or -knockdown brown adipocytes. (E,

831

F) Enrichment of H3K4me3 and H3K36me3 levels in the promoters of adipogenic genes on

832

the second day of brown adipocyte differentiation in the presence of dm-α-KG (n = 3). Data

833

represent the mean ± SD. Statistical analysis was performed using two-way ANOVA. **p <

834

0.005 and ***p < 0.0005 vs. control (Vehicle-treated control cells). #p < 0.05 and ###p <

835

0.0005. The data shown are representative results of at least three independent experiments.

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Fig. 7. α-KG administration decreases adipogenic gene expression in brown adipose

838

tissues of mice. (A) 8-week-old male C57BL/6 mice were injected intraperitoneally with

839

saline and 0.5, 1, and 2 g/kg of dm-α-KG (n = 4). The mice were sacrificed 3 hours post

840

injections to dissect brown adipose tissues. α-KG levels were measured in brown adipose

841

tissues of mice injected with saline or α-KG. Data represent the mean ± SD. Statistical

842

analysis was performed using one-way ANOVA. *p < 0.05 vs. control (Saline-administered

843

mice) (B) mRNA levels of adipogenic genes in brown adipose tissues after 3 days of daily

844

intraperitoneal injection with 1 g/kg α-KG into C57BL/6 mice (n = 5). Data represent the

845

mean ± SD. Statistical analysis was performed using a two-tailed Student's t test. **p < 0.005

846

and ***p < 0.0005 vs. control (Saline-administered mice). n.s., not significant. (C) Protein

847

levels of UCP1, PGC1α, PRDM16, and PPARγ were monitored by western blot in brown

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36

Journal Pre-proof 848

adipose tissues of mice injected with saline or α-KG for 3 days. (D) Levels of H3K4me3,

849

H3K9me3, H3K27me3, and H3K36me3 were measured in brown adipose tissues of saline or

850

α-KG administered mice. (E) Enrichment of H3K4me3 and H3K36me3 levels at the

851

promoters of brown adipocyte-specific genes in brown adipose tissues of mice injected with

852

saline or α-KG for 3 days. Data represent the mean ± SD. Statistical analysis was performed

853

using a two-tailed Student's t test. *p < 0.05 vs. control (Saline-administered mice). n.s., not

854

significant.

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Fig. 8. The AMPK activator metformin reduces brown adipogenesis. (A) Metformin (1

857

mM) was administered to brown preadipocytes, and AMPK and phosphorylated-AMPK

858

levels were measured by western blot. Relative amount of phosphorylated-AMPK was

859

quantified using ImageJ software and normalized to total AMPK. (B–D) The effects of

860

metformin on brown adipogenesis. Brown preadipocytes were treated with 1 mM metformin

861

every 48 h for 6 days. (B) α-KG levels were monitored in metformin-treated brown

862

adipocytes (n = 3). Data represent the mean ± SD. Statistical analysis was performed using

863

two-way ANOVA. **p < 0.005 and ***p < 0.0005 vs. control (Vehicle-treated cells on day 0).

864

##p < 0.005 and ###p < 0.0005. (C) Oil-Red-O staining of lipid droplets and quantification of

865

lipid accumulation in mature brown adipocytes in the presence or absence of metformin (n =

866

3). Data represent the mean ± SD. (D) mRNA levels of brown adipogenic genes in mature

867

adipocytes in the presence of metformin (n = 3). Data represent the mean ± SD. Statistical

868

analysis was performed using two-tailed Student's t test. *p < 0.05 vs. Vehicle. (E) Protein

869

levels in metformin-treated mature brown adipocytes. (F) Oil-Red-O staining of lipid droplets

870

and quantification of lipid accumulation in differentiated C3H10T1/2 MSCs after brown

871

adipogenesis (n = 3). Data represent the mean ± SD. Statistical analysis was performed using

872

a two-tailed Student's t test. n.s., not significant. (G) mRNA levels of brown adipogenic genes

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37

Journal Pre-proof 873

during brown adipogenesis of C3H10T1/2 MSCs treated with or without 1mM dm-α-KG (n =

874

3). Data represent the mean ± SD. Statistical analysis was performed using a two-way

875

ANOVA. *p < 0.05, *p < 0.005, and ***p < 0.0005 vs. control (Vehicle-treated cells on day

876

0). #p < 0.05 and ##p < 0.005. The data shown are representative of three independent

877

experiments.

878

Fig. 9. IDH1 and α-KG in brown adipose tissues is regulated by cold-induced

880

thermogenesis. (A) mRNA levels of Idh1 and thermogenic genes in brown adipose tissues

881

from 11-week-old C57BL/6 mice housed at room temperature (RT) or exposed to cold (5°C)

882

for the indicated time points (n = 3). Data represent the mean ± SD. Statistical analysis was

883

performed using one-way ANOVA. *p < 0.05 and **p < 0.005 vs. control (RT). (B) Protein

884

levels of IDH1, UCP1, and PGC1α from brown adipose tissues of mice exposed to cold (5°C)

885

for the indicated durations. (C) α-KG levels were measured in brown adipose tissues of mice

886

upon cold exposure. Data represent the mean ± SD. Statistical analysis was performed using

887

one-way ANOVA. **p < 0.005 vs. control (RT). (D) Levels of histone methylation, such as

888

H3K4me3, H3K9me3, H3K27me3, and H3K36me3, were analyzed in in brown adipose

889

tissues of mice during cold exposure.

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Journal Pre-proof Author contributions

892

HSK, JHL, K-JO, EWL, SCL, K-HB, and WKK conceived the study design, and data

893

interpretation. HSK, JHL, K-JO, EWL, K-YP, and JMS performed experiments. HSK, JHL,

894

JMS, K-HB, and WKK contributed to writing the manuscript. K-JO, EWL, BSH, J-KM, S-

895

WC, and SCL critically reviewed the study and gave the necessary suggestions. All authors

896

read and approved the final version of the manuscript.

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