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The regulation of glucose and lipid homeostasis via PLTP as a mediator of BAT –liver communication

2020; Springer Nature; Volume: 21; Issue: 9 Linguagem: Inglês

10.15252/embr.201949828

1469-3178

Carlos H. Sponton, Takashi Hosono, Junki Taura, Mark P. Jedrychowski, Takeshi Yoneshiro, Qiang Wang, Makoto Takahashi, Yumi Matsui, Kenji Ikeda, Yasuo Oguri, Kazuki Tajima, Kosaku Shinoda, Rachana Pradhan, Yong Chen, Zachary D. Brown, Lindsay S. Roberts, Carl C. Ward, Hiroki Taoka, Yoko Yokoyama, Mitsuhiro Watanabe, Hiroshi Karasawa, Daniel K. Nomura, Shingo Kajimura,

Lipid metabolism and biosynthesis

Article16 July 2020free access Source DataTransparent process The regulation of glucose and lipid homeostasis via PLTP as a mediator of BAT–liver communication Carlos H Sponton Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Takashi Hosono Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Junki Taura End-Organ Disease Laboratories, Daiichi-Sankyo Co., Ltd., Tokyo, Japan Search for more papers by this author Mark P Jedrychowski Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Takeshi Yoneshiro Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Qiang Wang Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Makoto Takahashi Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi-Sankyo Co., Ltd., Tokyo, Japan Search for more papers by this author Yumi Matsui Protein Production Research Group, Biological Research Department, Daiichi-Sankyo RD Novare Co., Ltd., Tokyo, Japan Search for more papers by this author Kenji Ikeda Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Yasuo Oguri Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Kazuki Tajima Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Kosaku Shinoda Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Rachana N Pradhan Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Yong Chen Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Zachary Brown Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Lindsay S Roberts Departments of Chemistry, Molecular and Cell Biology, and Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, CA, USA Search for more papers by this author Carl C Ward Departments of Chemistry, Molecular and Cell Biology, and Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, CA, USA Search for more papers by this author Hiroki Taoka Graduate School of Media and Governance, Keio University, Kanagawa, Japan Search for more papers by this author Yoko Yokoyama Graduate School of Media and Governance, Keio University, Kanagawa, Japan Search for more papers by this author Mitsuhiro Watanabe Graduate School of Media and Governance, Keio University, Kanagawa, Japan Search for more papers by this author Hiroshi Karasawa End-Organ Disease Laboratories, Daiichi-Sankyo Co., Ltd., Tokyo, Japan Search for more papers by this author Daniel K Nomura Departments of Chemistry, Molecular and Cell Biology, and Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, CA, USA Search for more papers by this author Shingo Kajimura Corresponding Author [email protected] orcid.org/0000-0003-0672-5910 Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Carlos H Sponton Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Takashi Hosono Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Junki Taura End-Organ Disease Laboratories, Daiichi-Sankyo Co., Ltd., Tokyo, Japan Search for more papers by this author Mark P Jedrychowski Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Takeshi Yoneshiro Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Qiang Wang Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Makoto Takahashi Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi-Sankyo Co., Ltd., Tokyo, Japan Search for more papers by this author Yumi Matsui Protein Production Research Group, Biological Research Department, Daiichi-Sankyo RD Novare Co., Ltd., Tokyo, Japan Search for more papers by this author Kenji Ikeda Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Yasuo Oguri Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Kazuki Tajima Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Kosaku Shinoda Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Rachana N Pradhan Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Yong Chen Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Zachary Brown Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Lindsay S Roberts Departments of Chemistry, Molecular and Cell Biology, and Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, CA, USA Search for more papers by this author Carl C Ward Departments of Chemistry, Molecular and Cell Biology, and Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, CA, USA Search for more papers by this author Hiroki Taoka Graduate School of Media and Governance, Keio University, Kanagawa, Japan Search for more papers by this author Yoko Yokoyama Graduate School of Media and Governance, Keio University, Kanagawa, Japan Search for more papers by this author Mitsuhiro Watanabe Graduate School of Media and Governance, Keio University, Kanagawa, Japan Search for more papers by this author Hiroshi Karasawa End-Organ Disease Laboratories, Daiichi-Sankyo Co., Ltd., Tokyo, Japan Search for more papers by this author Daniel K Nomura Departments of Chemistry, Molecular and Cell Biology, and Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, CA, USA Search for more papers by this author Shingo Kajimura Corresponding Author [email protected] orcid.org/0000-0003-0672-5910 Diabetes Center, University of California, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA Search for more papers by this author Author Information Carlos H Sponton1,2,3,†, Takashi Hosono1,2,3,†, Junki Taura4, Mark P Jedrychowski5, Takeshi Yoneshiro1,2,3, Qiang Wang1,2,3, Makoto Takahashi6, Yumi Matsui7, Kenji Ikeda1,2,3,†, Yasuo Oguri1,2,3, Kazuki Tajima1,2,3, Kosaku Shinoda1,2,3,†, Rachana N Pradhan1,2,3, Yong Chen1,2,3,†, Zachary Brown1,2,3, Lindsay S Roberts8, Carl C Ward8, Hiroki Taoka9, Yoko Yokoyama9, Mitsuhiro Watanabe9, Hiroshi Karasawa4, Daniel K Nomura8 and Shingo Kajimura *,1,2,3,† 1Diabetes Center, University of California, San Francisco, CA, USA 2Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, San Francisco, CA, USA 3Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA 4End-Organ Disease Laboratories, Daiichi-Sankyo Co., Ltd., Tokyo, Japan 5Department of Cell Biology, Harvard Medical School, Boston, MA, USA 6Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi-Sankyo Co., Ltd., Tokyo, Japan 7Protein Production Research Group, Biological Research Department, Daiichi-Sankyo RD Novare Co., Ltd., Tokyo, Japan 8Departments of Chemistry, Molecular and Cell Biology, and Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, CA, USA 9Graduate School of Media and Governance, Keio University, Kanagawa, Japan †Present address: Obesity and Comorbidities Research Center, Biology Institute, University of Campinas, Campinas, Brazil †Present address: Department of Chemistry and Life Science, Nihon University College of Bioresource Sciences, Fujisawa, Japan †Present address: Department of Molecular Endocrinology and Metabolism, Tokyo Medical and Dental University, Tokyo, Japan †Present address: Albert Einstein College of Medicine, Bronx, NY, USA †Present address: Department of Endocrinology at Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China †Present address: Harvard Medical School, Beth Israel Deaconess Medical Center, Division of Endocrinology, Diabetes & Metabolism, Boston, MA, USA *Corresponding author. Tel: +617 735 3289; E-mails: [email protected], [email protected] EMBO Rep (2020)21:e49828https://doi.org/10.15252/embr.201949828 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract While brown adipose tissue (BAT) is well-recognized for its ability to dissipate energy in the form of heat, recent studies suggest multifaced roles of BAT in the regulation of glucose and lipid homeostasis beyond stimulating thermogenesis. One of the functions involves interorgan communication with metabolic organs, such as the liver, through BAT-derived secretory factors, a.k.a., batokine. However, the identity and the roles of such mediators remain insufficiently understood. Here, we employed proteomics and transcriptomics in human thermogenic adipocytes and identified previously unappreciated batokines, including phospholipid transfer protein (PLTP). We found that increased circulating levels of PLTP, via systemic or BAT-specific overexpression, significantly improve glucose tolerance and insulin sensitivity, increased energy expenditure, and decrease the circulating levels of cholesterol, phospholipids, and sphingolipids. Such changes were accompanied by increased bile acids in the circulation, which in turn enhances glucose uptake and thermogenesis in BAT. Our data suggest that PLTP is a batokine that contributes to the regulation of systemic glucose and lipid homeostasis as a mediator of BAT-liver interorgan communication. Synopsis Phospholipid transfer protein (PLTP) released from BAT controls energy expenditure and systemic glucose/lipid homeostasis. The metabolic benefit of PLTP is mediated through a BAT-liver interorgan communication that involves the regulation of lipoproteins and bile acids. BAT secretes PLTP as a "batokine". PLTP increases the reverse transport of cholesterol to the liver, thereby stimulating the release of primary bile acids that activate BAT thermogenesis. Increased PLTP mitigates diet-induced obesity, glucose intolerance, and dyslipidemia in mice. Introduction Historically, brown adipose tissue (BAT) was viewed merely as a heat generation through the action of uncoupling protein 1 (UCP1), a mitochondrial protein that uncouples proton gradient across the mitochondrial membrane from ATP synthesis (Kajimura & Saito, 2014). However, emerging evidence suggests that the role of BAT is far more complex than stimulating thermogenesis. For instance, BAT serves as a significant metabolic sink for glucose, fatty acids, and branched-chain amino acid (BCAA), and such function significantly contributes to the regulation of systemic glucose and lipid homeostasis (Bartelt et al, 2011; Kajimura et al, 2015; Chondronikola et al, 2016; Yoneshiro et al, 2019). Notably, BAT maintains active glucose uptake even in UCP1 null mice (Ikeda et al, 2017; Olsen et al, 2017), indicating that the metabolic-sink function of BAT involves multiple mechanisms other than UCP1-mediated thermogenesis. A growing number of studies suggest that, besides its well-known thermogenic role, BAT functions as a secretory organ (Villarroya et al, 2017). For example, BAT secretes vascular endothelial growth factor A (VEGFA) and nerve growth factor (NGF) that act as paracrine/autocrine factors to enhance its thermogenic activity by remodeling of the vascular bed and sympathetic innervation (Nisoli et al, 1996; Mahdaviani et al, 2016). Furthermore, BAT secretes S100b that stimulates neurite outgrowth from sympathetic neurons (Zeng et al, 2019). Besides these paracrine/autocrine factors, BAT secretes endocrine molecules: Transplantation of BAT from healthy donor mice to obese or diabetic mice leads to an improvement in insulin sensitivity and glucose homeostasis, and such action is mediated in part by the secretion of IL6 from BAT (Stanford et al, 2013; Liu et al, 2015). BAT also secretes neuregulin 4 (Nrg4) that acts on the liver to inhibit hepatic lipogenic signaling (Wang et al, 2014), whereas BAT secretes myostatin that negatively controls skeletal muscle function (Kong et al, 2018). In addition to these polypeptides, BAT releases exosomal microRNAs and lipids, such as 12,13-diHOME, that control systemic glucose and lipid homeostasis (Lynes et al, 2017; Thomou et al, 2017). It is conceivable that multiple BAT-derived factors, a.k.a. batokine, including the above-mentioned factors and uncharacterized molecules, act in concert or independently to communicate with metabolic organs to regulate systemic glucose and lipid homeostasis. Thus, the comprehension of human batokines would be significant because the reconstitution of such factors may be a therapeutically tractable strategy to ameliorate glucose intolerance, insulin resistance, or dyslipidemia. To search for previously unappreciated batokines, we employed proteomics and transcriptomics in human differentiated adipocytes. To embark this project, we took advantage of clonally derived thermogenic adipocytes that we previously isolated from adult human BAT (Shinoda et al, 2015). We subsequently characterized the metabolic function of the identified human batokines in obese mouse models. Results Identification of human batokines by proteomics and transcriptomics We performed proteomics analyses of culture supernatants of clonally derived human thermogenic adipocytes (beige-like adipocytes) isolated from the supraclavicular BAT region and white adipocytes from subcutaneous white adipose tissue (WAT) (Shinoda et al, 2015) (Fig 1A). Subsequently, we combined the proteomics data to our RNA-sequencing database to validate the results. In the analyses, we chose the candidates that were highly expressed in beige adipocytes relative to white adipocytes and contained a secretory signal peptide, while lacking the transmembrane domains. The bioinformatic analysis identified 16 highly secreted candidates (fold change ≥ 5) from human beige adipocytes (Fig 1B). The candidates included several previously unappreciated secreted factors, such as the phospholipid transfer protein (PLTP), EGF-like domain multiple 7 (EGFL7), platelet-derived growth factor C (PDGFC), TIMP metallopeptidase inhibitor 4 (TIMP4), cysteine-rich EGF-like domains 1 (CRELD1), platelet-derived growth factor receptor like (PDGFRL), C1q and tumor necrosis factor-related protein 1 (C1QTNF1), secreted protein acidic and cysteine-rich-like protein 1 (SPARCL1), ephrin A5 (EFNA5), semaphorin 3c (SEMA3C), semaphorin 3f (SEMA3F), semaphorin 4b (SEMA4B), vascular growth factor (VGF), out at first homolog (OAF), fibrinogen-like 1 (FGL1) and the non-annotated BC028528. The majority of the candidates (87.5%, 14/16) were highly expressed in differentiated brown adipocytes relative to stromal cells (Table EV1). Figure 1. Identification of human batokines by proteomics and transcriptomics A. Schematic illustration of the experiment. Clonal human brown and white adipocytes were subjected to RNA sequencing and mass spectrometry. B. Bioinformatic analysis defined 16 secreted candidates (fold change ≥ 5) from brown compared to white adipocytes. C. Schematic illustration of the experiment. C57Bl/6 mice (11 weeks old) received adenovirus (2 × 109 PFU/mouse) for each candidate via tail-vein. GFP adenovirus was used as a control. Seven days after adenovirus infection, mice were euthanized and inguinal (iWAT) and epididymal (eWAT) white fat were weighed. n = 5 for all the candidates and control. D. Changes (Δg) in inguinal WAT (iWAT) mass of mice in (C). n = 5. *P < 0.05, **P < 0.01 relative to GFP by Student's t-test. N.S., not significant. E. Changes (Δg) in epididymal WAT (eWAT) mass of mice in (C). n = 5. *P < 0.05, **P < 0.01, ***P < 0.001 relative to GFP by Student's t-test. N.S., not significant. F. Expression of Pltp mRNA in indicated tissues. Data are shown by relative Pltp mRNA levels normalized by 18s expression. n = 3 for all tissues (except BAT and WAT n = 5, testis n = 2). *P < 0.05 Pltp expression in BAT vs other tissues (except testis and lung) by Student's t-test. G. Plasma PLTP activity from control (Ppargflox/flox) and BAT-less mice (PpargUCP1-KO, Ucp1-Cre × Ppargflox/flox). Following 6-h fasting, plasma was obtained from PpargUCP1-KO mice and littermate control. n = 3 for both groups. *P < 0.05 relative to control mice by Student's t-test. H. PLTP protein levels in the culture conditioned medium from primary differentiated inguinal adipocytes of wild-type (WT) and aP2-PRDM16 mice (Svensson et al, 2016). Peptide levels were determined by quantitative proteomics. n = 2 for both groups. I. Transcriptional regulation of the Pltp gene by PRDM16 and PPARγ. ChIP-seq data for PRDM16 and PPARγ were obtained from (Siersbaek et al, 2012; Harms et al, 2015), respectively. Data information: All the data were represented as mean ± SEM. Source data are available online for this figure. Source Data for Figure 1 [embr201949828-sup-0004-SDataFig1.xlsx] Download figure Download PowerPoint To screen the metabolic roles of these candidates in vivo, we next used a gain-of-function approach by injecting adenovirus (2 × 109 PFU/mouse) expressing each candidate via tail-vein to wild-type mice. Adenovirus expressing GFP was used as a control. This method generally results in robust expression of proteins in the liver and secretion to the plasma (Fig EV1A). As a readout of the assay, we measured tissue mass of the inguinal WAT (iWAT) and epididymal WAT (eWAT) of mice that expressed each candidate in the liver at significant levels (Fig 1C). The in vivo screening identified several candidates, including PLTP, TIMP4, and PDGFRL, that significantly reduced adipose tissue weight for both iWAT (Fig 1D) and eWAT (Fig 1E) compared to the GFP control group. Among these candidates, PLTP caught our attention because of its robust effect on adipose tissue mass, and also because single nucleotide polymorphism (SNP) at the PLTP gene locus, which increases PLTP transcripts, are associated with low levels of circulating triglycerides and high levels of HDL in humans (Kathiresan et al, 2009). Recent studies also found that PLTP is secreted from human and mouse brown fat (Ali Khan et al, 2018; Deshmukh et al, 2019). Notably, BAT expresses PLTP at a very high level relative to other tissues, including the skeletal muscle, liver, heart, and intestine (Fig 1F), while previous studies also reported PLTP expression in the ovary, thymus, placenta, and lung (Albers et al, 1995; Jiang & Bruce, 1995). Click here to expand this figure. Figure EV1. Expression of batokine candidates and regulation of PLTP expression A. Hepatic expression of batokine candidates in mice that received adenovirus expressing indicated genes. n = 5 for all groups. **P < 0.01, ***P < 0.001 relative to GFP by Student's t-test. B. Tissue mass of PpargUCP1 KO mice and control mice. n = 3 for control, n = 4 for PpargUCP1 KO. ***P < 0.001 relative to control by Student's t-test. N.S., not significant. C. Relative gene expression of thermogenic genes in the interscapular BAT from C57Bl/6 mice housed at room temperature (RT), cold (4°C), or thermoneutrality (30°C) for 2 weeks. 18s was used as an internal control. n = 5 for all groups. ANOVA followed by Tukey's test. *P < 0.05, **P < 0.01, ***P < 0.001. N.S., not significant. D. Relative gene expression of thermogenic genes in the iWAT from C57Bl/6 mice housed at room temperature (RT) exposure to cold (4°C) and thermoneutrality (30°C) for 2 weeks. 18s was used as an internal control. n = 5 for all groups. ANOVA followed by Tukey's test. *P < 0.05, **P < 0.01. N.S., not significant. Data information: All the data were represented as mean ± SEM. Source data are available online for this figure. Download figure Download PowerPoint To evaluate the contribution of BAT to the systemic levels of PLTP, we analyzed plasma PLTP activity in "BAT-less" mice in which PPARγ was deleted selectively by UCP1-Cre (PparγUCP1-KO mice, Ucp1-Cre × Pparγflox/flox), such that they lack multilocular brown adipocytes in the BAT (Yoneshiro et al, 2019). In BAT-less mice, the interscapular BAT mass was significantly lower than that in control mice, whereas no difference was seen in the tissue mass of WAT, liver, and muscle between the groups (Fig EV1B). BAT-less mice showed significantly lower levels of plasma PLTP activity than did littermate control mice (Pparγflox/flox; Fig 1G). In addition, high levels of PLTP protein were secreted from primary beige adipocytes derived from fat-selective PRDM16 transgenic mice when compared to white adipocytes isolated from wild-type control mice (Svensson et al, 2016) (Fig 1H). While a previous study suggests a compensatory increase in beige fat-selective gene expression, such as Ucp1, in the inguinal WAT of PparγUCP1-KO mice (Xiong et al, 2018), the possible contribution to circulating PLTP levels would be negligible because circulating PLTP levels in BAT-less mice was reduced by 58.1%, and thus, BAT is a significant source of circulating PLTP. Although increased PLTP activity in circulation is found in obesity and insulin resistance (Dullaart et al, 1994; Murdoch et al, 2000; Kaser et al, 2001), the regulatory mechanism of PLTP expression in physiology is less known. We found that PLTP expression in BAT and iWAT was not altered by cold exposure (Fig EV1C and D), whereas PLTP is regulated by the PRDM16-PPARγ complex, the master regulator of brown/beige fat development (Seale et al, 2007; Kajimura et al, 2008). By analyzing the ChIP-seq data from published studies (Siersbaek et al, 2012; Harms et al, 2015), we found that both PRDM16 and PPARγ are co-recruited on the regulatory regions of the Pltp gene locus in which high peaks of H3K27ac and H3K4me3 (i.e., active transcription marks) were found (Fig 1I). The data are consistent with our previous transcriptome data that PLTP expression is increased by the treatment with a synthetic PPARγ agonist (rosiglitazone) in adipocytes (Ohno et al, 2012) and also by PPARα (Bouly et al, 2001; Tu & Albers, 2001). PLTP prevents diet-induced body-weight gain by increasing whole-body energy expenditure The role of PLTP in lipoprotein metabolism has been well-appreciated (Tall et al, 1983; Albers et al, 1984; Nishida et al, 1997); however, its role in energy metabolism remains less known. The significant reduction in the WAT mass following increased PLTP expression prompted us to investigate the therapeutic potential of PLTP in obesity and insulin resistance. To explore this, we first developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based quantification method that quantifies circulating PLTP levels (Fig EV2A). We observed higher plasma PLTP levels in obese mice on a high-fat diet (HFD) than those in age-matched mice on a regular chow diet (RD; Fig EV2B). The data were in accordance with previous studies showing a positive association between obesity and plasma PLTP activity (Murdoch et al, 2000; Kaser et al, 2001). Overexpression of PLTP via tail-vain injection of adenovirus successfully increased plasma PLTP concentrations in mice on RD approximately by 9-fold throughout the 10 days examined (GFP: 8.2 ± 0.4 μg/ml, PLTP: 75.2 ± 10.8 μg/ml, P < 0.001; Fig EV2C). On the other hand, tail-vein injection of recombinant PLTP protein (rPLTP, 360 μg per mouse) was not able to sustain increased PLTP in the circulation, which was quickly reduced to a basal level within 4 h after rPLTP injection (Fig EV2D). Accordingly, we used adeno-associated virus vectors (AAV) to sustain high plasma levels of PLTP on diet-induced obese mice because AAV is considered suitable for long-term metabolic studies with minimal side effects (Lisowski et al, 2015). Click here to expand this figure. Figure EV2. Plasma PLTP levels following virally induced or recombinant PLTP administration A. Mouse PLTP coding sequence (https://www.ncbi.nlm.nih.gov/gene (gene ID:18830). Amino acid sequence of peptide FK18 (for mass spectrometry quantification) is highlighted. B. Plasma PLTP concentration of mice on regular chow diet (RD) and high-fat diet (HFD). n = 4 for both groups. ***P < 0.001 by Student's t-test. C. Plasma PLTP concentration of mice that received adenovirus expressing GFP or PLTP. PLTP protein levels were determined every day throughout 10 days. n = 4 for GFP and n = 5 for PLTP. Average bar graph of 10 days is shown in the right graph. ***P < 0.001 by Student's t-test. D. Plasma PL