2822 Diabetes Volume 66, November 2017 Tbx15 Defines a Glycolytic Subpopulation and White Adipocyte Heterogeneity Kevin Y. Lee,1,2,3 Rita Sharma,2,3 Grant Gase,2,3 Siegfried Ussar,1,4,5 Yichao Li,6 Lonnie Welch,6 Darlene E. Berryman,2,3 Andreas Kispert,7 Matthias Bluher,8 and C. Ronald Kahn1 Diabetes 2017;66:2822–2829 | https://doi.org/10.2337/db17-0218 Tbx15 is a member of the T-box gene family of mesoder- adipose tissue (WAT) is associated with insulin resistance and mal developmental genes. We have recently shown that increased risk of metabolic disease, whereas subcutaneous WAT Tbx15 plays a critical role in the formation and metabolic maybeprotectiveagainstthedevelopmentofmetabolic programming of glycolytic myofibers in skeletal muscle. disorders (1). We and others (2,3) have shown that differ- Tbx15 is also differentially expressed among white adi- ences in WAT depots are also marked by differential expres- pose tissue (WAT) in different body depots. In the current sion of developmental genes. study, using three independent methods, we show that Tbx15 is a member of the T-box gene family of develop- even within a single WAT depot, high Tbx15 expression mental genes and has an established role in skeletal forma- is restricted to a subset of preadipocytes and mature fi tion (4). We have recently shown that in skeletal muscle, white adipocytes. Gene expression and metabolic pro l- fi ing demonstrate that the Tbx15Hi preadipocyte and adipo- Tbx15 is highly and speci cally expressed in glycolytic fi fi cyte subpopulations of cells are highly glycolytic, whereas myo bers and regulates the metabolism of these myo bers Tbx15Low preadipocytes and adipocytes in the same de- (5). Furthermore, Tbx15 is differentially expressed among pot are more oxidative and less glycolytic. Likewise, in different WAT depots in both rodents and humans and is humans, expression of TBX15 in subcutaneous and vis- markedly downregulated in obesity (2). Genome-wide asso- OBESITY STUDIES ceral WAT is positively correlated with markers of glyco- ciation studies have shown that a single nucleotide poly- lytic metabolism and inversely correlated with obesity. morphism near the TBX15 gene correlates with adiposity in Furthermore, overexpression of Tbx15 is sufficient to re- patients of both European and African ancestry (6). duce oxidative and increase glycolytic metabolism in cul- In the current study, we show that even within a single tured adipocytes. Thus, Tbx15 differentially regulates WAT depot, the adipocytes and preadipocytes are hetero- oxidative and glycolytic metabolism within subpopula- geneous with high Tbx15 expression in only a subset of tions of white adipocytes and preadipocytes. This leads cells. Metabolic profiling demonstrates that these Tbx15Hi to a functional heterogeneity of cellular metabolism within preadipocytes and adipocytes are highly glycolytic, WAT that has potential impact in the understanding of whereas Tbx15Low preadipocytes and adipocytes are more human metabolic diseases. oxidative. Expression of TBX15 also correlates to markers ofglycolyticmetabolisminhumanfat.Finally,wedem- Over the past decade, it has become clear that adipose tissue onstrate that overexpression of Tbx15 is sufficient to drive and its contribution to metabolic syndrome are more complex an increase in glycolytic metabolism in cultured adipo- than originally believed. Accumulation of visceral white cytes. Thus, Tbx15 differentially regulates metabolism in 1Section on Integrative Physiology and Metabolism, Joslin Diabetes Center, Harvard 8Department of Molecular Endocrinology, University of Leipzig, Leipzig, Medical School, Boston, MA Germany 2 Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Corresponding author: C. Ronald Kahn, [email protected]. Ohio University, Athens, OH Received 22 February 2017 and accepted 20 August 2017. 3The Diabetes Institute, Ohio University, Athens, OH 4JRG Adipocytes and Metabolism, Institute for Diabetes and Obesity, Helmholtz This article contains Supplementary Data online at http://diabetes Diabetes Center at Helmholtz Center Munich, Neuherberg, Germany .diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0218/-/DC1. 5German Center for Diabetes Research, München-Neuherberg, Germany © 2017 by the American Diabetes Association. Readers may use this article as 6Russ College of Engineering and Technology, Ohio University, Athens, OH long as the work is properly cited, the use is educational and not for profit, and the 7Institut für Molekularbiologie, Medizinische Hochschule Hannover, Hannover, work is not altered. More information is available at http://www.diabetesjournals Germany .org/content/license. diabetes.diabetesjournals.org Lee and Associates 2823 Figure 1—Tbx15 is expressed in a subpopulation of white preadipocytes and is correlated with markers of glycolytic gene expression. Tbx15Low and Tbx15Hi cell lines are shown in blue and red, respectively. A: Expression level of Tbx15 mRNA was compared using qPCR of RNA isolated from subcutaneous and perigonadal fat of 8-week-old male C57BL/6 mice. Data are shown as mean 6 SEM of four samples. B: Schematic of FACS sorting for Tbx15Hi and Tbx15Low preadipocytes. C: Representative FACS plot of LacZ (+) preadipocytes from the subcutaneous and perigonadal fat depots of Tbx15-LacZ male mice at 8–10 weeks of age. D: Quantitation of LacZ(+) preadipocytes from WT and Tbx15-LacZ male mice at 8–10 weeks of age. Data are shown as mean 6 SEM of three to six animals per group. E: qPCR analysis for Tbx15, Pgc1a, Cox5b, Cox8b, Gapdh,andHk2 in RNA isolated from Tbx15Hi and Tbx15Low preadipocytes. Data are shown as mean 6 SEM of three to six animals per group. F:OilRedOStainingofTbx15Hi and Tbx15Low adipocytes after in vitro differentiation. The photographs were taken at 320 magnification. G:qPCRanalysisforTbx15, aP2, Adipoq,andPparg in RNA isolated from Tbx15Hi and Tbx15Low adipocytes after in vitro differentiation. Data are shown as mean 6 SEM of three to four animals per group. H:qPCRanalysisforPGC-1a, Cox8b,Cox5b,HK2,andGapdh in RNA samples from G. Asterisks indicate significant differences in all panels: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. PG, perigonadal; SC, subcutaneous; SVF, stromal vascular fraction; WT, wild type. 2824 Tbx15 in Fat Diabetes Volume 66, November 2017 white adipocytes and leads to a functional heterogene- Immunofluorescence and In Situ Hybridization ity in white fat cells similar to that found among muscle Immunofluorescence using an anti-Tbx15 antibody (8) and fibers. fluorescence in situ hybridization (9) were performed as de- scribed previously. Regions corresponding to 1,815–2,600 bp of – RESEARCH DESIGN AND METHODS Tbx15 mRNA and 39 796 bp of Adipoq mRNA were cloned into PCRII-TOPO as probe templates. The Tbx15 probe was la- Animals and Diets beled by incorporation of uridine-5’-triphosphate–digoxigenin, C57BL/6 mice (The Jackson Laboratory), Tbx15-LacZ mice and the Adipoq probe was labeled by incorporation of uridine- on a mixed genetic background (4), and Immortomouse (7) 5’-triphosphate–biotin. Probes were detected with Alexa Fluor were housed with a 12-h light/dark cycle in a temperature- 488 and 594 Tyramine signal amplification kits (Thermo Fisher). controlled room and allowed ad libitum access to water and food (Mouse Diet 9F 5020; PharmaServ). Animal care and Preadipocyte Isolation, Immortomouse Clonal Cell study protocols were approved by the Animal Care Com- Lines, and Cell Culture mittee of Joslin Diabetes Center in accordance with Na- Preadipocytes were isolated, grown, and differentiated as pre- tional Institutes of Health guidelines. viously described (10). Preadipocytes were plated, expanded, Figure 2—Clonal preadipocyte lines display differences in oxidative and glycolytic metabolism defined by Tbx15 expression. A: qPCR analysis for Tbx15 mRNA in Immortomouse subcutaneous preadipocyte clonal cell lines. Tbx15Low and Tbx15Hi cell lines are shown in blue and red, respectively. B:qPCRanalysisforTbx15 mRNA in Immortomouse preadipocyte clonal cell lines after 4 days of in vitro differentiation. Markers of adipocyte differentiation Adipoq and Pparg, markers of oxidative metabolism Pgc1a and Cox8b, and markers of glycolytic metabolism HK2 and Gapdh were measured. Data are shown as mean 6 SEM of 3–15 cell lines per group. C:qPCRanalysisforTbx15 mRNA in Immortomouse clonal preadipocyte cell lines after 4 days of in vitro differentiation. Markers of brown fat Ucp1, Tbx1,andPat2; markers of brite fat Tmem26 and P2RX5; and markers of white fat Leptin and Slc7a10 were measured. D: Representative picture of Immortomouse clonal preadipocyte cell lines and media 48 h after last media change. All wells are 100% confluent, and circled wells indicate Tbx15Hi Immortomouse clonal cell lines. E:Basal and maximal respiration of Tbx15Low and Tbx15Hi Immortomouse clonal cell lines after 4 days of in vitro differentiation were determined by calculating the area under the curve during measurements of OCR. Maximal OCR was measured after treatment with 1 mmol/L carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone. The whole experiment was repeated three times. Data are shown as mean 6 SEM of 3–15 cell lines per group. F: Basal and maximal ECARs of Tbx15Low and Tbx15Hi Immortomouse clonal cell lines after 4 days of in vitro differentiation were determined by calculating the area under the curve during measurements of basal and maximal ECAR. Maximal ECAR was measured after treatment with 1 mmol/L oligomycin. The whole experiment