Diabetes Mar 2017, db161079 

Id1 Promotes Obesity by Suppressing Brown Adipose Thermogenesis and White Adipose Browning

Mallikarjun Patil,Jinling Yuan & Ande Satyanarayan


Obesity results from increased energy intake or defects in energy expenditure. Brown adipose tissue (BAT) is specialized for energy expenditure, a process called adaptive thermogenesis. Peroxisome proliferator–activated receptor γ coactivator 1α (PGC1α) controls BAT-mediated thermogenesis by regulating the expression of Ucp1. Inhibitor of differentiation 1 (Id1) is a helix-loop-helix transcription factor that plays an important role in cell proliferation and differentiation. We demonstrate a novel function of Id1 in BAT thermogenesis and programming of beige adipocytes in white adipose tissue (WAT). We found that adipose tissue–specific overexpression of Id1 causes age-associated and high-fat diet–induced obesity in mice. Id1 suppresses BAT thermogenesis by binding to and suppressing PGC1α transcriptional activity. In WAT, Id1 is mainly localized in the stromal vascular fraction, where the adipose progenitor/precursors reside. Lack of Id1 increases beige gene and Ucp1 expression in the WAT in response to cold exposure. Furthermore, brown-like differentiation is increased in Id1-deficient mouse embryonic fibroblasts. At the molecular level, Id1 directly interacts with and suppresses Ebf2 transcriptional activity, leading to reduced expression of Prdm16, which determines beige/brown adipocyte cell fate. Overall, the study highlights the existence of novel regulatory mechanisms between Id1/PGC1α and Id1/Ebf2 in controlling brown fat metabolism, which has significant implications in the treatment of obesity and its associated diseases, such as diabetes.The aP2-Id1Tg+ transgenic expression vector and mice were generated by using the services of Cyagen Biosciences (Santa Clara, CA). Of four transgenic lines generated, one line showed strong expression of Id1 in WAT and BAT and was used for this study. The following primers were used to distinguish the aP2-Id1Tg+ transgenic from control aP2-Id1Tg− mice: Tg F: ATCTTTAAAAGCGAGTTCCCT; Tg R: CTCCGACAGACCAAGTACCA; internal control F: ACTCCAAGGCCACTTATCACC; and internal control R: ATTGTTACCAACTGGGACGACA. Endogenous mouse β-actin was used as the internal control. C57BL/6J background aP2-Id1Tg+, aP2-Id1Tg−, Id1+/+, and Id1−/− mice were used for this study. Mice were housed in a barrier facility under standard conditions with a 12-h light-dark cycle. Mice were handled in compliance with National Institutes of Health guidelines for animal care and use. All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Augusta University (Augusta, GA). Mice were fed a standard chow normal diet (ND) containing 6% crude fat (Harlan Teklad Rodent Diet 2918). For the HFD experiments, 1-month-old mice were switched from ND to HFD (60% kcal from fat, D12492; Research Diets, New Brunswick, NJ) and fed for 12 weeks. For cold exposure studies, mice were housed in standard cages without bedding, and the cages were placed in the cold room (4°C) for 4–12 h. Mice were then euthanized and tissues harvested.To investigate whether Id1 plays a role in adipose tissue metabolism, we analyzed the expression pattern of Id1 protein in mouse BAT, inguinal WAT (iWAT), epididymal WAT (eWAT), and retroperitoneal WAT (rWAT). Id1 was expressed in all the adipose tissues, and its expression was relatively stronger in BAT than in iWAT, eWAT, and rWAT (Fig. 1A and B). In addition, analysis of Id1 mRNA revealed a relatively higher Id1 mRNA in BAT and eWAT than in iWAT and rWAT (Fig. 1C). To investigate whether Id1 is required for brown adipocyte differentiation, we induced differentiation in the HIB1B brown preadipocyte cell line and detected a threefold induction of Id1 mRNA in day 4 differentiated cells compared with undifferentiated cells (Fig. 1D and E). The adipocyte differentiation marker aP2 and the brown adipocyte–specific thermogenic genes Pgc1α and Ucp1 also were induced in day 4 differentiated cells, as expected (Fig. 1E). Further analysis at the protein level revealed low levels of Id1 in undifferentiated HIB1B cells, and its expression was steadily elevated during the differentiation process (Fig. 1F). To further confirm that Id1 expression is induced in differentiated cells compared with nondifferentiated HIB1B cells, we performed Id1/aP2 costaining, which revealed increased Id1 staining in aP2-positive cells (Fig. 1G). These observations suggest that Id1 plays a role in brown adipocyte differentiation or in brown adipocyte–associated thermogenesis.Acknowledgments. The authors thank Dr. Jonathan Keller (Mouse Cancer Genetics Program, National Cancer Institute, Frederick, MD) for providing Id1−/− mice, Dr. Bruce Spiegelman (Department of Cell Biology, Harvard Medical School) for providing Gal4-PGC1α vectors and HIB1B cells, Dr. Patrick Seale (Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania) for providing Prdm16 luciferase reporter vectors, and Dr. Mark Christian (Division of Metabolic and Vascular Health, University of Warwick) for providing Ucp1 luciferase vector. The authors also thank Dr. Ali Arbab, Roxan Ara, and Chris Middleton (Tumor Angiogenesis Section, Georgia Cancer Center, Augusta University) for technical help with computed tomography scans; Dr. Jenfeng Pang (Molecular Oncology Program, Georgia Cancer Center, Augusta University) for help with Oxymax readings; and Dr. Rhea-Beth Markowitz (Georgia Cancer Center, Augusta University) for reviewing and editing the manuscript.Funding. This research was supported by the National Cancer Institute (grant K22-CA-168828 to A.S.) and the National Institute of Diabetes and Digestive and Kidney Diseases (grant DP2-DK-105565 to A.S.).Duality of Interest. No potential conflicts of interest relevant to this article were reported.Author Contributions. M.P., B.K.S., S.E., J.C., and S.K. performed experiments and collected and analyzed data. J.Y. maintained and provided animals for experiments. A.S. supervised the study. M.P. and A.S. analyzed and interpreted the data and wrote the manuscript. M.P. and A.S. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db16-1079/-/DC1.
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