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Cold-Inducible Klf9 Regulates Thermogenesis of Brown and Beige Fat

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Cold-Inducible Klf9 Regulates Thermogenesis of Brown and Beige Fat Diabetes Volume 69, December 2020 2603 Cold-Inducible Klf9 Regulates Thermogenesis of Brown and Beige Fat Heng Fan,1 Yujie Zhang,1 Jun Zhang,2 Qiyuan Yao,3 Yongfeng Song,4 Qiwei Shen,3 Jun Lin,5 Yuanxu Gao,6 Xiuyun Wang,7,8 Lei Zhang,1 Yinliang Zhang,1 Pingsheng Liu,9 Jiajun Zhao,4 Qinghua Cui,6 John Zhong Li,7,8 and Yongsheng Chang10 Diabetes 2020;69:2603–2618 | https://doi.org/10.2337/db19-1153 Promoting development and function of brown and beige expression of Pgc1a, a master regulator of fat thermo- fat may represent an attractive treatment of obesity. In genesis, by a direct binding to its gene promoter region, the current study, we show that fat Klf9 expression is subsequently promoting energy expenditure. The cur- markedly induced by cold exposure and a b-adrenergic rent study reveals a critical role for KLF9 in mediating agonist. Moreover, Klf9 expression levels in human white thermogenesis of brown and beige fat. adipose tissue (WAT) are inversely correlated with adi- posity, and Klf9 overexpression in primary fat cells stim- ulates cellular thermogenesis, which is Ucp1 dependent. METABOLISM Obesity results from a chronic imbalance between energy Fat-specific Klf9 transgenic mice gain less weight and intake and energy expenditure, which is closely associated have smaller fat pads due to increased thermogenesis of brown and beige fat. Moreover, Klf9 transgenic mice with many diseases, including cardiovascular diseases, type displayed lower fasting blood glucose levels and im- 2 diabetes, and nonalcoholic fatty liver disease. Tradition- proved glucose tolerance and insulin sensitivity under ally, adipocytes are divided into two types: unilocular white the high-fat diet condition. Conversely, Klf9 mutation in adipocytes and brown adipocytes. White adipose tissue brown adipocytes reduces the expression of thermogenic (WAT) is essential for triglyceride storage and endocrine genes, causing a reduction in cellular respiration. Klf9- signaling, while brown adipose tissue (BAT) dissipates energy mutant mice exhibited obesity and cold sensitivity due to to generate heat through uncoupled respiration mediated by impairments in the thermogenic function of fat. Finally, Ucp1 (1–3). Recent studies have identified another type of fat Klf9 deletion inhibits the b3 agonist–mediated induc- thermogenic adipocytes, namely, beige cells. Beige adipocytes tion of WAT browning and brown adipose tissue thermo- reside with white adipocytes and emerge in response to cold genesis. Mechanistically, cold-inducible Klf9 stimulates exposure or b-adrenergic receptor agonists (4,5). 1National Laboratory of Medical Molecular Biology, Institute of Basic Medical 9National Laboratory of Biomacromolecules, CAS Center for Excellence in Bio- Sciences, Chinese Academy of Medical Sciences and Peking Union Medical macromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, College, Beijing, China China 2Department of Basic Medicine, School of Medicine, Shihezi University, Xinjiang, 10Key Laboratory of Immune Microenvironment and Disease (Ministry of Educa- China tion), Tianjin Key Laboratory of Cellular Homeostasis and Disease, Department of 3Department of General Surgery, Huashan Hospital, Fudan University, Shanghai, Physiology and Pathophysiology, Tianjin Medical University, Tianjin, China China Corresponding author: John Zhong Li, [email protected], or Yongsheng 4 fi Department of Endocrinology, Shandong Provincial Hospital af liated to Shan- Chang, [email protected] dong First Medical University, Jinan, China Received 26 November 2019 and accepted 16 September 2020 5Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China This article contains supplementary material online at https://doi.org/10.2337/ 6Department of Biomedical Informatics, Department of Physiology and Patho- figshare.12988229. physiology, Center for Noncoding RNA Medicine, MOE Key Laboratory of Cardio- H.F. is currently affiliated with the Institute of Human Stem Cells, General Hospital vascular Sciences, School of Basic Medical Sciences, Peking University, Beijing, of Ningxia Medical University, Ningxia, China. China H.F., Y.Z., J. Zhang, and Q.Y. contributed equally. 7Key Laboratory of Rare Metabolic Disease, The Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing Medical University, Nanjing, © 2020 by the American Diabetes Association. Readers may use this article as fi China long as the work is properly cited, the use is educational and not for pro t, and the 8Department of Biochemistry and Molecular Biology, Nanjing Medical University, work is not altered. More information is available at https://www.diabetesjournals Nanjing, China .org/content/license. 2604 Klf9 and Thermogenesis of Brown and Beige Fat Diabetes Volume 69, December 2020 PGC1a is a central regulator in brown fat thermo- were housed and maintained in 12-h light and dark photo- genesis and is highly expressed in BAT. PGC1a expression periods. For DIO studies, 4-week-old male mice were fed in BAT is strongly induced by cold stress and b-adrenergic on a high-fat diet (HFD) (D12492; Research Diets) for signals, linking the physiological activator of brown fat 3 months. thermogenesis and the transcriptional machinery in brown adipocytes (6,7). Genetic ablation of Pgc1a results in Glucose and Insulin Tolerance Test reduced capacity for cold-induced thermogenesis in vivo The glucose tolerance tests (GTTs) and insulin tolerance and in a blunted response to cAMP signaling in brown fat tests (ITTs) were performed as previously described cells (8,9). (14,16). Krüppel-like factor 9 (KLF9) (also called basic transcrip- tion element binding protein 1), a member of the Krüppel- Body Weight, Body Temperature, Body Composition, and Energy Expenditure Measurement like family of zinc-finger domain transcription factors, Body weight was measured weekly. Body temperature was plays a key role in development (10–12). Interestingly, measured by rectal thermometer. Body composition (fat a human genetic study (genome-wide association study and lean mass) was determined by MRI (Echomri.Combo- [GWAS]) indicated that Klf9 is associated with BMI (13). 700). For metabolic studies, male mice were housed in- However, how KLF9 regulates obesity remains unclear. dividually in metabolic cages (Columbus Instruments) with Furthermore, we recently reportedthatKLF9promotes hepatic gluconeogenesis and hyperglycemia (14). Klf9 is free access to food and water. Oxygen consumption rate (OCR) was monitored for 48 h. Activity monitoring was ubiquitously expressed in many tissues (15). Neverthe- performed simultaneously with metabolic measurements. less, whether and how fat KLF9 regulates energy metab- olism remains unexplored. In the current study, we reveal Micro-Positron Emission Tomography/Computed the physiological function of KLF9 in adipose tissues. Tomography Glucose uptake of brown adipose tissue were determined RESEARCH DESIGN AND METHODS by positron emission tomography/computed tomography Ethics Compliance Statement (PET/CT) as previously described (17). Studies involving human specimens were approved by the ethics committees of Huashan Hospital, Fudan University, Histology Analysis and Shihezi University School of Medicine. Human omen- For hematoxylin-eosin (H-E) staining, Oil Red staining, tal adipose tissues specimens were collected after informed and UCP1 immunohistochemistry, inguinal WAT (iWAT) consent was obtained, and the study was approved by the and epididymal WAT (eWAT) and BAT tissues were treated institutional review board of Huashan Hospital (no. 2015- as previously described (17). For transmission electron 145). All animal experiments were approved by the In- microscopy, BAT sections were treated as previously de- stitute of Basic Medical Sciences and Peking Union Medical scribed (17). College. All animal experiments were conducted under protocols approved by the Institutional Animal Care Use Stromal Vascular Fraction Isolation and Differentiation & Welfare Research Committee, the Institute of Basic of Primary Brown Adipocytes Medical Sciences, Chinese Academy of Medical Sciences Isolation of brown fat stromal vascular fraction (SVF) and and Peking Union Medical College (ACUC-A01-2014-033 differentiation of primary brown preadipocytes were per- and ACUC2011A02-293). formed as previously described (17), with minor modifi- cations. Briefly, the digested brown adipose tissue was Animal Treatment filtered through a 60-mesh nylon screen and centrifuged Male mice were used during this study. Global Klf9 mutant (1,000 3 rpm) for 10 min to collect the preadipocytes. mice were obtained from The Jackson Laboratory (no. 012909). Klf9 transgenic mice were generated at Beijing Oxygen Consumption Assays of Brown Adipocytes and Biocytogen Co., Ltd. The full-length coding sequence of Fat mouse Klf9 was amplified from hepatic RNA by PCR. The For determination of cellular oxygen consumption, isolated 5.4-kb adiponectin promoter was kindly provided by Dr. brown preadipocytes were plated in an XF24-well microplate Philipp E. Scherer (Department of Internal Medicine, (Seahorse Bioscience) and differentiated into mature brown University of Texas Southwestern Medical Center), which adipocytes, followed by OCR measurement at 37°C with an was inserted into pBluescript vector. The Klf9 cDNA was XF24 analyzer (Seahorse Bioscience) in accordance with the inserted into vector containing the 5.4-kb adiponectin manufacturer’s instructions. Oligomycin (2 mmol/L), car-
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