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 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 , 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 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 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- promoter. Mouse oocytes were injected with this construct bonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) at Beijing Biocytogen Co., Ltd. Global Ucp1 mutant mice (2 mmol/L), and rotenone/antimycin (0.5 mmol/L) were were kindly provided by Dr. Yifu Qiu (Institute of Molec- delivered to detect the uncoupled respiration, maximal res- ular Medicine, Peking University). C57BL/6J, ob/ob, and piration, and nonmitochondrial respiration, respectively. Ox- db/db mice were purchased from the Model Animal Re- ygen consumption of brown fat tissues was measured as search Center of Nanjing University (Nanjing, China) and previously described (18). diabetes.diabetesjournals.org Fan and Associates 2605

Chromatin Immunoprecipitation Assay CL 316,243 compound (Supplementary Fig. 1A–C). Nota- Chromatin immunoprecipitation (ChIP) assay was per- bly, we observed that KLF9, a , is also formed as previously described (14,17). Antibodies specific induced by CL 316,243 (Supplementary Fig. 1A). Our real- for KLF9 (ab227920; Abcam) or unspecific IgG (sc-2027; time PCR and Western blotting analyses confirmed CL Santa Cruz Biotechnology) was used for ChIP assay. The 316,243–mediated increases in Klf9 expression in BAT and purified DNA was used to amplify the KLF9 regulatory iWAT (Fig. 1A and B). We also examined Klf9 expression element on the mouse Pgc1a promoter by a real-time PCR levels in different fat pads (including BAT, iWAT, and reaction. Primers directed at upstream or downstream of eWAT). Klf9 is highly expressed in BAT, with lower ex- the binding site were used as a negative control. The pression levels in iWAT and eWAT (Supplementary Fig. sequences of primers are shown in Supplementary Table 1. 1D). Cold exposure also induces adaptive thermogenesis in Immunoblotting Analysis brown and beige fat via stimulating norepinephrine secre- Immunoblotting was performed with the following primary tion. For exploration of the potential roles of KLF9 in cold- antibodies: KLF9 (A7196; ABclonal), PGC1a (AB3242; Milli- induced thermogenesis, mice were maintained at 4°C. As pore), UCP1 (ab10983; Abcam), and b-Tubulin (AC021; a result, Klf9 mRNA and protein levels in both BAT and fi ABclonal). Target protein bands were quanti ed with subcutaneous iWAT were elevated after cold exposure ImageJ software. compared with those at room temperature. Moreover, the expression of thermogenic genes including Pgc1a and Ucp1 RNA Extraction and Quantitative Real-time PCR was also induced by cold exposure (Fig. 1C and D). Total RNA from either the mouse adipose tissue or the We next examined KLF9 expression in BAT and iWAT primary adipocytes was extracted using the TRIzol-based from db/db and HFD-fed mice. Western blotting analysis method (Invitrogen). Real-time PCR was performed as showed that KLF9 protein abundance is lower in db/db previously described (14). The sequences of primers are diabetic mice and mice with HFD-induced obesity than in shown in Supplementary Table 1. their respective control db/m and chow diet–fed mice (Fig. 1E and F). Metabolite Measurement Moreover, a recent human genetic study (GWAS) sug- Metabolites were measured as previously described (14). gested that a single nucleotide polymorphism in the KLF9 Statistical Analysis promoter region (rs11142387) is associated with BMI (13). The quantitative data are represented as the mean 6 the Thus, we tested whether KLF9 expression in adipose tissue SEM of three independent experiments. In most of the samples was associated with BMI. Omental fat tissue cases for in vivo experiments in mice, an n 5 5 was the biopsies were collected from 39 obese individuals and minimum amount used. A two-tailed, unpaired Student t 11 normal weight individuals. Regression analysis revealed test was used for pairwise comparison of genotypes or a reverse association between KLF9 mRNA levels in fat and 2 5 , treatments. One-way ANOVA and two-way ANOVA were BMI (r 0.5139, P 0.0001) (Fig. 1G). These results used in comparison of three or more groups, as indicated in implicate Klf9 in obesity and thermogenesis of brown and the figure legends and otherwise. Analysis was performed beige fat, and decreased adipose KLF9 expression may using Microsoft Excel and/or GraphPad Prism. P , 0.05 impair systemic energy homeostasis. was considered significant, as indicated in the figure legends. Transgenic Klf9 Expression in Adipose Tissue Stimulates Brown Fat Thermogenesis and WAT Data and Resource Availability Browning The data sets generated and/or analyzed during the cur- We recently reported that hepatic KLF9 stimulates gluco- rent study are available from the corresponding author on neogenesis via induction of PGC1a (14), and PGC1a has reasonable request. No applicable resources were gener- been shown to be a master regulator of brown fat thermo- ated during the current study. genesis (19). Thus, we hypothesized that KLF9 might regulate fat thermogenesis via PGC1a. For testing this RESULTS hypothesis, primary adipocytes differentiated from the Adipose Klf9 Expression Is Regulated by b3-Adrenergic SVF from the brown fat pads of wild-type (WT) mice Agonist and Other Physiological Stimuli were transduced with an adenovirus expressing Klf9 To identify the key signaling molecules mediating the (Ad-Klf9). The overexpression of Klf9 in adipocytes beiging and thermogenesis induced by b3-adrenergic ag- induced thermogenic genes, including Ucp1, Dio2, and onist in fat, we first performed mRNA microarray analysis Pgc1a (Supplementary Fig. 2A). Based on these prelimi- of subcutaneous iWAT from normal C57 mice intraperi- nary data, we generated fat-specific Klf9 transgenic mice toneally injected with b3-adrenergic agonist CL 316,243 or (Supplementary Fig. 2B), driven by the 5.4-kb adiponectin saline (control). As expected, preliminary analysis of the promoter (20). We selected two independent founder microarray data indicated that genes involved in energy lines, tg-1 and tg-2, with tg-1 having a higher level of metabolism, glucose and lipid metabolism, are induced by Klf9 expression in brown fat (Fig. 2A). 2606 Klf9 and Thermogenesis of Brown and Beige Fat Diabetes Volume 69, December 2020

Figure 1—Adipose Klf9 expression is regulated by b3-adrenergic agonist and related to obesity. A: Quantitative PCR analysis of Klf9 mRNA levels in iWAT and BAT of C57BL/6J mice injected daily with saline or CL 316,243 (1 mg/kg/day) for 4 days (n 5 6/group). B: Representative Western blot analysis of protein levels of KLF9, PGC1a, and UCP1 in iWAT and BAT of mice described in A (left panel), and quantification of the target protein bands relative to tubulin control using ImageJ software (right panel). C: Quantitative PCR analysis of Klf9 mRNA levels in iWAT and BAT of C57BL/6J mice housed at room temperature or 4°C for 48 h (n 5 6/group). D: Representative Western blot analysis of protein levels of KLF9, PGC1a, and UCP1 in iWAT and BAT of mice described in C (left panel) and quantification of the target protein bands using ImageJ software (right panel). E and F: Western blot analysis of KLF9 protein levels in iWAT and BAT of db/db (E) and HFD-fed (F) mice, and their respective control mice (left panel), and quantification of the target protein bands relative to tubulin control was performed using ImageJ software (right panel). G: Linear regression analysis between BMI and Klf9 mRNA levels in human omental adipose tissue (n 5 50). Throughout, data are mean 6 SEM. *P , 0.05, **P , 0.01, ***P , 0.001 by two-tailed Student t test (A–F). diabetes.diabetesjournals.org Fan and Associates 2607

Figure 2—Adipose-specific Klf9 transgenic mice are lean and display enhanced energy expenditure. A: Western blot analysis of KLF9 protein levels in iWAT and BAT of WT and adipose-specific Klf9 transgenic mice line 1 (Tg1) and line 2 (Tg2) (left panel), and quantification of the target protein bands relative to tubulin control using ImageJ software (right panel). B: The growth curve of WT, Tg1, and Tg2 mice fed a chow diet (n 5 6/group). C: Gross morphology of chow diet–fed WT, Tg1, and Tg2 mice at 14 months of age. D: Body weight of WT, Tg1, and Tg2 mice described in C (n 5 6/group). E: Gross appearance of interscapular BAT and inguinal and epididymal fat pads from WT, Tg1, and Tg2 mice described in C. F: MRI analysis of body composition of mice described in C (n 5 4/group). G: H-E staining of paraffin-embedded interscapular BAT, iWAT, and eWAT sections of mice described in C. H and I: OCR of 3-month-old WT, Tg1, and Tg2 mice (n 5 4/group). J and K:CO2 production rates of 3-month-old WT, Tg1, and Tg2 mice (n 5 4/group). L: Basal OCR in interscapular BAT of 3-month-old WT, Tg1, and Tg2 mice (n 5 3/group). M: Rectal temperature of 3-month-old mice during acute cold exposure (4°C) (n 5 4/group). N: ChIP assay performed as described in RESEARCH DESIGN AND METHODS showing enhanced KLF9 binding to Pgc1a gene promoter region containing Klf9 2608 Klf9 and Thermogenesis of Brown and Beige Fat Diabetes Volume 69, December 2020

Figure 2—Continued. binding site in Klf9 transgenic mice relative to WT mice described in C. O and P: Quantitative PCR analysis of mRNA levels of genes involved in thermogenesis, fatty acid oxidation, and mitochondrial energy metabolism in the iWAT (O) and BAT (P) of mice described in C (n 5 4/group). Q: Western blot analysis of PGC1a and UCP1 in the BAT of mice in C (upper panel) and quantification of the target protein bands using ImageJ software (bottom panel). R: Representative images of UCP1 immunohistochemistry (IHC) of BAT from mice treated as in C. Throughout, data are mean 6 SEM. IP, immunoprecipitation. *P , 0.05, **P , 0.01, ***P , 0.001 (Tg1 vs. WT), #P , 0.05 (Tg2 vs. WT) by two-tailed Student t test (A, B, D, F, I, K–M, and O–Q); **P , 0.01, ***P , 0.001 by one-way ANOVA (N).

As a result, under a normal chow diet, the transgenic (Fig. 2B–D and Supplementary Fig. 2C). eWAT, iWAT, and mice gained less body weight as they aged, though they interscapular BAT from transgenic mice were markedly consumed an amount of food similar to that of controls smaller and weighed less than those from control mice diabetes.diabetesjournals.org Fan and Associates 2609

Figure 3—Adipose-specific Klf9 transgenic mice are resistant to HFD-induced obesity. A: The growth curve of WT and adipose-specific Klf9 transgenic mice fed an HFD starting at 4 weeks of age (n 5 5/group). B: Gross morphology of WT and Klf9 transgenic mice fed an HFD for 3 months. C: Gross appearance of interscapular BAT and inguinal and epididymal fat pads from mice in B. D: MRI assay of body composition of mice in B (n 5 5/group). E: H-E staining of paraffin-embedded BAT and inguinal and epididymal fat pad sections from the mice in B. F–H: Quantification of adipocyte size of eWAT (F), iWAT (G), and BAT (H) of the mice in B. (Data were collected from H-E–stained sections from five individual mice, three fields per mouse, 10–15 cells per field in each group, using Image J software.) I and J: Quantitative PCR analysis of thermogenic genes of BAT (I) and iWAT (J) of the mice in B (n 5 4/group). K: Representative Western blot analysis of thermogenic genes in BAT of the mice described in B (left panel) and quantification of the target protein bands using ImageJ software (right panel). L: Representative images of UCP1 immunohistochemistry (IHC) of BAT from mice treated as in B. M–O: Serum concentrations of FFAs (M), triglyceride (N), and hepatic triglyceride (O) in mice described in B (n 5 5/group). P and Q: Basal blood glucose levels of 6-h-fasted (P) and 16-h-fasted (Q) mice in 2610 Klf9 and Thermogenesis of Brown and Beige Fat Diabetes Volume 69, December 2020

Figure 3—Continued. B (n 5 4/group). R and S: Blood glucose levels during GTT (R) and ITT (S) in the mice in B (n 5 4/group). Scale bar, 100 mm. Throughout, data are presented as mean 6 SEM. *P , 0.05, **P , 0.01 (Tg1 vs. WT), #P , 0.05 (Tg2 vs. WT) by two-tailed Student t test (A, D, I–K, and M–S).

(Fig. 2E and Supplementary Fig. 2D). MRI examination by housing mice in metabolic cages. Considering that the confirmed less fat mass in transgenic mice, while the lean explanation of causality in metabolic cage analysis is mass remained unaltered compared with that from control clearer in groups of mice with similar body weights mice (Fig. 2F). Consistently, histological examination (H-E (21,22), we used body weight–matched 3-month-old Klf9 staining) of these fat depots revealed a reduction in size of transgenic mice and controls. The Klf9 transgenic mice the lipid droplets and adipocytes in eWAT, iWAT, and showed no changes in locomotor activity compared with interscapular BAT of Klf9 transgenic mice (Fig. 2G and controls, while energy expenditure, measured by respira- – Supplementary Fig. 2E G). Transmission electron micros- tory oxygen consumption and carbon dioxide (CO2) pro- copy reveal that Klf9 transgene increased the number of duction, was higher in transgenic mice during both day and mitochondria in brown adipocytes but did not markedly night cycles (Fig. 2H–K). Moreover, Klf9 transgene pro- affect mitochondrial structure (Supplementary Fig. 2H). moted OCR in BAT of mice (Fig. 2L). When subjected to To test whether the Klf9 transgene affects energy acute cold exposure, these transgenic mice showed cold expenditure, we monitored gas exchange and activity levels resistance (Fig. 2M). diabetes.diabetesjournals.org Fan and Associates 2611

Figure 4—Klf9 overexpression activates cellular respiration and thermogenesis of primary brown fat cells, dependent on Ucp1. A: Quantitative PCR analysis of mRNA levels of genes involved in thermogenesis and mitochondrial oxidative phosphorylation in differentiated primary brown cells from Klf9 transgenic and WT mice (n 5 3/group). B: Representative Western blot analysis of protein levels of KLF9, PGC1a, and UCP1 in differentiated brown cells described in A (left panel) and quantification of the target protein bands using ImageJ software (right panel). C and D: OCRs of differentiated brown cells described in A was measured under basal conditions, following the addition of oligomycin, FCCP, and antimycin A 1 rotenone (n 5 3/group). E: Quantitative PCR analysis of mRNA levels of Pgc1a and Dio2 in differentiated brown cells from WT and Ucp1-deficient mice infected with the indicated adenoviruses (Ad-GFP and Ad-Klf9) (n 5 3/group). F– H: OCRs of differentiated brown cells described in E (n 5 3/group). I: Quantitative PCR analysis of genes involved in thermogenesis and mitochondrial energy metabolism in differentiated primary brown cells from WT and Klf9-mutant mice (n 5 3/group). J: Representative Western blot analysis of protein levels of KLF9, PGC1a, and UCP1 in differentiated brown cells described in I (left panel), and quantification of 2612 Klf9 and Thermogenesis of Brown and Beige Fat Diabetes Volume 69, December 2020

We previously demonstrated that KLF9 activates Pgc1a Klf9 Stimulates Thermogenesis and Cellular gene transcription through direct binding to its promoter Respiration of Fat Cells via Ucp1 region in hepatocytes (14). As expected, ChIP assays using For exploration of whether the effects of KLF9 on fat mouse BAT extracts confirmed that the Klf9 transgene thermogenesis are cell autonomous, the SVF isolated from enhanced Klf9 protein binding to the Pgc1a gene promoter the brown fat pads from Klf9 transgenic mice was induced region (Fig. 2N). Correspondingly, the Klf9 transgene in- into adipogenic differentiation. Real-time PCR and West- creased the expression of genes involved in thermogenesis, ern blotting analyses confirmed that the Klf9 transgene in fatty acid oxidation, and mitochondrial energy metabo- primary adipocytes also enhanced the expression of Pgc1a lism in iWAT, interscapular BAT, and eWAT (Fig. 2O–Q and its target gene (Fig. 4A and B). Upon using a Seahorse and Supplementary Fig. 2I). Immunohistochemical analy- XF-24 Extracellular Flux Analyzer, we observed that basal sis also revealed more intense UCP1 immunoreactivity in mitochondrial respiration and maximal mitochondrial re- BAT of Klf9 transgenic mice (Fig. 2R), while UCP1 protein spiratory capacity increased in Klf9 transgenic adipocytes, levels in iWAT and eWAT of these old mice were below the as did oligomycin-dependent uncoupled cellular respiration limit of detection (Supplementary Fig. 2J). However, Klf9 (Fig. 4C and D). transgene did not influence the expression of Zfp423 Since Pgc1a is a direct target gene of Klf9 (14) (Fig. 2N), (Supplementary Fig. 2K), a critical factor maintaining we next explored whether PGC1a is required for a white adipocyte identity through suppression of thermo- KLF9-induced thermogenic program. Indeed, knockdown of genic gene program (23). Pgc1a largely abolished the stimulatory We also examined glucose and lipid metabolism in Klf9 effectsonthermogenicgenes(SupplementaryFig.4A). transgenic mice. Blood glucose levels in transgenic mice To test whether the ability of KLF9 to stimulate ther- were markedly lower than those in control mice under mogenesis in primary brown adipocytes is Ucp1 depen- short-term fasting conditions (Supplementary Fig. 2L). dent, we treated brown adipocytes derived from Ucp1 The GTTs indicated that the Klf9 transgene enhanced knockout (KO) mice and WT mice with Ad-Klf9 and glucose tolerance, and the ITTs indicated improved insulin Ad-GFP (control). Although the thermogenic gene expres- sensitivity in transgenic mice (Supplementary Fig. 2M and sion program induced by Ad-Klf9 was similar in Ucp1 KO N). Finally, biochemical analysis revealed a significant de- adipocytes and WT cells, the ability of KLF9 to increase crease in serum triglycerides, free fatty acids (FFAs), and cellular respiration was lost in Ucp1 KO cells (Fig. 4E–H). hepatic triglyceride levels in the transgenic mice (Supple- These data suggest that Ucp1 is required for Klf9-mediated mentary Fig. 2O–Q). stimulation of thermogenesis in fat cells. We also examined these mice under an HFD condition and obtained results similar to those above (Fig. 3). Klf9 Klf9 Deficiency in Fat Cells Leads to Decreased Cell transgenic mice are resistant to HFD-induced obesity (Fig. Respiration 3A and B). The transgenic mice weighed less at 9 weeks For further study of KLF9 physiological function in fat after HFD feeding. The sizes of three different fat depots cells, the SVF isolated from brown fat from Klf9 KO mice in transgenic mice were smaller compared with those in and WT mice were differentiated into mature adipocytes. control mice (Fig. 3C). MRI analysis also confirmed re- Notably, depletion of Klf9 did not affect morphological duced fat mass in Klf9 transgenic mice (Fig. 3D). Like- differentiation or change the expression of adipocyte wise, the sizes of the lipid droplets and adipocytes in general markers (Supplementary Fig. 4B and C). However, BATandWATofKlf9 transgenic mice were smaller the expression of thermogenic and mitochondrial genes than those in control mice (Fig. 3E–H). Consistent was decreased in Klf9-mutant cells (Fig. 4I and J). Oxygen with the increased expression of genes involved in consumption experiments also revealed decreased total the thermogenesis of BAT and iWAT (Fig. 3I–L and respiration and uncoupled respiration (Fig. 4K and L). Supplementary Fig. 3A), transgenic mice had decreased These data indicate that KLF9 is required for the main- serum FFA, serum triglyceride, and hepatic triglyceride tenance of brown adipocyte identity and function. How- levels (Fig. 3M–O). Notably, after 3 months of HFD ever, KLF9 is not required for adipogenesis per se. feeding, transgenic mice had lower fasting blood glucose levels and improved glucose tolerance and insulin sen- Loss of Klf9 Function Predisposes Mice to Obesity and sitivity compared with control littermates (Fig. 3P–S). Causes Reduced Whole-Body Energy Expenditure and These results suggest that Klf9 overexpression in fat Impaired Thermogenic Function of Fat protects against HFD-induced obesity and improves To further examine KLF9 function in energy metabolism glucose metabolism. in vivo, we first employed global Klf9 mutant mice (11).

the target protein bands using ImageJ software (right panel). K and L: OCRs of differentiated brown cells described in I were measured under basal conditions, following the addition of oligomycin, FCCP, and antimycin A 1 rotenone (n 5 3/group). Throughout, data are presented as mean 6 SEM. *P , 0.05, **P , 0.01, ****P , 0.0001 by two-tailed Student t test (A, B, D, J, and L), two-way ANOVA (E and H). diabetes.diabetesjournals.org Fan and Associates 2613

Figure 5—Global Klf9-mutant mice display reduced energy expenditure and are prone to obesity with age. A: Western blot analysis of KLF9 protein levels in iWAT and BAT from WT and global Klf9-mutant mice (left panel) and quantification of the target protein bands relative to tubulin control using ImageJ software (right panel). B: The body weight curve of the WT and global Klf9-mutant mice on a chow diet (n 5 6/ group). C: Gross morphology of 5-month-old WT and global Klf9-mutant mice on a chow diet. D: MRI assay of body composition of mice described in C (n 5 6/group). E: Gross appearance of interscapular BAT and inguinal and epididymal fat pads from the mice described in C. F: H-E staining of paraffin-embedded interscapular BAT and eWAT and eWAT sections from the mice described in C. G and I: OCR of 3-month- old WT and Klf9 mutant mice (n 5 4/group). H and J:CO2 production rates of WT and Klf9 mutant mice in G (n 5 4/group). K: Basal OCR in interscapular BAT of 3-month-old WT and KO mice (n 5 3/group). L: Rectal temperature of mice described in H during acute cold exposure (4°C) (n 5 4/group). M: PET-CT assessing the metabolic activity of BAT of WT and global Klf9-mutant mice described in C. N: ChIP assay showing that endogenous KLF9 proteins in BAT of WT mice bind to Pgc1a gene promoter, while Klf9 mutation abolished these effects (n 5 4/ group). O: Western blot analysis of PGC1a and UCP1 in the BAT of mice described in C (left panel) and quantification of the target protein bands using ImageJ software (right panel). P and Q: Quantitative PCR analysis of genes involved in thermogenesis, fatty acid oxidation, and mitochondrial energy metabolism in the BAT (P) and iWAT (Q) of mice described in C (n 5 5/group). Scale bar, 100 mm. Throughout, data are presented as mean 6 SEM. ID/g, injected dose per gram; IP, immunoprecipitation. *P , 0.05, **P , 0.01, ***P , 0.001, ****P , 0.0001 by two- tailed Student t test (A, B, D, I–L, and O–Q); **P , 0.01 by one-way ANOVA (N). 2614 Klf9 and Thermogenesis of Brown and Beige Fat Diabetes Volume 69, December 2020

Figure 5—Continued.

Western blotting analysis confirmed the lack of expression Klf9-mutant mice (Fig. 5F and Supplementary Fig. 5C–E). of KLF9 in BAT and iWAT of these KO mice (Fig. 5A). On Notably, many unilocular fat cells appeared in the BAT of a chow diet, the body weight of mice began to diverge at these mutant mice, indicating that the Klf9 mutation 15 weeks of age, with mutant mice gaining more weight induces a brown-to-white fat switch (Fig. 5F). Transmis- than control mice, although mutant mice and control mice sion electron microscopy revealed that Klf9 deficiency had similar food intake per day (Fig. 5B and C and decreased the number of mitochondria in brown adipo- Supplementary Fig. 5A). MRI examination confirmed cytes (Supplementary Fig. 5F). that mutant mice had more fat mass than control mice Metabolic cage experiments using body weight–matched (Fig. 5D). We dissected and weighed different fat pads and 3-month-old Klf9 mutant mice and controls suggested that organs (including the liver, kidney, and spleen) and found the Klf9 mutation did not affect locomotor activity, although that the individual fat pads of the mutant mice were it decreased energy expenditure, as measured by respiratory markedly larger than those of control mice (Fig. 5E and oxygen consumption and CO2 production (Fig. 5G–J). Fur- Supplementary Fig. 5B). Histological analysis (H-E stain- thermore, Klf9 deficiency decreased OCR of BAT of mice (Fig. ing) revealed adipocyte hypertrophy in BAT and WAT in 5K). At room temperature, Klf9 mutant mice had core body diabetes.diabetesjournals.org Fan and Associates 2615

Figure 6—KLF9 is required for b3-adrenergic agonist–induced thermogenesis. A: Gross appearance of interscapular BAT and inguinal and epididymal fat pads from chow diet–fed WT and global Klf9 KO mice injected daily with saline or CL 316,243 (1 mg/kg/day) for 4 days. B: H-E staining of paraffin-embedded BAT sections from the mice described in A. C and D: H-E staining and UCP1 immunohistochemistry of paraffin-embedded iWAT sections from the mice described in A. E and F: The OCR of 8-week-old WT and global Klf9 KO mice treated with CL 316,243 (1 mg/kg) for 2 h (n 5 4/group). G and H: ChIP assay performed as described in RESEARCH DESIGN AND METHODS showing that both CL 316,243 injection and cold exposure promote endogenous KLF9 proteins in BAT of WT mice binding to the Pgc1a gene promoter (n 5 4/ group). I–K: Quantitative PCR (I and J) and Western blot (K) analysis of thermogenic genes in interscapular BAT and iWAT from the mice described in A (n 5 6/group). L: Based on the current study and the previous reports, we proposed a model of cold exposure stimulation of thermogenesis of brown and beige fat. Scale bar, 100 mm. Throughout, data are presented as mean 6 SEM. *P , 0.05, **P , 0.01, ***P , 0.001, ****P , 0.0001 by two-tailed Student t test (E and F), one-way ANOVA (G and H), two-way ANOVA (I and J). RT, room temperature.

temperatures comparable with those of WT mice. However, promoter region, while Klf9 mutation abolished these when subjected to acute cold exposure, Klf9 mutant mice effects (Fig. 5N). As expected, Klf9 deficiency led to a de- displayedcoldintoleranceat4°C(Fig.5L). Furthermore, we crease in PGC1a and its downstream target genes (Fig. analyzed the in vivo BAT function of Klf9 mutant mice. 5O–Q and Supplementary Fig. 5G). Klf9 mutant mice had PET-CT imaging data suggested a marked decrease in [18F]- elevated serum and hepatic triglyceride levels. Moreover, fluorodeoxyglucose uptake in the BAT of Klf9 mutant mice serum FFA levels were also higher in Klf9 mutant mice (Fig. 5M). (Supplementary Fig. 5H–J). Collectively, these data sug- ChIP assays confirmed that the endogenous KLF9 gest that loss of KLF9 function impairs whole-body proteins in BAT of WT mice bind to the Pgc1a gene metabolism. 2616 Klf9 and Thermogenesis of Brown and Beige Fat Diabetes Volume 69, December 2020

Figure 6—Continued.

Klf9 Is Also Required for WAT Browning and BAT Molecular mechanism studies suggest that CL 316,243 Thermogenesis Induced by a b-Adrenergic Agonist injection or cold exposure promotes KLF9 proteins binding Since both cold stress and b-adrenergic agonists stimulate to Pgc1a gene promoter (Fig. 6G and H). As expected, fat Klf9 expression, we explored whether KLF9 functions treatment with the b3-adrenergic agonist CL 316,243 in thermogenesis induced by a b-adrenergic agonist. We induced thermogenic gene expression in BAT and iWAT first observed reduced size and mass of BAT, eWAT, and of control mice (Fig. 6I–K). However, these effects were iWAT of WT mice following 4 days of b-adrenergic agonist largely impaired in global Klf9 KO mice (Fig. 6I–K). These treatment. The subcutaneous iWAT of CL 316,243–treated data indicate that KLF9 is also required for adipose ther- control mice appeared browner than that of vehicle- mogenesis induced by a b-adrenergic agonist. treated control mice (Fig. 6A). Histological analysis (H-E Based on these data, we proposed a model of cold staining) also revealed reduced lipid droplet size in BAT exposure induction of the thermogenesis of brown and and eWAT of CL 316,243–treated control mice (Fig. 6B and beige fat (Fig. 6L). In response to cold exposure, sympa- C). Furthermore, CL 316,243 treatment of control mice thetic neurons secrete catecholamines, which bind to led to the occurrence of abundant UCP1-positive beige b-adrenoreceptors, leading to activation of adenylyl cy- adipocytes with multilocular lipid droplets, as revealed by clase and increased cAMP and PKA activity, thereby stim- immunohistochemical staining (Fig. 6D). We further an- ulating Klf9 expression in adipocytes. Furthermore, induced alyzed in vivo thermogenic fat function by measuring KLF9 promotes browning of WAT and thermogenesis of whole-animal oxygen consumption before and after in- brown and beige fat via a direct binding to Pgc1a gene jection of CL 316,243. CL 316,243 injection immediately promoter to activate its transcription (Fig. 6L). stimulated oxygen consumption; however, Klf9 mutation significantly impaired the compound-stimulated oxygen DISCUSSION consumption, clearly suggesting that KLF9 is required for Previously, a human genetic study (GWAS) suggested that the thermogenic function of brown and beige fat in vivo the rs11142387 single nucleotide polymorphism located in (Fig. 6E and F). the promoter region of KLF9 is associated with obesity in diabetes.diabetesjournals.org Fan and Associates 2617 the East Asian population. However, the nature of KLF9 studies are required to clarify the molecular mechanism affecting human BMI was not explored. In the current underlying the differences in function between KLF9 and study, we show that fat KLF9 regulates energy metabolism PGC1a. by stimulating the expression of Pgc1a, a master regulator In summary, the results of the current study suggest of oxidative phosphorylation and thermogenesis. Global that KLF9 is a critical regulator of thermogenesis of fat. Klf9-deficient mice exhibited obesity; conversely, Klf9 transgenic mice gained less weight and were resistant to obesity induced by an HFD. Of note, although our in vitro Funding. This work was supported by the National Key Research and and in vivo data indicate KLF9 stimulates fat cell thermo- Development Program of China (2018YFA0800601), the National Natural Science Foundation of China (grants 81730024, 81825004, 81670749, and 81471079), genesis, we cannot rule out the possibility that the Klf9 fi and the Natural Science Foundation of Ningxia (2020AAC03422). de ciency in other tissues also contributes to the obesity in fl fi Duality of Interest. No potential con icts of interest relevant to this article global Klf9-de cient mice. Of note, in our study the body were reported. – weight of WT mice at 21 57 weeks of age on a chow diet is Author Contributions. Y.C., H.F., Yu.Z., and J.Z.L. contributed to de- higher than that in other studies in the field. The possible signing research studies. H.F., Yu.Z., J. Zhang, and Q.Y. contributed to conducting reason is that fat in the chow diet in this study accounts for experiments. H.F., Yu.Z., J. Zhang, Q.Y., Y.S., Q.S., J.L., Y.G., X.W., L.Z., and Yi.Z. 13.8% of total calories (cat. no. 1010010; Xietong Organ- contributed to acquiring data. H.F., Yu.Z., J. Zhang, Q.Y., Y.S., Q.S., P.L., J. Zhao, ism, Jiangsu, China), which is a little higher than that in and Q.C. contributed to analyzing data. J. Zhao, Q.C., and J.Z.L. contributed regular diet from Research Diets (10% of total calories to providing reagents. Y.C. wrote the manuscript. Y.C. is the guarantor of from fat, cat. no. D12450J). this work and, as such, had full access to all the data in the study and Moreover, we observed a reverse association between takes responsibility for the integrity of the data and the accuracy of the data analysis. Klf9 mRNA levels in fat and human BMI. These data confirm its physiologically thermogenic role and con- References servation of KLF9 function between different species. 1. Rosen ED, Spiegelman BM. What we talk about when we talk about fat. 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