A Small Molecule, UAB126, Reverses Diet-Induced Obesity and Its Associated Metabolic Disorders

Diabetes Volume 69, September 2020 2003

A Small Molecule, UAB126, Reverses Diet-Induced Obesity and its Associated Metabolic Disorders

Guang Ren,1,2 Teayoun Kim,1,2 Hae-Suk Kim,1,2 Martin E. Young,2,3 Donald D. Muccio,4 Venkatram R. Atigadda,5,6 Samuel I. Blum,2,7 Hubert M. Tse,2,7 Kirk M. Habegger,1,2 Sushant Bhatnagar,1,2 Tatjana Coric,8 Mary-Ann Bjornsti,8 Anath Shalev,1,2 Stuart J. Frank,1,2 and Jeong-a Kim1,2

Diabetes 2020;69:2003–2016 | https://doi.org/10.2337/db19-1001

Targeting retinoid X receptor (RXR) has been proposed Obesity in the U.S. is an epidemic, affecting more than one- as one of the therapeutic strategies to treat individuals third of American adults. Obesity elevates risk for a wide with metabolic syndrome, as RXR heterodimerizes with range of health problems, including diabetes and cardiovas- THERAPEUTICS AND PHARMACOLOGY multiple nuclear receptors that regulate genes involved cular diseases (1). Modulation of nuclear receptors (NRs) has in metabolism. Despite numerous efforts, RXR ligands been suggested as one of the most attractive therapeutic (rexinoids) have not been approved for clinical trials to strategies against obesity-associated cardiometabolic diseases treat metabolic syndrome due to the serious side effects (2). Targeting retinoid X receptor (RXR) may be especially such as hypertriglyceridemia and altered thyroid hor- useful to modulate multiple NRs simultaneously because RXR mone axis. In this study, we demonstrate a novel rexinoid- dimerizes with other NRs, including peroxisome proliferator– like small molecule, UAB126, which has positive activated receptors (PPARs), liver X receptor (LXR), retinoic effects on metabolic syndrome without the known acid receptor, and farnesoid X receptor (2). Moreover, these side effects of potent rexinoids. Oral administration of NRs regulate carbohydrate and lipid metabolism (2). There UAB126 ameliorated obesity, insulin resistance, hepatic are three RXR isotypes—RXRa,-b,and-g (NR2B1–3)—in steatosis, and hyperlipidemia without changes in food mammals that are encoded by distinct genes (3). These intake, physical activity, and thyroid hormone levels. RNA-sequencing analysis revealed that UAB126 reg- isotypes of RXR are differentially expressed in various tissues. ulates the expression of genes in the liver that are RXRa is predominantly expressed in mouse liver, while RXRb modulated by several nuclear receptors, including is preferentially expressed in brain (4). RXRg is expressed in peroxisome proliferator–activated receptor a and/or skeletal and cardiac muscle (4). 9-Cis-retinoicacidisapotent liver X receptor in conjunction with RXR. Furthermore, ligand for RXR in vitro, but has not been found in vivo (5). UAB126 not only prevented but also reversed obesity- Thus, RXR is an orphan NR. Several compounds that selec- associated metabolic disorders. The results suggest that tively bind to RXR (rexinoid) have been developed and have optimized modulation of RXR may be a promising strat- been shown to have glucose-lowering, insulin-sensitizing, and egy to treat metabolic disorders without side effects. antiobesity effects (6–8). However, most of these rexinoids Thus, the current study reveals that UAB126 could be an exhibit serious side effects, including hypertriglyceridemia, attractive therapy to treat individuals with obesity and its hepatomegaly, and alterations of the thyroid hormone axis comorbidities. (6). These side effects may be attributed to the potent and

1Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, 8Department of Pharmacology and Toxicology, University of Alabama at Birming- University of Alabama at Birmingham, Birmingham, AL ham, Birmingham, AL 2 UAB Comprehensive Diabetes Center, University of Alabama at Birmingham, Corresponding author: Jeong-a Kim, [email protected] Birmingham, AL Received 4 October 2019 and accepted 18 June 2020 3Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL This article contains supplementary material online at https://doi.org/10.2337/ 4Department of Biochemistry and Molecular Genetics, University of Alabama at figshare.12505421. Birmingham, Birmingham, AL © 2020 by the American Diabetes Association. Readers may use this article as 5Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL long as the work is properly cited, the use is educational and not for profit, and the 6Department of Dermatology, University of Alabama at Birmingham, Birmingham, work is not altered. More information is available at https://www.diabetesjournals AL .org/content/license. 7Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 2004 A Novel Treatment for Obesity and Diabetes Diabetes Volume 69, September 2020 nonselective stimulation of RXR (9). Therefore, optimal Body Composition Measurement modulation of RXR activities may advance the therapeutic These experiments were conducted by the UAB Small Animal potential of rexinoids. Using our understanding of the Phenotyping Core facility funded by our Nutrition Obesity structural interactions between rexinoids and RXR, we Research Center. In vivo body composition (total body fat designed and synthesized a novel rexinoid-like small mol- and lean tissue) of mice was determined using an EchoMRI ecule, UAB126, from already known rexinoid UAB30 (10). 3-in-1 quantitative magnetic resonance machine (Echo Med- In this study, we introduce a novel compound, UAB126, ical Systems, Houston, TX). Quantification of results was which reduces obesity, insulin resistance, and dyslipidemia standardized by conducting a system test using a known fat without changes in thyroid hormone level (6). standard prior to experimental measurements being taken.

RESEARCH DESIGN AND METHODS Glucose Tolerance Test and Insulin Tolerance Test Synthesis of UAB126 Intraperitoneal glucose tolerance test (GTT) and insulin UAB126 was synthesized in two steps starting from naph- tolerance test (ITT) were performed after 6 h fasting. During thalene boronic acid using Suzuki reaction conditions. Cou- GTT, tail-vein blood glucose level was determined at 0, 15, pling of boronic acid 1 with p-bromobenzaldehyde in the 30, 60, and 120 min with a hand-held glucometer (FreeStyle; presence of Tetrakis palladium gave the desired aldehyde Abbott, Abbott Park, IL) after intraperitoneal injection of 3 in 70% yield after purification by chromatography. Knoe- 2 g/kg body weight glucose. During ITT, glucose level was venagel-type condensation of aldehyde 3 with malonic acid determined 0, 15, 30, 60, and 90 min after intraperitoneal provided the UAB126 as a single isomer. Purity of the injection of 0.5 units/kg body weight insulin. UAB126 was assessed by high-performance liquid chromatography (HPLC) analysis using the Agilent 1260 Infinity II Indirect Calorimetry instrument. HPLC analysis was performed using an Agilent Respirationrate,foodintake,energyexpenditure,andphys- Technologies column (poroshell 120, C18, 2.7 mm; 4.6 3 ical activity were accurately quantified as previously described 100 mm). The eluent was 65% acetonitrile/35% water and using the Comprehensive Laboratory Animal Monitoring 0.1% formic acid with a flow rate of 1 mL/min (isocratic). System (Columbus Instruments Inc., Columbus, OH) (11). Fluorescence (excitation 230, emission 460) and diode array Respiratory exchange ratio (RER) was calculated as CO2 detectors were used to detect the compound. generation/O2 consumption. Analysis of Serum Samples Measurement of Serum Parameters Six-week-old male C57BL/6J mice were purchased from The Mice were fasted overnight, and the blood was collected Jackson Laboratory (Bar Harbor, ME) and acclimated for during euthanization. The serum was collected after centri- 1 week before the experiments. UAB126 was dissolved in fugation (2,200g for 10 min at 4°C) of clotted blood. Serum 1:9 ethanol/PEG400, and then vehicle or UAB126 (100 mg/kg insulin level was assessed by an ultrasensitive mouse insulin body weight/day) was administered orally by gavage for 5 days. ELISA (Crystal Chem Inc., Elk Grove Village, IL). Free fatty One hour after the finaldose,serumwascollectedandsub- acids were assessed using the NEFA-HR(2) kit (Wako, Rich- jected to HPLC analysis. A total of 100 mL of serum samples mond, VA). Triglyceride (TG) levels were assessed using was diluted with HCl (0.5N, 100 mL) and 0.5 mL of ethyl Pointe Scientific Inc. (Pittsburgh, PA) TG liquid reagents. acetate. Samples were vortexed and centrifuged (10,000 rpm Cholesterol level was assessed using Thermo Fisher Scientific for 10 min), and the supernatant liquid was separated and total cholesterol reagents (Pittsburgh, PA). The ELISA Mul- evaporated to dryness under a stream of nitrogen. Finally, the tiplex kit (Millipore Sigma-Aldrich, St. Louis, MO) was residue was dissolved in 0.5 mL of acetonitrile. Blank serum used according to the manufacturer’s instructions to mea- samples were also analyzed following the above conditions. sure triiodothyronine (T3) and thyroid-stimulating hormone Vehicle-treated serum samples spiked with UAB126 prior to (TSH) levels in serum. HOMA of insulin resistance (HOMA- extraction were used as a positive control for detection of IR) and QUICKI were calculated as previously described (12). UAB126.

Animal Housing and Maintenance Preparation of Tissue Lysates and Immunoblotting All animal procedures were performed in accordance with Mice were fasted for 6 h, and insulin (1 unit/kg) was the rules of and approved by the Animal Use and Care injected intraperitoneally 10 min before euthanasia. Skel- Committee at the University of Alabama at Birmingham etal muscle, liver, and epididymal adipose tissue were (UAB). C57BL/6J mice were purchased from The Jackson collected and frozen in liquid nitrogen until used. Tissue Laboratory. All animals were maintained in a temperature- homogenates were prepared in the cell lysis buffer and controlled facility with a 12:12-h light/dark cycle. Mice were processed according to the manufacturer’s instructions for fed low-fat diet (LFD) (10% calories from fat, D132021801; a TissueLyser II (Qiagen, Germantown, MD). Cell lysate Research Diets, Inc., New Brunswick, NJ) or high-fat diet was subjected to an immunoblotting with antibodies as (HFD) (60% calories from fat, D13021802; Research Diets, described previously (13) and visualized and quantiﬁed Inc.). Diets were mixed without or with UAB126 (1.2 g/kg using the ChemiDoc imaging system and Image Lab 5.0 of diet). Mice had free access to food and water. software (Bio-Rad Laboratories). diabetes.diabetesjournals.org Ren and Associates 2005

Immunohistochemistry buffer, treated with Fc block (#553142; BD Biosciences), Tissues were excised at the time of sacrifice and then fixed in and then stained with fluorochrome-conjugated antibodies 10% formaldehyde/PBS solution overnight. Paraffinblocks specific for Live/Dead (Thermo Fisher Scientific), indicated were prepared by the UAB Pathology Core Research Labo- surface markers, and rat IgG1 isotype controls. Cells were ratory. Tissue sections were deparaffinized, hydrated, and analyzed with the Attune NxT Flow Cytometer (Thermo then treated at 95°C to retrieve antigens in a plastic slide jar Fisher Scientific) with ;1,000,000 events collected for containing 10 mmol/L citrate buffer (pH 6.0) for 20 min. each sample and analyzed with FlowJo (10.6.2) software The sections were immersed in 0.3% hydrogen peroxide for (BD Biosciences). The flow cytometry gating scheme is 30 min, blocked with ABC blocking serum (catalog number shown in Supplementary Fig. 3. PK-6105; Vector Laboratories, Burlingame, CA) for 1 h, and then incubated overnight with an anti-FGF21 antibody Brown Adipocyte Differentiation (catalog number AF3057; R&D Systems, Minneapolis, Brown adipocyte differentiation of SVCs from inguinal MN). The tissue sections were processed using a Vectastain adipose tissue was performed as described previously (17). Elite ABC Kit (Vector Laboratories) according to the man- The cells were incubated with induction media containing ufacturer’s instructions to visualize the staining of FGF21. dexamethasone (2 mg/mL), 3-isobutyl-1-methylzanthine In some cases, the tissue sections were then stained with (0.5 mmol/L), insulin (5 mg/mL), and T3 (1 nmol/L) for hematoxylin and eosin and then mounted with Permount. 2 days and then with maintenance media containing in- The slides were photographed and analyzed by light sulin (5 mg/mL) and T3 (1 nmol/L) for an additional microscopy. 6 days. The maintenance media was changed every 2 days.

Quantitative Real-Time PCR Statistical Analysis fi Tissues were collected, snap-frozen in liquid nitrogen, and Statistical signi cance was assessed by two-tailed Student then stored until subjected to an analysis. Total RNA was t test or one-way ANOVA followed by Newman-Keuls or prepared by homogenizing tissues in TRIzol (Invitrogen, two-way ANOVA by Tukey or Bonferroni post hoc tests. fi Carlsbad, CA) according to the manufacturer’s instructions. Differences were considered to be statistically signi cant if , One microgram of total RNA was used for cDNA synthesis by the P value was 0.05. Statistical analyses were performed using an Omniscript RT kit (Qiagen). Then, the cDNA was using Prism Version 8 (GraphPad Software, San Diego, CA). subjected to real-time quantitative PCR (qPCR) analysis by Data and Resource Availability using iQ SYBR Green Supermix (Bio-Rad Laboratories). Each All primary data and animal models used in the study are gene expression was normalized to b-actin or cyclophilin. available to investigators upon reasonable request. RNA-Sequencing Analysis RESULTS Livers were isolated from HFD- and HFD plus UAB126– treated mice after a 12-week feeding (n 5 2). Total RNA Oral Administration of UAB126 Prevents HFD-Induced was isolated as described above and then subjected to an Obesity and Increases Fatty Acid Oxidation RNA-sequencing (RNA-seq) analysis. RNA-seq analysis was We designed and synthesized UAB126 from a known rexi- performed by the UAB Genomics Core facility. NextSeq500 noid, UAB30 (10,18) (Fig. 1A and B). The purity of the fi was used for the analysis. A total of 1,334 genes were UAB126 was evaluated by HPLC analysis. We con rmed that . selected for the over 62-fold differential expression. These UAB126 was 99% pure (Fig. 1C). To determine whether genes were subjected to an online gene enrichment tool, UAB126 has an effect on body weight, 6-week-old male Enrichr (14,15), and Ingenuity. C57BL/6J mice were fed LFD or HFD with or without UAB126 (1.2 mg/g diet). Mice fed HFD with UAB126 gained Isolation of Adipose Stromal Vascular Cells and Flow significantly less body weight compared with the mice fed Cytometry HFD without UAB126 (Fig. 2A). Attenuation of HFD-induced Stromal vascular cells (SVCs) were isolated from epididy- weight gain was mainly due to reduction in fat mass (45% mal white adipose tissues (WATs) as described previously reduction) (Fig. 2B), but not in lean mass (Fig. 2C). To (16). Briefly, epididymal fat pads were harvested from mice examine whether UAB126 alters energy metabolism and fuel and chopped into small pieces in PBS containing 2% BSA, source utilization, metabolic parameters were evaluated by and then collagenase type 2 and DNase I were added to the indirect calorimetry. UAB126-treated mice displayed in- tissue suspension. After the suspension was incubated for creased fat oxidation rate (indicated by decreased RER in 20 min at 37°C with shaking, the cells were filtered through HFD-fed mice (Fig. 2D). The effect of UAB126 on metabolic a 100-mm nylon mesh and centrifuged at 1,000 rpm for rate in HFD-fed mice was manifested by the enhanced 5 min. Five minutes before completion of the digestion, 0.1 oxygen consumption (VO2) and carbon dioxide production mol/L EDTA was added. The red blood cells were lysed, and (VCO2)(Fig.2E and F), whereas no difference in food in- single cells were resuspended in DMEM containing 10% take or physical activity was observed in response to FBS and incubated for 4 h at 37°C with BD GolgiPlug UAB126 supplementation (Fig. 2G and H). While UAB126 (1:1,000) to measure intracellular cytokine synthesis. Fol- did not affect RER in LFD-fed mice (Supplementary Fig. lowing the 4-h incubation, cells were resuspended in FACS 1A), VO2 and VCO2 were still increased in LFD-fed mice 2006 A Novel Treatment for Obesity and Diabetes Diabetes Volume 69, September 2020

(Supplementary Fig. 1B and C). To understand the mecha- UAB126-treated groups compared with the untreated nism by which UAB126 regulates metabolic rate, we exam- groups. UAB126 enhanced the phosphorylation of insulin ined whether UAB126 regulates thermogenic genes in brown signaling pathway molecules, including insulin receptor sub- adipose tissue (BAT). UAB126 reduced the lipid accumula- strate-1 (IRS-1), protein kinase B (Akt), glycogen synthase tion in BAT (Fig. 2I) and increased the gene expression of kinase 3b (GSK3b), and p70S6 kinase (p70S6K) in skeletal Ucp1, Cytb, Cpt1a, Cpt1b, Cidea, Elov3,andPrdm16 that are muscle, liver, and WAT in HFD-fed mice (Fig. 4F–V). While associated with thermogenesis and fatty acid oxidation (Fig. UAB126 also enhanced insulin signaling in skeletal muscle 2J). Furthermore, UAB126 did not affect the circulating (Fig. 4F–K), its effect was only modest in liver (Fig. 4L–Q) thyroid hormone level (Fig. 2K). The results suggest that and WAT (Fig. 4R–V) of LFD-fed mice. Interestingly, the UAB126 prevents HFD-induced weight gain by increasing protein level of IRS-1 in liver was increased by UAB126 in fat utilization without altering food intake, physical activity, both LFD- and HFD-fed mice (Fig. 4Q). Taken together, and T3 level. these data suggest that UAB126 enhances insulin sensitivity in metabolic tissues and improves glucose homeostasis. UAB126 Modulates Genes That Are Involved in Lipolysis and Proinflammatory Responses in WAT UAB126 Prevents HFD-Induced Hepatic Steatosis and Hyperlipidemia The size of adipocytes in epididymal fat pad was decreased Nonalcoholic fatty liver disease is prevalent in obesity and when UAB126 was supplemented (Fig. 3A). Consistent type 2 diabetes (20). We examined whether UAB126 has an with the reduced size of WAT, treatment with UAB126 effect on HFD-induced hepatic steatosis. Analysis of liver increased the expression of genes associated with lipolysis histology and TG content revealed that supplementation (hormone-sensitive lipase), insulin sensitivity (adiponectin), and fatty acid oxidation (PPARa and acyl coenzyme with UAB126 decreased TG accumulation in the liver tissue (Fig. 5A and B). Moreover, UAB126 lowered the circulating oxidase) in the WAT (Fig. 3B). Increased inflammatory TG, nonesterified fatty acids (NEFA), and cholesterol levels response and macrophages in WAT are the typical feature (Fig. 5C–E). The lipid-lowering effects of UAB126 were of obesity (19). As such, adipose tissue crownlike structure consistent with the decreased lipogenic gene expression in and the expression of proinflammatory genes were in- the liver (Fig. 5F). Thus, our data suggest that UAB126 may creased in HFD-fed obese mice compared with those in prevent the HFD-induced nonalcoholic fatty liver disease by LFD-fed mice (Fig. 3C). These chronic inflammatory modulation of genes that are involved in lipid metabolism. markers were reduced when UAB126 was supplemented in HFD-fed mice (Fig. 3C and D). To confirm the anti- UAB126 Modulates Gene Expression Profiles Involved inflammatory effect of UAB126 in adipose tissue, FACS in Glucose and Lipid Metabolism analyses were performed on stromal vascular cells isolated To further examine the hepatic gene expression regulated by from epididymal adipose tissue (gating strategy is shown in UAB126, we performed RNA-seq analysis on the livers Supplementary Fig. 2, and the representative FACS plots are isolated from mice fed HFD with or without UAB126. shown in Supplementary Fig. 3). The populations of myeloid Because UAB126 was developed as a potential rexinoid, cells, monocytes, and macrophages were increased in HFD- we investigated whether UAB126 can affect the genes fed mice, whereas they were decreased when UAB126 was that are regulated by RXR and other NRs that heterodi- supplemented (Fig. 3E–G). Furthermore, the population of merize with RXR. The genes that were increased or decreased 1 proinflammatory myeloid cells, TNF-a–positive CD11b cells, more than twofold by UAB126 were selected for Ingenu- was increased in HFD-fed mice, but it was decreased when ity Pathway and Enrichr (https://amp.pharm.mssm.edu/ UAB126 was added to the HFD (Fig. 3H). The populations of Enrichr) chromatin immunoprecipitation enrichment anal- MHC class II–positive macrophages and TNF-a–positive M1 ysis (14,15). Chromatin immunoprecipitation enrichment macrophages were increased in HFD-fed mice, whereas they analysis demonstrated that RXR, LXR, and PPARa are the were decreased when UAB126 was supplemented in HFD- major transcription factors involved in UAB126-mediated fed mice (Fig. 3I and J). In contrast, UAB126 did not affect hepatic gene regulation. Gene enrichment analysis showed the populations of myeloid cells in LFD-fed mice. These that a total of 342 genes were regulated by RXR, LXR, or suggest that UAB126 stimulates lipolysis and decreases PPARa (Fig. 6A). Among the 342 genes, 58 genes were proinflammatory response in WAT. commonly regulated by all three NRs (RXR, LXR. and PPARa). The enriched genes regulated by RXR were sub- UAB126 Prevents HFD-Induced Insulin Resistance jected to a gene ontology test, which indicated that the RXR Next, we examined whether UAB126 prevents HFD-induced target genes regulated by UAB126 in the liver were involved glucose intolerance. UAB126 improved glucose tolerance in in lipid and glucose homeostasis, hepatic steatosis, and HFD-fed mice only (Fig. 4A), while UAB126 improved insulin hepatokines implicated in adipocyte browning (14,15) (Table tolerance in both LFD-fed and HFD-fed mice (Fig. 4B). 1). The genes enriched for mammalian metabolic phenotypes UAB126 lowered fasting glucose levels only in HFD-fed are demonstrated by a heat map (Fig. 6B). Interestingly, mice (Fig. 4C), while UAB126 lowered fasting insulin levels fibroblast growth factor 21 (Fgf21) was the most common in both HFD-fed and LFD-fed mice (Fig. 4D). HOMA-IR (Fig. gene that appeared in the top selected categories. Thus, 4E) indicates that insulin sensitivity was improved in the FGF21 may be an important mediator of metabolic effects of diabetes.diabetesjournals.org Ren and Associates 2007

Figure 1—UAB126 was synthesized by modifications of the previously known rexinoid UAB30. A: UAB126 was designed based upon the previously known rexinoid UAB30. B: UAB126 was synthesized in two steps starting from naphthalene boronic acid (1) using Suzuki reaction conditions. Coupling of boronic acid 1 with p-bromobenzaldehyde (2) in presence of Tetrakis palladium gave the desired aldehyde 3 in 70% yield after purification by chromatography. Knoevenagel-type condensation of aldehyde 3 with malonic acid provided the UAB126 as a single isomer. C: Purity of the UAB126 assessed by HPLC analysis. UAB126 was detected by diode array detectors (DAD) (top panel) and fluorescence (FLD) (excitation [Ex] 230 and emission [Em] 460) (bottom panel). The major single peaks from the both detection methods demonstrate that UAB126 is .99% pure. Ref, reference; Sig, signal. 2008 A Novel Treatment for Obesity and Diabetes Diabetes Volume 69, September 2020

Figure 2—Oral administration of UAB126 prevents HFD-induced obesity and increases fatty acid oxidation. A: Six-week-old male mice were fed LFD or HFD (60% calories from fat) for 12 weeks. Body weight was monitored weekly. Two-way ANOVA: **P , 0.01, ***P , 0.001, ****P , 0.0001 (n 5 12). Fat mass (B) and lean mass (C) of HFD-fed mice were measured by a quantitative magnetic resonance test (n 5 6). D: RER in HFD-fed mice was measured by indirect calorimetry (n 5 5). Oxygen consumption (E) and carbon dioxide production (F) in HFD-fed mice were measured by an indirect calorimetry (n 5 5). Food intake (G) and physical activity (H) were measured by using indirect calorimetry. The mice were fed HFD and housed at 25°C. The data are shown as average of 48 h (n 5 5). I: Representative histology of BAT (hematoxylin and eosin staining). J: mRNA expression of thermogenic genes in BAT (n 5 5). K: Serum T3 levels were measured after overnight fasting. (n 5 6). Data are presented as mean 6 SEM. *P , 0.05; **P , 0.01. Black circles, LFD; red circles, LFD 1 UAB126; black squares, HFD; red squares, HFD 1 UAB126. hr, hours; V, vehicle; wk, weeks. diabetes.diabetesjournals.org Ren and Associates 2009

Figure 3—UAB126 modulates genes that are involved in lipolysis and proinﬂammatory response in WAT. A: WAT isolated from HFD-fed mice with or without UAB126 (n 5 6). B: mRNA levels of Hsl, Adiponectin, Ppara, and Acox in WAT (n 5 6). C: Representative histology of epididymal fat tissue (hematoxylin and eosin staining). Arrows indicate the crownlike structure. D: mRNA expression of the proinﬂammatory responsive genes in WAT by qPCR analyses (n 5 6). E–J: SVCs were isolated from epididymal WAT as described in RESEARCH DESIGN AND METHODS. SVCs were subjected to FACS analyses with the indicated antibodies (n 5 5–7). Data are presented as mean 6 SEM. **P , 0.01; ***P , 0.001; ****P , 0.0001. Black circles, LFD; red circles, LFD 1 UAB126; black squares, HFD; red squares, HFD 1 UAB126. V, vehicle. 2010 A Novel Treatment for Obesity and Diabetes Diabetes Volume 69, September 2020

Figure 4—UAB126 prevents HFD-induced insulin resistance. A (left): GTT. The mice were fed LFD (n 5 6) or HFD (n 5 6) without or with UAB126 supplementation for 13 weeks. Glucose (2 g/kg) was intraperitoneally injected after 6 h fasting, and then blood glucose was measured. A (right): The area under the curve (AUC) for intraperitoneal GTT (n 5 11). **P , 0.001, ***P , 0.0001 HFD vs. HFD 1 UAB126 (n 5 11). B: ITT. Insulin (0.5 units/kg) was intraperitoneally injected after overnight fasting, and the blood glucose was measured. The slope for the ﬁrst 30 min was calculated as Kg30.*P , 0.05, **P , 0.001, ***P , 0.0001 (n 5 5–6). C: Overnight-fasting serum glucose levels (n 5 5). D: Overnight-fasting insulin levels (n 5 5). E: HOMA-IR (n 5 5). F–V: Insulin (1 unit/kg) was intraperitoneally injected after 6 h fasting. The mice were euthanized 5 min after the injection. Skeletal muscle (F–K), liver (L–Q), and epididymal adipose tissue (R–V) were harvested and subjected to immunoblotting with the indicated antibodies. The band intensity was normalized by unphosphorylated proteins. GAPDH was used as a loading control. The images of Western blot were quantiﬁed by using Image Lab software (n 5 5). Data are presented as mean 6 SEM. *P , 0.05; **P , 0.01; ***P , 0.001; ****P , 0.0001. Black circles, LFD; red circles, LFD 1 UAB126; black squares, HFD; red squares, HFD 1 UAB126. HF, HFD; LF, LFD; p, phosphorylated; V, vehicle. diabetes.diabetesjournals.org Ren and Associates 2011

Figure 5—UAB126 prevents HFD-induced hepatic steatosis and hyperlipidemia. A: Representative histology of liver hematoxylin and eosin staining. B: Hepatic TG contents (n 5 5). Overnight-fasting serum levels of TG (C), NEFA (D), and cholesterol (E)(n 5 5–6). F: qPCR analysis for the genes that are involved in lipogenesis. (n 5 4–5). The quantiﬁed data are presented as mean 6 SEM. *P , 0.05; **P , 0.01; ****P , 0.0001. Black circles, LFD; red circles, LFD 1 UAB126; black squares, HFD; red squares, HFD 1 UAB126. V, vehicle. 2012 A Novel Treatment for Obesity and Diabetes Diabetes Volume 69, September 2020

UAB126. Next, we examined whether UAB126 regulates for 12 weeks prior to administration of UAB126, and then the FGF21 expression. Treatment with UAB126 elevated the ex- mice were treated with or without UAB126. Treatment with pression of Fgf21 protein and mRNA in the liver (Fig. 6C and D) UAB126 induced weight loss (Fig. 7A and B). UAB126 improved and the circulating levels of serum FGF21 (Fig. 6E). The results glucose and insulin tolerance (Fig. 7C–F) as well as reduced suggest that UAB126 regulates genes that are involved in fasting serum glucose and insulin levels (Fig. 7G and H). An glucose and lipid metabolism by modulation of NRs. index of insulin sensitivity, HOMA-IR, was also signiﬁcantly improved by UAB126 (Fig. 7I). Furthermore, UAB126 lowered UAB126 Reverses HFD-Induced Obesity, Insulin circulating and hepatic TG levels (Fig. 7J and K)andcirculating Resistance, and Hyperlipidemia cholesterol level (Fig. 7L). UAB126 did not alter the serum T3 To test the therapeutic ability of UAB126, we examined and TSH levels (Fig. 7M and N). These data suggest that whether UAB126 can reverse obesity, insulin resistance, and UAB126 provides a promising therapeutic approach in the hyperlipidemia. Obesity was induced by feeding mice with HFD settings of obesity with insulin resistance and hyperlipidemia.

Figure 6—UAB126 modulates gene expression proﬁles involved in glucose and lipid metabolism. A: The major NRs that are modulated by UAB126 and the number of genes that are regulated by each NR. B: A heat map of genes that are involved in mammalian metabolic phenotype. C: A representative immunohistochemistry of liver. FGF21 expression was assessed by immunohistochemistry with an anti- FGF21 antibody. D: Fgf21 mRNA expression by a qPCR analysis on livers (n 5 5–6). E: Serum FGF21 levels. Overnight fasting serum was subjected to an ELISA for FGF21 (n 5 5–6). The quantiﬁed data are presented as mean 6 SEM. **P , 0.01; ***P , 0.001. Black circles, LFD; red circles, LFD 1 UAB126; black squares, HFD; red squares, HFD 1 UAB126. V, vehicle. diabetes.diabetesjournals.org Ren and Associates 2013

Table 1—Ontology with enriched genes for RXR Terms Genes Lipid homeostasis FABP1, EHHADH, ST3GAL5, TXNIP, IRS2, CYP4A14, THRSP, LPIN1, ADIPOR2, AGPAT2, ACACB, FGF21 Circulating cholesterol level LIPG, APOC1, APOA2, SAA1, APOA1, TXNIP, IRS2, DBI, FGF21, GNMT, CYP17A1 Hepatic steatosis IGFBP1, PHYH, FGL1, TXNIP, AHR, MAT1A, LPIN1, PCK1, AGPAT2, FGF21, GNMT Glucose homeostasis SOCS2, FABP1, TREH, ST3GAL5, TXNIP, IRS2, LPIN1, ADIPOR2, AGPAT2, ACACB, FGF21 Circulating TG level LIPG, APOC1, APOA1, TXNIP, IRS2, LPIN1, PCK1, AGPAT2, ACACB, FGF21 Liver TG level EGR1, FGL1, APOC1, DBI, LPIN1, AGPAT2, FGF21 Brown fat cell morphology FGL1, PCK1, ADIPOR2, FGF21 Circulating glucose level SOCS2, ONECUT1, AFMID, LPIN1, ADIPOR2, AGPAT2, FGF21 Circulating insulin level PHYH, FGL1, UCP3, APOA2, AGPAT2, FGF21

UAB126 Requires In Vivo Metabolism for its Activity and hyperlipidemia without causing hypertriglyceridemia or To further analyze the molecular mechanisms by which noticeable adverse effects on the thyroid hormone axis. UAB126 may confer its beneficial metabolic effects, we Previously known rexinoids such as LG1069 (bexaro- tested whether it also regulated gene transcription in vitro. tene), LG100268, AGN194204, and LG101506 do not have Interestingly, we found that while UAB30, a proven rexi- an effect on body weight (6), but demonstrate glucose- noid (10), induced RXR element–driven luciferase activity lowering effects (6,27,29,30). One study demonstrated (21) and endogenous Ucp-1 gene expression in cell culture that administrations of LG100268 by gavage or intrace- (22), UAB126 did not (Supplementary Fig. 4A and B). rebral ventricular injection decrease food consumption and Together with the pronounced effects observed in vivo, body weight (27,31–33). Although we have not tested the this raised the possibility that UAB126 has to be metab- effects of UAB126 on the central nervous system, UAB126 olized in vivo for its activity. Therefore, we analyzed the does not affect food intake or physical activity (Fig. 2G and UAB126-treated serum samples using HPLC. Indeed, H). Instead, UAB126 stimulated fat utilization (Fig. 2D) HPLC spectra demonstrated that the serum of vehicle- and increased metabolic rate (Fig. 2E and F). Thus, the treated mice spiked with UAB126 showed the same profile mechanism by which UAB126 regulates body weight seems with a peak at 3.96 min (Supplementary Fig. 4C) as that of to be distinct compared with that of the known rexinoids. the purified compound (Fig. 1). In contrast, the serum Interestingly, UAB126 stimulated the expression of genes from UAB126-treated mice showed a shifted profile with that are involved in thermogenesis in BAT (Fig. 2J) and the major peak at 3.24 min (Supplementary Fig. 4D). These lipolysis in WAT (Fig. 3B). The increased fat utilization in results suggest that UAB126 is metabolized in vivo and UAB126-treated animals may be due to FGF21, which is may act as a prodrug that must be metabolized either by a known factor that increases fat oxidation (34,35). Pre- intestinal or hepatic enzymes to give rise to an active vious reports have shown that the promoter of Fgf21 is compound. directly regulated by RXR-PPARa or RXR-LXR heterodimers (36,37). Thus, it is likely that as a rexinoid-like DISCUSSION molecule, UAB126 regulates Fgf21 expression via RXR. Nuclear receptors play important roles in energy homeosta- Furthermore, our RNA-seq analysis supports this idea sis, glucose and lipid metabolism, and nutrient absorption (Fig. 6). However, our mechanistic studies revealed that (2). Although RXR can function as a homodimer, it hetero- unlike UAB30, a proven rexinoid (10), UAB126 was not dimerizes with other NRs and exhibits diverse physiological active in vitro and metabolized in vivo (Supplementary Fig. and pathophysiological roles (2,23). The known ligand for 4). This suggests that UAB126 acts as a prodrug and must RXR is 9-cis retinoic acid, a metabolite of vitamin A. How- be metabolized in vivo to exert its metabolic functions. In ever, it is controversial whether 9-cis retinoic acid is an fact, this feature may have contributed to the favorable endogenous ligand or not because 9-cis retinoic acid is not safety profile observed with UAB126 as opposed to other found in vivo (24–26). Due to the important roles of RXR in rexinoids. Obviously, future studies will have to define the metabolism, several synthetic RXR ligands (rexinoids) have exact metabolites of UAB126 and their exact functions. been developed and suggested as treatments for diabetes Although liver is the primary contributor to the circu- (6,27,28). However, most of the previously developed rexi- lating FGF21 levels (38), other tissues such as adipose noids have side effects, including elevation of TGs, hepato- tissue and skeletal muscle may also contribute to the megaly, and an alteration of the thyroid hormone axis (6). In serum FGF21 levels (39,40). However, Fgf21 gene expres- the current study, we report a novel rexinoid-like small sion was not stimulated by UAB126 in other tissues (data molecule (Fig. 1) that is a potential treatment for metabolic not shown). The data suggest that UAB126 regulates Fgf21 disease, including obesity, insulin resistance, hyperglycemia, gene expression in a tissue-specific manner possibly by 2014 A Novel Treatment for Obesity and Diabetes Diabetes Volume 69, September 2020

Figure 7—UAB126 reverses HFD-induced obesity, insulin resistance, and hyperlipidemia. Mice were fed HFD for 12 weeks and then divided into two groups. The mice were treated without UAB126 (n 5 5) or with UAB126 (1.2 g/kg diet; n 5 6) for the additional 8 weeks. A: Body weight was monitored weekly. The arrow indicates the time when the mice were treated with vehicle or UAB126. B: Fat mass was measured by a quantitative magnetic resonance. C and D: Intraperitoneal GTT (ipGTT). C: Serum glucose levels were measured during ipGTT (2 g/kg) after overnight fasting. D: The area under the curve (AUC) for ipGTT. E and F: ITT. E: Serum glucose levels were measured during intraperitoneal ITT (0.5 units/kg) after 6 h fasting. F:Kg30 indicates the slope during the first 30 min. G: Overnight-fasting serum glucose levels. H: Overnight-fasting insulin levels. I: HOMA-IR. Overnight-fasting serum levels of TG (J), NEFA (K), and cholesterol (L). M: Serum T3 levels were measured by an ELISA. N: Serum TSH levels were measured by an ELISA. Data are presented as mean 6 SEM. *P , 0.05, **P , 0.01, ***P , 0.001 vehicle, ****P , 0.0001 (n 5 5) vs. UAB126 (n 5 6). Black squares, HFD; red squares, HFD 1 UAB126. V, vehicle; wk, weeks. diabetes.diabetesjournals.org Ren and Associates 2015 differential recruitment of coactivators to the transcrip- have been developed and have antidiabetic effects without tion sites. This hypothesis warrants further studies re- demonstrating hypertriglyceridemia or hypothyroidism (9). garding tissue-specific UAB126 activities. Thus, RXR seems to be a promising target if the specificity Because RXR plays important roles in insulin sensitiv- and potency are optimally modulated. ity, cholesterol metabolism, and macrophage differentiation, In the current study, we introduce a novel rexinoid-like UAB126 may also modulate macrophage differentiation and molecule that not only prevents but also reverses diet- affect the microenvironment of WAT (16,41,42). Indeed, induced obesity, insulin resistance, steatosis, and hyperlip- UAB126 increases lipolysis and suppresses proinflammatory idemia without altering food intake and physical activity. responsesinWAT(Fig.3). Thus, UAB126 may be a powerful pharmacological treatment UAB126 significantly improved glucose tolerance and option for obesity-associated cardiometabolic disorders. insulin sensitivity, although the effects on glucose tolerance seemed to be milder. This could be explained by an in- complete suppression of hepatic glucose production as a re- Acknowledgments. The authors thank the members of the UAB Com- prehensive Diabetes Center for useful discussions. sult of the dramatically decreased insulin levels observed in Funding. This study was supported by the UAB Diabetes Research Center– UAB126-treated mice (Fig. 4D) in response to the increased sponsored pilot and feasibility program supported by National Institutes of Health/ – insulin sensitivity in skeletal muscle (Fig. 4F K). This is also National Institute of Diabetes and Digestive and Kidney Diseases (P30-DK- consistent with the data showing that reduction of fasting 079626), UAB Center for Clinical and Translational Science pilot and feasibility insulin (Fig. 4D) by UAB126 is greater than fasting glucose program by the NIH Clinical Center (UL1-TR-003096), UAB Comprehensive Di- (Fig. 4C). Another possibility is that UAB126 increases fat abetes Center and National Heart, Lung, and Blood Institute (R01-HL-128695 to oxidation (Fig. 2D), so the fuel source utilization favors fat J.-a.K.), National Institute on Aging (R03-AG-058078 to J.-a.K.), National Institute rather than glucose independent of insulin sensitivity. of Diabetes and Digestive and Kidney Diseases (R01-DK-112934 to K.M.H., R00- The biggest drawback from using potent RXR agonists DK-95975 and 1R01-DK-120684 to S.B., and DK-099550 to H.M.T.), and National as treatments for metabolic diseases is that they cause Institute of General Medical Sciences (T32-GM-109780 to S.I.B.). fl central hypothyroidism in humans and rodents (43,44). Duality of Interest. No potential con icts of interest relevant to this article were reported. Potent RXR agonists decrease TSH and T3 levels, and RXR Author Contributions. J.-a.K. devised and coordinated the project. G.R., directly suppresses thyrotropes, which is also related to the T.K., H.-S.K., and S.I.B. performed experiments. M.E.Y. performed indirect calorimetry. increased lipid levels (45). By contrast, UAB126 did not D.D.M. and V.R.A. invented and synthesized UAB126. M.E.Y. and J.-a.K. designed alter the levels of T3 (Fig. 7M) and TSH (Fig. 7N). Despite experiments. G.R., T.K., M.E.Y., D.D.M., V.R.A., H.M.T., K.M.H., M.E.Y., S.B., T.C., unaltered thyroid hormone levels, UAB126 is able to de- M.-A.B., A.S., S.J.F., and J.-a.K., provided expertise and wrote the manuscript. J.-a.K. is crease TG and cholesterol levels. Moreover, RNA-seq analysis the guarantor of this work and as such, had full access to all of the data in the study and does not indicate any thyroid hormone receptor activity by takes responsibility for the integrity of the data and the accuracy of the data analysis. UAB126. Thus, it is unlikely that thyroid hormone mediates Prior Presentation. 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