TGFBI remodels adipose metabolism by regulating the Notch-1 signaling pathway

TGFBI remodels adipose metabolism by regulating the Notch-1 signaling pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article TGFBI remodels adipose metabolism by regulating the Notch-1 signaling pathway Ju-Ock Nam, Seul Gi Lee, Seon Min Woo, Seung Un Seo, Ha-Jeong Kim, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1739900/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Mar, 2023 Read the published version in Experimental & Molecular Medicine → Version 1 posted 5 You are reading this latest preprint version Abstract Extracellular matrix proteins are associated with metabolically healthy adipose tissue and regulate inflammation, fibrosis, angiogenesis, and subsequent metabolic deterioration. In this study, we demonstrated that transforming growth factor-beta (TGFBI), an extracellular matrix (ECM) component, plays an important role in adipose metabolism and browning during high-fat diet-induced obesity. TGFBI KO mice were resistant to adipose tissue hypertrophy, liver steatosis, and insulin resistance. Furthermore, adipose tissue from TGFBI KO mice presented a large population of CD11b + macrophages, which control adipokine secretion through paracrine mechanisms. Mechanistically, we showed that inhibiting TGFBI-stimulated release of adipsin by Notch-1-dependent signaling resulted in adipocytes browning. TGFBI was physiologically bound to Notch-1 and stimulated its activation in adipocytes. Our findings revealed a novel protective effect of TGFBI deficiency in obesity that is realized via the activation of the Notch-1 signaling pathway. adipocyte browning extracellular matrix macrophage obesity TGFBI Notch-1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Obesity is a major health concern worldwide that is associated with several diseases, including cerebrovascular disease, hypertension, hyperlipidemia, and type II diabetes 1 . In most countries, the incidence of obesity and obesity-induced metabolic diseases has been increasing 2 , 3 . White adipose tissue (WAT) plays an important role in maintaining energy balance, including glucose homeostasis and lipid metabolism, by storing calories and secreting adipokines, such as leptin and resistin 4 , 5 . Several WATs contain thermogenic adipocytes (brite or beige adipocytes) that transform energy-storing white adipocytes into heat-producing beige adipocytes upon acute cold exposure 6 , 7 . Thus, browning of WAT is considered a potential strategy for treating obesity. Adipocytes are in constant contact with a network of insoluble proteins and polysaccharides that are part of the extracellular matrix (ECM) 8 . Changes in ECM deposition exhibit the characteristics of hyperplastic development in adipose tissue and are associated with reduced tissue plasticity 9 . The ECM microenvironment unlocks adipocyte differentiation in adult human bone marrow mesenchymal stem cells (BM-MSCs) 10 . Transforming growth factor-beta-induced protein (TGFBI, also known as βig-H3 or keratoepithelin) is an ECM protein, whose expression is upregulated by TGF-β1, a regulator of angiogenesis and new blood vessel formation in various tumors. We recently reported that TGFBI deletion promotes adipose angiogenesis by regulating essential processes in endothelial cells 11 . Angiogenic factors affect metabolic diseases, such as obesity and diabetes 12 , 13 . Based on the contribution of TGFBI to adipose angiogenesis, we sought to determine whether TGFBI functions as a regulator of chronic processes, such as adipose expansion and inflammation, and whether it can control metabolic health during obesity. Notch-1 activation is an important process in tumor angiogenesis that regulates the responsiveness of endothelial cells to vascular endothelial growth factor (VEGF) 14 . Several studies have indicated that Notch-1 signaling is sufficient to induce the differentiation and tumorigenic transformation of mature adipocytes 15 , 16 ; however, the precise mechanisms underlying the ability of Notch-1 to regulate gene expression remain unclear. In the present study, we determined the role of TGFBI in adipose tissue expansion and related metabolic disorders using a mouse model of high-fat diet (HFD)-induced obesity. We found that TGFBI KO mice were protected against obesity as characterized by limited adipose expansion and improved insulin/glucose homeostasis. Mechanistically, TGFBI directly bound to Notch-1 and subsequently regulated adipsin secretion. Our findings establish the importance of TGFBI in regulating adipose metabolism in obesity and present the mechanisms underlying this effect. Materials And Methods Generation of TGFBI KO mice Generation and genotyping of TGFBI −/− (KO) mice of C57/BL6 background was performed as previously described 11 . The TGFBI-targeting vector was constructed with sites introduced upstream and downstream of exon 3, as shown in Supplementary Fig. 1A. The target plasmid was microinjected into C57BL/BL6 blastocysts, and the FLP recombinase target-flanked PGK-neomycin cassette was removed by Flp-mediated recombination. Genotypes were determined by tail biopsy using polymerase chain reaction (PCR). The following primers were used: sense (5'-CCATACTCTGACTTCCAGGTTATTA-3') and antisense (5'-TGGCAGACTAGCAAG GGTTT-3'). All animals were maintained in a controlled environment with 10–20% humidity, 24 ± 1°C temperature, and a 12-h light/dark cycle. The mice were provided free access to water and fed either a standard chow diet or a 60% HFD. All animal experiments were approved by the Institutional Animal Care Committee of Kyungpook National University (approval number: KNU 2017-0059). The study was performed in compliance with the ARRIVE guidelines, and all methods were implemented according to relevant guidelines and regulations. GTT and ITT After 10 h of fasting, glucose and insulin tolerance test (GTT and ITT, respectively) were performed on 20-week-old mice fed a HFD for 12 weeks. For the GTT and ITT, the mice were injected intraperitoneally with either D-glucose (1 g/kg) or insulin (1 unit/kg). Glucose levels were measured from tail bleeds at 0, 15, 30, 60, 90, and 120 min using an AccuChek-EZ glucose monitor (Roche Molecular Biochemicals, IN, Indiana). Recombinant TGFBI Recombinant TGFBI expression was induced and extracted from engineered Escherichia coli as previously described 17 , 18 . The TGFBI cDNA was inserted into the EcoRV and EcoRI sites of pET-29b (Novagen, Madison, WI, USA). A clone was selected, cultured, and induced with IPTG (Isopropyl ß-D-1-thiogalactopyranoside) to express recombinant TGFBI protein. The recombinant protein was purified using Ni-NTA column, dialyzed, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and stained with Coomassie brilliant blue staining solution. Culture and differentiation on 3T3-L1 adipocytes Mouse 3T3-L1 preadipocytes were purchased from the Korea Cell Line Bank (Seoul, Korea). The cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% NBCS and antibiotics (1% penicillin/streptomycin) at 37°C in a humidified incubator supplied with 5% CO 2 . After reaching 100% confluence, the cells were maintained for another 2 days and then exposed to a differentiation medium (MDI) consisting of DMEM supplemented with 10% FBS, 0.5 mM IBMX, 10 µg/ml insulin, 1 µM dexamethasone, and 100 µM indomethacin. After 2 days, the culture medium was replaced with DMEM supplemented with 10% FBS and insulin, and changed every 2 days thereafter (from day 2 to day 8). Transfection Lentivirus-expressing small interfering RNAs (siRNAs) against TGFBI (Cat. no. 4659409) and lentiviral scrambled siRNA (Cat. no. LV015-G) were purchased from ABM. Then, 293FT cells were transfected with a lentiviral vector plasmid together with each of the packaging plasmids. After transfection, the viruses were harvested and passed through a 0.4 - µm filter for infection. 3T3-L1 cells were infected with the virus in the presence of polybrene in 6-well plates. After two days, the medium containing the virus was removed and replaced with fresh medium. Subsequently, the cells were selected continuously using puromycin (1–2 µg/ml). Isolation of BM-MSC and BM-DM BM-MSC and bone marrow-derived macrophages (BM-DM) were isolated from the hind legs of 6–8-week-old WT and TGFBI KO mice as previously described 19 , 20 , with some modifications (BM isolation and BM-DM isolation). For BM-MSC differentiation into beige adipocytes, the isolated cells were treated with an adipogenic cocktail (0.5 mM IBMX, 170 nM insulin, 5 µM dexamethasone, 125 µM indomethacin, 2 nM T3, and 1 µM rosiglitazone). After two days, the differentiation medium was replaced with DMEM supplemented with 170 nM insulin, 2 nM T3, and 1 µM rosiglitazone for 10 days. Isolated BM-DM were cultured in DMEM containing 30% L929 conditioned medium and 20% FBS. A week after isolation, the BM-DM culture medium was collected and centrifuged at 500 × g for 10 min at 4°C to remove cell debris. The resulting macrophage conditioned medium (M-CM) was used at a 1:1 ratio with a growth medium containing an adipogenic cocktail. Mature adipocytes from BM-MSCs cultured without M-CM were used as controls. Flow cytometry BM-DM cells were isolated from the hind legs of WT and TGFBI KO mice, as described above, and then treated with Fc-block and stained with CD11b antibody for 2 h at RT. Cells were washed with PBS and analyzed using an Attune FACS system (Life Technologies, Darmstadt, Germany). Notch binding assay Recombinant Notch-1 protein (2 g/ml) in PBS was coated onto 96-well polystyrene microplates at room temperature (RT) overnight. The plates were washed and blocked with 1% bovine serum albumin (BSA) in PBS for 1 h at RT. The blocking solution was removed and binding proteins Jagged-1 or TGFBI were added at concentrations ranging from 0 to 2000 ng/ml. After incubation, biotinylated antibodies were added to the plate and incubated for 1 h at RT. The plates were washed, and then streptavidin-horseradish peroxidase (HRP) was added for 20–30 min at RT. The plates were washed again, the substrate was added, and the OD at 450 nm was measured. Kd (nM) was calculated using the following equation: ED50 (ng/mL)/molecular weight of binding protein. Oxygen consumption rate assay 3T3-L1 adipocytes were seeded into XF96-well plates (Agilent Technologies, Santa Clara, CA, USA) coated with 0.1% gelatin (3 × 10 3 cells per well). After three days, the medium was replaced with Seahorse XF assay medium (Agilent) containing 1 mM pyruvate, 1 mM glutamine, and 0.25 mM glucose. The oxygen consumption rate (OCR) was measured under basal conditions and in response to 1 µM oligomycin, 2 µM FCCP (carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone), and 1 µM antimycin and rotenone exposure using an XF96 Extracellular Flux Analyzer (Agilent). Adhesion assay BSA (20 µg/ml) or TGFBI was coated onto 96-well plates and incubated for 2 h at 37°C. 3T3-L1 preadipocytes were pretreated with Notch-1 antibody (5 µg/ml) or control IgG for 15 min at 37°C and then plated at a density of 3,000 cells per well. After incubation for 30 min at 37°C, the cells were washed and fixed with 4% paraformaldehyde in PBS. Adherent cells were imaged under a microscope and counted. Immunofluorescence staining Recombinant TGFBI or galectin-3 was coated onto 6-well chamber slides, which were maintained for 24 h at 4°C. Next, 3T3-L1 preadipocytes were plated into precoated wells and incubated. The following day, the cells were fixed in 4% PFA, permeabilized with 25% Triton X-100, and blocked with blocking buffer containing 1% BSA in PBS. The cells were incubated with a Notch-1 specific primary antibody (ab52627) for 2 h at RT and stained with a secondary antibody (Alexa Fluor® 488) under the same conditions. The samples were then mounted using DAPI-containing mounting solution (Vector Laboratories, Burlingame, CA, USA) and imaged using a Leica DM IL LED microscope (Leica, Germany). Quantitative real time polymerase chain reaction (qRT-PCR) RNA extraction, complementary DNA (cDNA) synthesis, and quantitative real-time PCR (qPCR) were performed as previously described 11 . PCR was performed on an iCycler iQ™ Real-Time PCR Detection System (Bio-Rad Laboratories, USA) using SYBR Green Master Mix (TOYOBO, Japan). The relative gene expression levels were determined using the 2 −ΔΔCt method and expressed as the fold-change increase compared with that of wild-type mice. The primer sequences are listed in the Supplementary Table; β-actin was used as the control gene. Western blot analysis Protein extraction and western blot analyses were performed as previously described 21 , 22 . Briefly, total protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes. The following primary antibodies were used: anti-GLUT-2 (Cat. sc-9117, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PGC-1α (Cat. ab54481; Abcam, Cambridge, UK), anti-PPARγ (cat. ab19481; Abcam), anti-UCP-1 (Cat. UCP11-A; Alpha Diagnostics, San Antonio, TX, USA), anti-cleaved Notch-1 (Cat. D3B8; Cell Signaling Technology (CST), Danvers, MA, USA), anti-Notch-1 (Cat. ab52627; Abcam), anti-Hes-1 (Cat.D6P2U; CST), C/EBPα (Cat. ab15048; Abcam), anti-tubulin (Cat. 2148;CST), anti-lamin B1 (cat. 12586;CST), and anti-β-actin (Cat. sc-47778; Santa Cruz Biotechnology) antibodies. The signals were quantified using a Fusion Solo Detector (Vilber Lourmat, Marne La Vallee, France). Immunoprecipitation 3T3-L1 preadipocytes were treated with recombinant TGFBI (20 µg/ml) for 24 h, and total protein was extracted in RIPA buffer containing 10 mM nethylmaleimide (EMD Millipore, Darmstadt, Germany). Immunoprecipitation was carried out overnight at 4°C with primary antibody, followed by the addition of Protein G agarose beads, and incubation for 1 h. Beads were collected by pulse centrifugation and washed with lysis buffer. The precipitated proteins were analyzed by western blotting as described above. Immunohistochemistry Inguinal white adipose tissue (iWAT) and liver tissue were fixed with 4% paraformaldehyde, processed, and embedded in paraffin. Tissue sections (5–7 µm thick) were stained with hematoxylin and eosin (H&E). The size of the adipocytes in three mice per genotype was measured using ImageJ software (NIH). Statistical analysis Statistical analyses for all experiments were performed using SPSS ver.20.0 (SPSS Inc., Chicago, IL, USA). Statistical differences between groups were analyzed using two-sided t-test, and statistical significance was set at p < 0.05. Results TGFBI KO mice exhibit resistance to high-fat diet-induced obesity A genetically modified animal model was used to determine whether TGFBI affected diet-induced obesity. The PCR analysis confirmed the deletion of the TGFBI gene, and the 376-bp product was compared with the 1099-bp product for the WT mice (Supplementary Fig. 1B). The deletion of TGFBI in the plasma of WT and KO mice was also confirmed by ELISA. The whole body weight of 11-week-old HFD-fed KO mice was significantly lower than that of WT mice of the same age (Fig. 1 A). The 20-week-old HFD-fed KO mice showed a dramatic decrease in body weight gain compared with the WT mice. These differences were also observed between the ND-fed WT and KO mice, although a smaller difference was observed compared with that for the HFD-fed groups. A significant decrease was observed in the inguinal WAT (iWAT), retroperitoneal WAT (rWAT), epididymal WAT (eWAT), and liver mass of the HFD-fed KO mice, whereas the brown adipose tissue (BAT) and subcutaneous WAT (sWAT) mass were unaltered compared with those of the HFD-fed WT mice (Fig. 1 B-C). The masses of the heart, lungs, kidneys, and spleen tissue were comparable between the WT and TGFBI KO mice (Fig. 1 C). As expected, with suppressed adipose expansion, the size of the iWAT adipocytes decreased in the HFD-fed KO mice compared with that of the HFD-fed WT mice (Fig. 1 D). These results suggest that TGFBI KO mice exhibit resistance to HFD-induced body weight gain and adipose tissue expansion. HFD-induced ECM remodeling was apparent not only in adipose tissue but also in the liver, and HFD-fed mice exhibited increased expression of collagen type I and III in the liver 18 , 23 . This remodeling process was also closely associated with insulin resistance 24 . Therefore, we determined whether the lack of TGFBI contributed to improved homeostasis and liver function in HFD-fed mice. HFD-fed KO mice exhibited a marked reduction in lipid composition, triacylglycerol (TG) content, and free fatty acid (FFA) content in the liver compared with that in the HFD-fed WT mice (Fig. 1 E-G). HFD-fed KO mice showed increased expression of gluconeogenesis-related genes, including those encoding GLUT-2, PGC-1α, and PPARα (Fig. 1 H). Glucose tolerance and insulin sensitivity improved in the HFD-fed KO mice compared with those in the HFD-fed WT mice (Fig. 1 I-J). To determine whether differences occurred between sexes, we evaluated the phenotype of HFD-fed female KO mice. Similar to that of the HFD-fed male KO mice, the female KO mice also exhibited lower body weight and iWAT mass and improved glucose homeostasis than the female WT mice (Supplementary Fig. 2A-F). Taken together, these results suggest that TGFBI KO mice had improved capacity for obesity-induced metabolic changes. TGFBI KO induces expression of proteins involved in the PPAR γ signaling pathway To explore the mechanisms underlying the protective effects of TGFBI during obesity, we examined the PPARγ signaling pathway, which is closely involved in adipocyte differentiation, in iWAT from ND- and HFD-fed WT and TGFBI KO mice. We observed that the protein and mRNA expression levels of PPARγ, C/EBPα, and adiponectin did not change in the ND-fed WT and KO mice (Fig. 2 A-B). Interestingly, the protein and mRNA expression levels of PPARγ and C/EBPα were significantly lower in the HFD-fed TGFBI KO mice than the WT mice (Fig. 2 C-D). In addition, the serum leptin levels were decreased in the TGFBI KO mice (Fig. 2 E). Notably, the TGFBI KO mice showed improved food efficiency, although their food intake was comparable with that of the WT mice (Fig. 2 F). These results indicated that the obesity resistance of TGFBI KO mice was not affected by the reduction of fat in the diet. Taken together, these results demonstrate that the lack of TGFBI may lead to the suppression of excess adipose expansion by inhibiting the PPARγ signaling pathway. Knockdown of TGFBI inhibits adipogenic differentiation We determined whether TGFBI plays a role in preadipocyte differentiation into mature adipocytes. We confirmed the adipogenic capacity of TGFBI-knockdown cells and compared it with that of the control cells. TGFBI knockdown cells exhibited reduced Oil Red O staining and intracellular TG accumulation, which suggest reduced differentiation capacity compared with the control siRNA cells (Fig. 3 A-C). The knockdown efficacy was confirmed by ELISA (Fig. 3 D). TGFBI knockdown cells showed suppressed expression of adipogenesis-related genes, including those encoding PPARγ and C/EBPα (Fig. 3 E). These proteins also showed reduced expression in TGFBI knockdown cells compared with that in the control cells (Fig. 3 F-G). Furthermore, maximum oxygen consumption was significantly higher in the TGFBI knockdown cells than the control cells (Fig. 3 H-I). In contrast, no differences were observed in the basal state. TGFBI KO mice exhibit over-secretion of adipsin and higher macrophage populations To completely understand the mechanism through which metabolism benefits HFD-fed TGFBI KO mice, we performed an RNA sequence analysis of iWAT from these mice (Fig. 4 A). Among the gene processes that were modulated in TGFBI KO mice, we focused on the significantly enriched complement and coagulation cascades. Adipsin is a regulatory protein of the complement cascade whose expression is downregulated in mice with acquired metabolic diseases 25 . Consistently, the expression of adipsin was reduced in HFD-fed obese mice compared with ND-fed normal mice (Supplementary Fig. 3A-B). We observed that the mRNA, protein, and secretory levels of adipsin were upregulated in the HFD-fed TGFBI KO mice, whereas no difference was observed between the ND-fed WT and KO mice (Fig. 4 B-C). Next, we hypothesized that primary adipocyte-derived BM-MSCs could alter adipsin secretion in an autocrine manner. However, the adipsin levels of differentiated BM-MSCs were comparable between those isolated from TGFBI KO mice or WT mice (Fig. 4 D). Previous studies have indicated that fat-produced adipsin regulates inflammation and adipose tissue macrophage properties in adipsin KO mice 26 , 27 . To determine the contribution of immune cells, such as macrophages, to the processes observed in the present study, we measured the macrophage population in the adipose tissues. We detected CD11b-positive macrophages in the adipose tissue and found a higher population of CD11b + macrophages in the TGFBI KO mice compared with the WT mice (Fig. 4 E-F). In addition, the levels of the pro-inflammatory cytokines IL-6 and TNF-α were decreased in the TGFBI KO mice (Fig. 4 G). Moreover, significant differences were not observed in the expression of CD206 and CD86 between the WT and TGFBI KO mice (Fig. 4 H). These data suggest that TGFBI facilitates the differentiation of distinct populations of macrophages and modulates the inflammatory status of obese adipose tissue. Adipose macrophages regulate the browning and adipsin secretion of adipocytes Because increased numbers of macrophages were present in TGFBI KO mice, we sought to determine whether adipsin expression was regulated by macrophages under indirect coculture conditions (Fig. 5 A). We found that adipocytes cultured with TGFBI KO macrophages contained brown-like adipocytes that featured multiple smaller lipid droplets in their cytoplasm (Supplementary Fig. 4A). Consistent with these morphological differences, adipsin, UCP-1, and PGC-1α were mainly detected in adipocytes cocultured with macrophages isolated from TGFBI KO mice compared with those isolated from WT mice (Fig. 5 B-D and Supplementary Fig. 4B). Next, we confirmed the effects of TGFBI on adipsin expression under macrophage coculture conditions. TGFBI treatment ameliorated adipsin expression in adipocytes cultured with TGFBI KO macrophages (Fig. 5 E-G). Interestingly, we observed that TGFBI was primarily secreted by the small vascular fraction containing macrophages rather than by adipocytes. In contrast, adipsin levels were significantly higher in adipocytes (Fig. 5 H-I). This result suggests that these macrophages play important roles in the regulation of TGFBI-mediated adipsin secretion from adipocytes. Additionally, TGFBI is largely derived from macrophages and may influence adipsin expression in adipocytes in a paracrine manner, and which closely associated with adipocyte characteristics and browning. TGFBI induces Notch-1 signaling activation in adipocytes. Notch-1-induced transcriptional regulator Hes-1 downregulates adipsin expression in adipocytes 28 and Notch-1 signaling promotes obesity progression and ameliorates adipose browning 29 . However, the mechanisms underlying the activation of these processes and whether these processes are related to ECM remodeling in HFD-induced obese mice have not been clarified. HFD-feeding induced the overexpression of Notch-1 and Hes-1 in WAT obtained from WT mice compared with that from lean mice (Fig. 6 A-B). Interestingly, Notch-1 protein expression was significantly reduced in TGFBI KO mice, whereas differences at the mRNA level relative to that in WT mice were not observed (Fig. 6 C). In contrast, Hes-1 mRNA and protein levels decreased by 1.5- to 2.0-fold in TGFBI KO mice. To determine whether exogenous treatment with TGFBI affected Notch-1 activation, we measured the expression of Notch-1 and Hes-1 in TGFBI-treated adipocytes. The TGFBI treatment increased the protein expression of Hes-1 and Notch-1 in 3T3-L1 adipocytes (Fig. 6 D). Consistent with the in vivo data, downregulation of Hes-1 mRNA expression was observed, while changes in Notch-1 expression were not observed (Fig. 6 E). These data suggest that TGFBI controls Notch-1 abundance at the post-transcriptional level. Next, we postulated that TGFBI may facilitate the nuclear translocation of Notch-1. The results showed that the TGFBI treatment induced nuclear translocation of Notch-1, which was similar to that observed for the positive control galectin-3 (Fig. 6 F). Furthermore, the content of Notch-1 intracellular domain increased in the nuclear fractions of TGFBI-treated 3T3-L1 adipocytes, whereas Notch-1 activation was blocked by a Notch-1 inhibitor in cells cotreated with TGFBI (Fig. 6 G). Furthermore, Notch-1 expression remained unchanged in the cytoplasmic fractions. These results suggest that TGFBI activates nuclear translocation of Notch-1 in adipocytes. Notch-1 binds to TGFBI and regulates cell adhesion of adipocytes Next, we determined whether TGFBI binds to Notch-1 in 3T3-L1 adipocytes. Notch-1 was physically associated with TGFBI in 3T3-L1 adipocytes (Fig. 7 A). In a non-cell-based system, TGFBI exhibited binding affinity for Notch-1 (Kd = 3.18 nM), although its affinity was weaker than that of jagged-1, which is a strong ligand for Notch-1 (Fig. 7 B). The ECM facilitates cellular attachment and mediates cellular mobility, adhesion, proliferation, and differentiation 30 . To determine whether TGFBI controls adipocyte adhesion via Notch-1, we performed a cell adhesion assay using TGFBI-coated plates (Fig. 7 C). Notch-1 inhibition markedly suppressed the adhesion capacity of 3T3-L1 adipocytes on TGFBI-coated plates, although cytotoxicity was not observed (Fig. 7 D-F). These results indicated that TGFBI is functionally active and directly binds to Notch-1 in adipocytes. TGFBI suppresses adipocyte browning and adipsin secretion via activating Notch-1 signaling To establish the physiological significance of TGFBI, we examined whether it regulates adipocyte browning by activating Notch-1. The TGFBI treatment markedly reduced multilocular lipid formation and browning-related gene expression in differentiated 3T3-L1 cells compared with that in control cells (Fig. 8 A-B). Notably, this TGFBI-induced reduction was restored by cotreatment with a Notch-1 inhibitor, and significantly elevated the expression of UCP-1 and PGC-1α (Fig. 8 B-C). Accordingly, adipsin expression and secretion were significantly elevated by cotreatment of the cells with the Notch inhibitor and TGFBI compared to that under treatment with TGFBI alone (Fig. 8 C-E). Moreover, the adipsin levels were unchanged by the treatment with the Notch-1 inhibitor alone. These findings indicate that TGFBI ameliorates adipsin-mediated browning via a Notch-1 dependent mechanism. Finally, we determined whether Notch-1 regulated the expression of adipsin and browning markers. Notch-1 has intracellular domains that include RAM (Rbpj interacting domain) and NLS (nuclear localization signal) (Fig. 8 F). Deletion of the NLS domain upregulated the mRNA and protein expression of UCP-1 and adipsin (Fig. 8 G-I), while deletion of RAM did not induce the expression of these proteins. The promoter activity of adipsin was repressed by TGFBI (Fig. 8 J); however, this was restored by deleting the NLS domain in the presence of TGFBI (Fig. 8 K). These results indicate that deletion of the NLS domain directly enhances adipsin promoter activity and markedly increases the expression of browning-related genes. Discussion Obesity is characterized by abnormal adipose expansion and fibrosis accompanied with the overaccumulation of ECM 31 . A recent study indicated that remodeling is required to maintain a healthy adipose tissue condition 32 . However, limitations have been observed in studies that primarily focused on major ECM proteins, such as collagen and fibronectin. TGFBI has been implicated in the pathogenesis of cancer and diabetic retinopathy 33 , 34 . Moreover, previous studies have detected TGFBI in adipose tissue and associated TGFBI polymorphisms with insulin levels and body mass 11 , 35 . However, the gain- and loss-of-function mechanisms of TGFBI are not completely understood. In the present study, we demonstrated that the ECM component TGFBI plays a role in HFD-induced obesity and improves several physiological conditions, such as glucose/insulin resistance, adipose expansion, liver steatosis, and adipocyte differentiation. Indeed, weight loss resulting from TGFBI deficiency occurs not only in HFD-fed mice but also in ND-fed mice. A previous study demonstrated that TGFBI regulates periosteal bone formation, including the bone mass, size, and strength 36 . TGFBI KO mice show reduced bone mass and 8–20% lower body weight than WT mice during postnatal development. However, why TGFBI KO mice exhibit a slight decline in body weight gain due to ND-feeding remains elusive. In the present study, we found that HFD-fed TGFBI KO mice exhibited a greater reduction in adipose and liver mass and lipid accumulation. Future studies are warranted to address the tissue-specific and direct targeted effects of TGFBI using engineered mice. Inconsistent with our data, a previous study reported that TGFBI knockdown accelerated human preadipocyte adipogenesis 37 ; however, the use of human preadipocytes isolated from subcutaneous adipose tissue or visceral adipose tissue likely led to the observed discrepancy in the results. Adipose-derived stem cells undergo multilineage changes and then differentiate into different type of adipocyte, such as white, beige, and brown adipocytes 38 , 39 . These adipocytes have characteristic differences in their anatomical location and function. The 3T3-L1 mouse cell line used in our study shows characteristics typical of white adipocytes 40 . Based on these points, we assumed that the role of TGFBI in each type of adipocyte may differ, which may have caused the discrepancy in the results between the previous study and our study. In fact, in our results based on BM-MSC-derived adipocytes, TGFBI did not affect adipogenic capacity, but significantly promoted adipose browning. TGFBI overexpression is detected in fibroblasts, peritoneal cells, macrophages, and T cells 41 , 42 , among which, macrophages are predominant producers of TGFBI. In the present study, we found that TGFBI was largely produced by small vascular fractions containing various immune cells and also secreted from adipocytes. Indeed, significant changes were not observed in the upregulated adipsin expression between the BM-MSC-derived adipocytes cultured alone and those from the WT mice following TGFBI KO. This suggests that additional metabolically stimulatory conditions or factors may contribute to adipsin secretion from adipocytes. Therefore, we propose that TGFBI acts on obesity and adipose tissue in both autocrine and autocrine manners. Macrophages are the primary inflammatory cells found in inflamed adipose tissue that control adipose homeostasis and energy expenditure 43 , 44 . Several reports have indicated the involvement of cytokines secreted from adipose tissue macrophages in beige adipocyte formation and WAT browning 45 . Consistent with these findings, our results provided several lines of evidence supporting that adipose-associated macrophages regulate the expression of inflammatory cytokines and browning markers in adipose tissues obtained from TGFBI KO mice. We speculate that the different phenotypes detected in our study occurred in a distinct population of CD11b + M1-like macrophages in TGFBI KO mice compared to those in WT mice. Interestingly, adipsin levels were strongly elevated in adipose tissue and adipocytes cocultured with macrophages isolated from TGFBI KO mice. We confirmed that adipsin expression was positively correlated with the expression of adipose browning markers UCP-1 and PGC-1α. In TGFBI KO mice, adipsin was secreted in response to macrophages; thus, we concluded that increased adipsin expression is closely associated with adipose browning and other metabolic benefits in TGFBI KO mice. The Notch-1 signaling pathway has been implicated in various biological processes, such as tumorigenesis, apoptosis, and proliferation 46 . Recently, Notch signaling was also found to be important in adipogenesis 47 . Inhibition of Notch-1 and its signaling mediators results in adipose browning and regulates adipsin expression 48 , 49 , which is consistent with our observation that the pharmacological effects and genetic mutations of Notch-1 resulted in elevated adipose browning. Furthermore, we discovered that Notch-1 represses adipsin expression and the Notch-1 intracellular domain NLS is required for the inhibition of adipsin transcription. Notably, we discovered that TGFBI acts as a new ligand for Notch-1, and this signaling activation is required for suppression of adipsin secretion and adipose browning. In summary, the present study revealed that the deletion of the ECM protein TGFBI plays a protective role against HFD-induced metabolic disorders. Mechanistically, TGFBI activates Notch-1 signaling and subsequently regulates adipsin expression, which may explain the upregulation of adipose browning related-gene expression and the beneficial metabolic effects observed in TGFBI KO mice. Taken together, our findings offer new insights for the development of new therapeutic approaches for obesity and related disorders via TGFBI regulation. Declarations ACKNOWLEDGMENTS This study was supported by the National Research Foundation of Korea (NRF) funded by the Korean government (NRF-2017R1A2B4011003, NRF-2019R1A2C1090619, 2021R1A6A3A01087252, and NRF-2019R1A2C2005921). AUTHOR CONTRIBUTIONS S.G.L., T.K.K., and J.-O.N. designed the project; S.G.L., J.C., S.M.W., and S.U.S. performed the experiments; H.-J.K., H.-S.P., and D.D.S. provided consultation and guidance; I.-S.K. provided the transgenic mice and guidance; T.-K.K. and J.-O.N. supervised the project; and S.G.L., T.-K.K., and J.-O.N. wrote the manuscript. COMPETING INTERESTS The authors declare that they have no competing interests References Kaila, B. & Raman, M. Obesity: a review of pathogenesis and management strategies. Canadian Journal of Gastroenterology and Hepatology 22 , 61–68 (2008). Peirson, L. et al. Treatment for overweight and obesity in adult populations: a systematic review and meta-analysis. CMAJ open 2 , E306 (2014). Han, T.S. & Lean, M.E. A clinical perspective of obesity, metabolic syndrome and cardiovascular disease. JRSM cardiovascular disease 5 , 2048004016633371 (2016). Guilherme, A., Virbasius, J.V., Puri, V. & Czech, M.P. Adipocyte dysfunctions linking obesity to insulin resistance and type 2 diabetes. Nature reviews Molecular cell biology 9 , 367 (2008). Scherer, P.E. Adipose tissue: from lipid storage compartment to endocrine organ. Diabetes 55 , 1537–1545 (2006). Shabalina, I.G. et al. UCP1 in brite/beige adipose tissue mitochondria is functionally thermogenic. Cell reports 5 , 1196–1203 (2013). Wu, J. et al. Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human. Cell 150 , 366–376 (2012). Vaicik, M.K. et al. Laminin alpha4 deficient mice exhibit decreased capacity for adipose tissue expansion and weight gain. PloS one 9 , e109854 (2014). Sun, K., Kusminski, C.M. & Scherer, P.E. Adipose tissue remodeling and obesity. The Journal of clinical investigation 121 , 2094–2101 (2011). Lee, M.H. et al. ECM microenvironment unlocks brown adipogenic potential of adult human bone marrow-derived MSCs. Scientific reports 6 , 21173 (2016). Lee, S.G. et al. Endothelial angiogenic activity and adipose angiogenesis is controlled by extracellular matrix protein TGFBI. Scientific reports 11 , 9644 (2021). Gealekman, O. et al. Depot-specific differences and insufficient subcutaneous adipose tissue angiogenesis in human obesity. Circulation 123 , 186–194 (2011). Zafar, M.I. et al. Association between the expression of vascular endothelial growth factors and metabolic syndrome or its components: a systematic review and meta-analysis. Diabetology & metabolic syndrome 10 , 62 (2018). Nijhawans, P., Behl, T. & Bhardwaj, S. Angiogenesis in obesity. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 126 , 110103 (2020). Yamaguchi, K. et al. Notch1 haploinsufficiency in mice accelerates adipogenesis. Scientific reports 11 , 1–14 (2021). Garcés, C. et al. Notch-1 controls the expression of fatty acid-activated transcription factors and is required for adipogenesis. The Journal of biological chemistry 272 , 29729–29734 (1997). Bae, J.S. et al. Transforming growth factor β-induced protein promotes severe vascular inflammatory responses. American journal of respiratory and critical care medicine 189 , 779–786 (2014). Kim, J.E. et al. A TGF-beta-inducible cell adhesion molecule, betaig-h3, is downregulated in melorheostosis and involved in osteogenesis. Journal of cellular biochemistry 77 , 169–178 (2000). Soleimani, M. & Nadri, S. A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. Nature protocols 4 , 102–106 (2009). Toda, G., Yamauchi, T., Kadowaki, T. & Ueki, K. Preparation and culture of bone marrow-derived macrophages from mice for functional analysis. STAR protocols 2 , 100246 (2021). Kim, J.S. et al. Eupatilin inhibits adipogenesis through suppression of PPARgamma activity in 3T3-L1 cells. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 103 , 135–139 (2018). Lee, S.G., Kim, J.S., Min, K., Kwon, T.K. & Nam, J.O. Hispidulin inhibits adipogenesis in 3T3-L1 adipocytes through PPARgamma pathway. Chemico-biological interactions 293 , 89–93 (2018). Williams, A.S. et al. Integrin alpha1-null mice exhibit improved fatty liver when fed a high fat diet despite severe hepatic insulin resistance. The Journal of biological chemistry 290 , 6546–6557 (2015). Bonner, J.S. et al. Relaxin treatment reverses insulin resistance in mice fed a high-fat diet. Diabetes 62 , 3251–3260 (2013). Ryu, K.Y. & Jeon, E.J. Regulation of Adipsin Expression by Endoplasmic Reticulum Stress in Adipocytes. 10 (2020). Lo, J.C. et al. Adipsin is an adipokine that improves β cell function in diabetes. Cell 158 , 41–53 (2014). Li, Y. et al. Fat-Produced Adipsin Regulates Inflammatory Arthritis. Cell reports 27 , 2809–2816.e2803 (2019). Searfoss, G.H. et al. Adipsin, a biomarker of gastrointestinal toxicity mediated by a functional gamma-secretase inhibitor. The Journal of biological chemistry 278 , 46107–46116 (2003). Bi, P. et al. Inhibition of Notch signaling promotes browning of white adipose tissue and ameliorates obesity. 20 , 911–918 (2014). Koh, K.H. et al. Nonimmune cell-derived ICOS ligand functions as a renoprotective αvβ3 integrin-selective antagonist. The Journal of clinical investigation 129 , 1713–1726 (2019). Spencer, M. et al. Adipose Tissue Extracellular Matrix and Vascular Abnormalities in Obesity and Insulin Resistance. The Journal of Clinical Endocrinology & Metabolism 96 , E1990-E1998 (2011). Ruiz-Ojeda, F.J., Méndez-Gutiérrez, A., Aguilera, C.M. & Plaza-Díaz, J. Extracellular Matrix Remodeling of Adipose Tissue in Obesity and Metabolic Diseases. International journal of molecular sciences 20 , 4888 (2019). Ozawa, D. et al. TGFBI Expression in Cancer Stromal Cells is Associated with Poor Prognosis and Hematogenous Recurrence in Esophageal Squamous Cell Carcinoma. Annals of surgical oncology 23 , 282–289 (2016). Betts-Obregon, B.S. et al. TGFβ induces BIGH3 expression and human retinal pericyte apoptosis: a novel pathway of diabetic retinopathy. Eye (London, England) 30 , 1639–1647 (2016). Park, K.S. et al. Genetic polymorphisms in the transforming growth factor beta-induced gene associated with BMI. Human mutation 25 , 322 (2005). Yu, H., Wergedal, J.E., Zhao, Y. & Mohan, S. Targeted disruption of TGFBI in mice reveals its role in regulating bone mass and bone size through periosteal bone formation. Calcified tissue international 91 , 81–87 (2012). Hirata, K. et al. Role of leukotriene B(4) receptor signaling in human preadipocyte differentiation. Biochemical and biophysical research communications 429 , 197–203 (2012). Zheng, B., Cao, B., Li, G. & Huard, J. Mouse adipose-derived stem cells undergo multilineage differentiation in vitro but primarily osteogenic and chondrogenic differentiation in vivo. Tissue engineering 12 , 1891–1901 (2006). Park, A., Kim, W.K. & Bae, K.H. Distinction of white, beige and brown adipocytes derived from mesenchymal stem cells. World journal of stem cells 6 , 33–42 (2014). Morrison, S. & McGee, S.L. 3T3-L1 adipocytes display phenotypic characteristics of multiple adipocyte lineages. Adipocyte 4 , 295–302 (2015). Lecker, L.S. et al. TGFBI production by macrophages contributes to an immunosuppressive microenvironment in ovarian cancer. Cancer research 81 , 5706–5719 (2021). Steitz, A.M. et al. Tumor-associated macrophages promote ovarian cancer cell migration by secreting transforming growth factor beta induced (TGFBI) and tenascin C. Cell death & disease 11 , 1–15 (2020). Wolf, Y. et al. Brown-adipose-tissue macrophages control tissue innervation and homeostatic energy expenditure. Nature immunology 18, 665–674 (2017). Kotzbeck, P. et al. Brown adipose tissue whitening leads to brown adipocyte death and adipose tissue inflammation. J Lipid Res 59, 784–794 (2018). Keuper, M. On the role of macrophages in the control of adipocyte energy metabolism. Endocrine connections 8, R105-r121 (2019). Li, D., Masiero, M., Banham, A.H. & Harris, A.L. The notch ligand JAGGED1 as a target for anti-tumor therapy. Frontiers in oncology 4, 254 (2014). Shan, T., Liu, J., Wu, W., Xu, Z. & Wang, Y. Roles of Notch Signaling in Adipocyte Progenitor Cells and Mature Adipocytes. Journal of cellular physiology 232, 1258–1261 (2017). Bi, P. et al. Inhibition of Notch signaling promotes browning of white adipose tissue and ameliorates obesity. Nature medicine 20, 911–918 (2014). Searfoss, G.H. et al. Adipsin, a biomarker of gastrointestinal toxicity mediated by a functional γ-secretase inhibitor. Journal of Biological Chemistry 278, 46107–46116 (2003). Additional Declarations (Not answered) Supplementary Files supplementaryinformation.docx Supplementary information Cite Share Download PDF Status: Published Journal Publication published 01 Mar, 2023 Read the published version in Experimental & Molecular Medicine → Version 1 posted Editorial decision: revise 17 Jul, 2022 Submission checks completed at journal 14 Jun, 2022 Unknown event 13 Jun, 2022 First submitted to journal 08 Jun, 2022 Editor assigned by journal 08 Jun, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1739900","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":121773295,"identity":"3bf5115f-b8d5-4dd0-a3d0-f66a64dc9835","order_by":0,"name":"Ju-Ock Nam","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtUlEQVRIiWNgGAWjYJACA4aKBCjzANFazpCqhYGxjRQt5uynEwo+zkvLMzjA/PADw5l7hLVY9uRuMJy5LafY4ACbsQTDjWLCWgwO5G4w5t1WkbjhAIMZA8OHBII6GAzOvwVqmQPSwv6NSC03QLY05AC18ABtuUGUlrcbDGccS0uceZinWCIBHtp4HZa7zeBDTXJi3/H2jR8+HCNCCxCwGYApZiAmTgNQ7QMiFY6CUTAKRsFIBQCW7D6KQVsOGgAAAABJRU5ErkJggg==","orcid":"","institution":"Kyungpook National University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ju-Ock","middleName":"","lastName":"Nam","suffix":""},{"id":121773296,"identity":"0b54c357-9f65-43fd-bfe4-fa9bddb8c8c1","order_by":1,"name":"Seul Gi Lee","email":"","orcid":"","institution":"Kyungpook National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Seul","middleName":"Gi","lastName":"Lee","suffix":""},{"id":121773297,"identity":"3f32547c-0c48-4aab-930e-66232eb5314e","order_by":2,"name":"Seon Min Woo","email":"","orcid":"","institution":"Keimyung University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Seon","middleName":"Min","lastName":"Woo","suffix":""},{"id":121773298,"identity":"dc29cc4a-4787-44b0-bdf5-8e1b22184542","order_by":3,"name":"Seung Un Seo","email":"","orcid":"","institution":"Keimyung University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Seung","middleName":"Un","lastName":"Seo","suffix":""},{"id":121773299,"identity":"2024cf1f-89cb-4969-93d5-077b6af40b04","order_by":4,"name":"Ha-Jeong Kim","email":"","orcid":"","institution":"Kyungpook National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ha-Jeong","middleName":"","lastName":"Kim","suffix":""},{"id":121773300,"identity":"82d362f9-4333-4f21-bf72-ec5c3a5a6f91","order_by":5,"name":"David D. Schlaepfer","email":"","orcid":"","institution":"University of California","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"David","middleName":"D.","lastName":"Schlaepfer","suffix":""},{"id":121773301,"identity":"4af902b2-0523-4aad-a325-6dcf524db0f9","order_by":6,"name":"In-San Kim","email":"","orcid":"","institution":"Korea University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"In-San","middleName":"","lastName":"Kim","suffix":""},{"id":121773302,"identity":"8eb44589-c597-451a-ad68-651f4dc1d213","order_by":7,"name":"Hee-Sae Park","email":"","orcid":"","institution":"Chonnam National University Medical School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hee-Sae","middleName":"","lastName":"Park","suffix":""},{"id":121773303,"identity":"d08c5480-e078-43b1-a2b4-a7af6eb8014a","order_by":8,"name":"Taeg Kyu Kwon","email":"","orcid":"","institution":"Keimyung University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Taeg","middleName":"Kyu","lastName":"Kwon","suffix":""}],"badges":[],"createdAt":"2022-06-09 01:50:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1739900/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1739900/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s12276-023-00947-9","type":"published","date":"2023-03-01T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":27207561,"identity":"6bf126de-006f-4a67-b04c-e45391acf11e","added_by":"auto","created_at":"2022-09-30 19:04:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":142340,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTGFBI-deficient mice are resistant to high-fat diet-induced obesity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) WT and KO mice (8-week-old) were fed a high-fat diet (HFD) or normal diet (ND) for 12 weeks, and body weight was recorded every week (left). On the final day of experiment, representative photos were taken of the HFD-fed WT and KO mice (middle). The gain in body weight was calculated using the following formula: Final weight (20-week-old) – Initial weight (8-week-old). (B-C) Indicated organ weights from the above mice. (D) Representative H\u0026amp;E staining of iWAT obtained from ND- or HFD-fed WT and KO mice (left). Diameter distribution of adipocytes in the indicated adipose tissues (right). (E) Representative H\u0026amp;E staining of the livers from 20-week-old HFD-fed WT and KO mice. The bottom panel shows representative livers from the above mice. (F-G) TG and FFA content from HFD-fed WT and KO mice. (H) Relative mRNA expression of the indicated genes in the livers of HFD-fed WT and KO mice. (I-J) GTT and ITT results. Error bars represent the ± SEM. *p \u0026lt; 0.05 by two-sided t-test.\u003c/p\u003e","description":"","filename":"Slide1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1739900/v1/70e5bd3a9d9f42b885a13b73.jpg"},{"id":27207596,"identity":"b15c3068-fb63-4b50-aa53-0c64b14df362","added_by":"auto","created_at":"2022-09-30 19:09:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":89169,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHFD-fed TGFBI-deficient mice show reduced expression of adipogenic-related genes\u003c/strong\u003e (A-D) Protein and mRNA expression of the indicated genes in iWAT obtained from ND-fed (A, B) or HFD-fed (C, D) WT and KO mice. The expression levels were normalized to those of β-actin. (E) Relative plasma leptin levels of HFD-fed WT and KO mice. (f) Feed intake was calculated using the following formula: Total feed intake (entire experimental period) / days (entire experimental period, 83 days). The feed efficiency ratio was calculated using the following formula: Body weight gain (g) / Amount of feed provided (g) × 100. Error bars represent the ± SEM. *p \u0026lt; 0.05 by two-sided t-test.\u003c/p\u003e","description":"","filename":"Slide2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1739900/v1/5d29adeec431ae5a2ab01f40.jpg"},{"id":27207554,"identity":"5dd2b66b-1a78-4de6-aed9-08d72e8211b8","added_by":"auto","created_at":"2022-09-30 19:04:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":94318,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTGFBI knockdown inhibits adipogenic differentiation of 3T3-L1 preadipocytes \u003c/strong\u003e(A-C). 3T3-L1 preadipocytes were transfected with lentiviruses encoding either TGFBI siRNA or control siRNA. After infection, the cells were induced to differentiate into mature adipocytes and stained with ORO solution (A-B), and the intracellular TG content was measured (C). (D) TGFBI expression was confirmed by ELISA in control siRNA- and TGFBI siRNA-transfected cells. (E-G) Relative mRNA (E) and protein expression (F-G) of the indicated genes in differentiated control siRNA- and TGFBI siRNA-transfected cells. (H-I) OCR measured with the substrate in the absence (basal) or presence of oligomycin, (stressed) FCCP, and rotenone. Error bars represent the ± SEM. *p \u0026lt; 0.05 by two-sided t-test.\u003c/p\u003e","description":"","filename":"Slide3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1739900/v1/852968a7a81c2cd315d0a96a.jpg"},{"id":27207598,"identity":"4ad298ea-ee93-424e-9058-89482c3063d6","added_by":"auto","created_at":"2022-09-30 19:09:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":110391,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTGFBI KO mice show increased adipsin expression and macrophage infiltration in adipose tissue \u003c/strong\u003e(A)\u003cstrong\u003e \u003c/strong\u003eExpression heat map (left), volume plots (middle), and gene set enrichment analysis (right) in iWAT obtained from 20-week-old HFD-fed WT and KO mice. (B-C) Adipsin expression level were verified by performing western blot analysis (upper), qRT-PCR (bottom, left), and ELISA (bottom, right) in iWAT obtained from ND-fed (B) and HFD-fed (C) WT and KO mice. (D) Secretory (left) and mRNA expression of adipsin (right) in differentiated BM-MSC isolated from WT and KO mice. (E-F) CD11b\u003csup\u003e+\u003c/sup\u003e macrophages isolated from iWAT of WT and KO mice. (G-H) Relative mRNA of the indicated genes in iWAT obtained from the above mice. Error bars represent the ± SEM. *p \u0026lt; 0.05 by two-sided t-test.\u003c/p\u003e","description":"","filename":"Slide4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1739900/v1/da4c7030466f90344373e8dd.jpg"},{"id":27207557,"identity":"ffc0a162-dd95-4b8f-a56b-62ae3340a1ce","added_by":"auto","created_at":"2022-09-30 19:04:40","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":86702,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTGFBI-deficient macrophages show increased adipsin and browning-related protein expression in a paracrine manner. \u003c/strong\u003e(A) Schematic of the experimental design. BM-MSCs were isolated from WT mice and induced to differentiate with M-CM obtained from WT or KO mice. (B) Expression of the indicated proteins in differentiated BM-MSCs cultured with WT or KO macrophages. (C-D) Secretory and mRNA expression of adipsin in the above cells. (E-G) BM-MSCs were either unstimulated or treated with 10 µg recombinant TGFBI under coculture conditions with TGFBI KO macrophages. Expression of the indicated proteins (E), and secretory (F) and mRNA (G) levels of adipsin in the above cells. (H-I) Adipocyte and SVF fractions were isolated in iWAT obtained from WT and KO mice. mRNA expression of TGFBI (H) and adipsin (I) were measured in the indicated fractions. Error bars represent the ± SEM. *p \u0026lt; 0.05 by two-sided t-test.\u003c/p\u003e","description":"","filename":"Slide5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1739900/v1/8d7d4000668ea8c0287d4454.jpg"},{"id":27207846,"identity":"eb92002b-dff5-4bbf-8c0a-b98b0f81f82b","added_by":"auto","created_at":"2022-09-30 19:14:40","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":105948,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTGFBI regulates the activation of Notch-1 in adipocytes. \u003c/strong\u003e(A) Notch-1 and Hes-1 protein expression in iWAT obtained from ND- and HFD-fed WT mice. (B-C) Protein and mRNA expression of the indicated genes in iWAT obtained from HFD-fed WT and KO mice. (D-E) 3T3-L1 adipocytes were treated with SHAM1 (Notch-1 inhibitor) in the presence or absence of recombinant TGFBI. (F) Localization of Notch-1 in 3T3-L1 cells treated with either recombinant TGFBI or galectin-3. (G) Indicated protein expression in the cytoplasm (left) and nucleus (right).\u003c/p\u003e","description":"","filename":"Slide6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1739900/v1/6e53e40c54171aa6b4e95eb1.jpg"},{"id":27207600,"identity":"4b8620e1-6e93-436f-a384-9530f65d8177","added_by":"auto","created_at":"2022-09-30 19:09:40","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":101406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTGFBI physiologically interacts with Notch-1. \u003c/strong\u003e(A) IP analysis of the interaction of Notch-1 and TGFBI. (B) Non-cell-based binding assay. Recombinant Notch-1 protein was precoated and allowed to bind to TGFBI or Jagged-1 at the indicated concentrations. (C) Schematic of the experimental design for the cell adhesion assay (D-E). 3T3-L1 adipocytes were pretreated with either control IgG or Notch-1 specific antibody (abNotch-1) and then allowed to adhere on recombinantTGFBI-coated plates. (F) Cytotoxicity of abNotch-1 at the indicated concentrations was tested using an MTT assay. Error bars represent the ± SEM. *p \u0026lt; 0.05 by two-sided t-test.\u003c/p\u003e","description":"","filename":"Slide7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1739900/v1/931f9c38a8e269e80413f1ff.jpg"},{"id":27207558,"identity":"1a48f328-0505-4284-9fb1-ee54b0099611","added_by":"auto","created_at":"2022-09-30 19:04:40","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":202960,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNotch-1 represses adipsin promoter activity and browning-related protein expression. \u003c/strong\u003e3T3-L1 adipocytes were treated with Notch-1 inhibitor throughout the differentiation period in the presence or absence of recombinant TGFBI. (A) Representative images of ORO-stained cells in each indicated group. (B-C) mRNA and protein expression of the indicated genes. (D-E) Adipsin mRNA and secretory levels of each indicated group. (F) 3T3-L1 adipocytes were transfected with the indicated WT and mutant Notch-1 (RAM and NLS). (G-I) Protein (G-H) and mRNA (I) expression of the indicated genes in transfected cells. (J). HEK293 cells were transfected with an Adipsin-Luc vector (−430 bp) in the presence or absence of recombinant TGFBI. (K) HEK293 cells were cotransfected with the indicated Notch-1 construct and Adipsin-Luc vector. Error bars represent the ± SEM. *p \u0026lt; 0.05 by two-sided t-test.\u003c/p\u003e","description":"","filename":"Slide8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1739900/v1/04003e256071754a65374dab.jpg"},{"id":33608454,"identity":"4f1ce019-b99b-4a8f-b1ba-948785ee9303","added_by":"auto","created_at":"2023-03-01 08:07:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1226443,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1739900/v1/05cea893-77a8-452d-80c2-b39db9feb4b3.pdf"},{"id":27207599,"identity":"3a3414c6-8f7a-451d-80a3-762421ef56b0","added_by":"auto","created_at":"2022-09-30 19:09:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":557206,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary information\u003c/p\u003e","description":"","filename":"supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-1739900/v1/7c2fae51076355fcb042fd38.docx"}],"financialInterests":"(Not answered)","formattedTitle":"TGFBI remodels adipose metabolism by regulating the Notch-1 signaling pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eObesity is a major health concern worldwide that is associated with several diseases, including cerebrovascular disease, hypertension, hyperlipidemia, and type II diabetes \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In most countries, the incidence of obesity and obesity-induced metabolic diseases has been increasing \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. White adipose tissue (WAT) plays an important role in maintaining energy balance, including glucose homeostasis and lipid metabolism, by storing calories and secreting adipokines, such as leptin and resistin \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Several WATs contain thermogenic adipocytes (brite or beige adipocytes) that transform energy-storing white adipocytes into heat-producing beige adipocytes upon acute cold exposure \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Thus, browning of WAT is considered a potential strategy for treating obesity.\u003c/p\u003e \u003cp\u003eAdipocytes are in constant contact with a network of insoluble proteins and polysaccharides that are part of the extracellular matrix (ECM) \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Changes in ECM deposition exhibit the characteristics of hyperplastic development in adipose tissue and are associated with reduced tissue plasticity \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The ECM microenvironment unlocks adipocyte differentiation in adult human bone marrow mesenchymal stem cells (BM-MSCs) \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTransforming growth factor-beta-induced protein (TGFBI, also known as βig-H3 or keratoepithelin) is an ECM protein, whose expression is upregulated by TGF-β1, a regulator of angiogenesis and new blood vessel formation in various tumors. We recently reported that TGFBI deletion promotes adipose angiogenesis by regulating essential processes in endothelial cells \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Angiogenic factors affect metabolic diseases, such as obesity and diabetes \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Based on the contribution of TGFBI to adipose angiogenesis, we sought to determine whether TGFBI functions as a regulator of chronic processes, such as adipose expansion and inflammation, and whether it can control metabolic health during obesity.\u003c/p\u003e \u003cp\u003eNotch-1 activation is an important process in tumor angiogenesis that regulates the responsiveness of endothelial cells to vascular endothelial growth factor (VEGF) \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Several studies have indicated that Notch-1 signaling is sufficient to induce the differentiation and tumorigenic transformation of mature adipocytes \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e; however, the precise mechanisms underlying the ability of Notch-1 to regulate gene expression remain unclear.\u003c/p\u003e \u003cp\u003eIn the present study, we determined the role of TGFBI in adipose tissue expansion and related metabolic disorders using a mouse model of high-fat diet (HFD)-induced obesity. We found that TGFBI KO mice were protected against obesity as characterized by limited adipose expansion and improved insulin/glucose homeostasis. Mechanistically, TGFBI directly bound to Notch-1 and subsequently regulated adipsin secretion. Our findings establish the importance of TGFBI in regulating adipose metabolism in obesity and present the mechanisms underlying this effect.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of TGFBI KO mice\u003c/h2\u003e \u003cp\u003eGeneration and genotyping of TGFBI \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e (KO) mice of C57/BL6 background was performed as previously described \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The TGFBI-targeting vector was constructed with sites introduced upstream and downstream of exon 3, as shown in Supplementary Fig.\u0026nbsp;1A. The target plasmid was microinjected into C57BL/BL6 blastocysts, and the FLP recombinase target-flanked PGK-neomycin cassette was removed by Flp-mediated recombination. Genotypes were determined by tail biopsy using polymerase chain reaction (PCR). The following primers were used: sense (5'-CCATACTCTGACTTCCAGGTTATTA-3') and antisense (5'-TGGCAGACTAGCAAG GGTTT-3'). All animals were maintained in a controlled environment with 10\u0026ndash;20% humidity, 24\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C temperature, and a 12-h light/dark cycle. The mice were provided free access to water and fed either a standard chow diet or a 60% HFD. All animal experiments were approved by the Institutional Animal Care Committee of Kyungpook National University (approval number: KNU 2017-0059). The study was performed in compliance with the ARRIVE guidelines, and all methods were implemented according to relevant guidelines and regulations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eGTT and ITT\u003c/h2\u003e \u003cp\u003eAfter 10 h of fasting, glucose and insulin tolerance test (GTT and ITT, respectively) were performed on 20-week-old mice fed a HFD for 12 weeks. For the GTT and ITT, the mice were injected intraperitoneally with either D-glucose (1 g/kg) or insulin (1 unit/kg). Glucose levels were measured from tail bleeds at 0, 15, 30, 60, 90, and 120 min using an AccuChek-EZ glucose monitor (Roche Molecular Biochemicals, IN, Indiana).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRecombinant TGFBI\u003c/h2\u003e \u003cp\u003eRecombinant TGFBI expression was induced and extracted from engineered \u003cem\u003eEscherichia coli\u003c/em\u003e as previously described \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The TGFBI cDNA was inserted into the \u003cem\u003eEcoRV\u003c/em\u003e and \u003cem\u003eEcoRI\u003c/em\u003e sites of pET-29b (Novagen, Madison, WI, USA). A clone was selected, cultured, and induced with IPTG (Isopropyl \u0026szlig;-D-1-thiogalactopyranoside) to express recombinant TGFBI protein. The recombinant protein was purified using Ni-NTA column, dialyzed, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and stained with Coomassie brilliant blue staining solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCulture and differentiation on 3T3-L1 adipocytes\u003c/h2\u003e \u003cp\u003eMouse 3T3-L1 preadipocytes were purchased from the Korea Cell Line Bank (Seoul, Korea). The cells were maintained in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM) supplemented with 10% NBCS and antibiotics (1% penicillin/streptomycin) at 37\u0026deg;C in a humidified incubator supplied with 5% CO\u003csub\u003e2\u003c/sub\u003e. After reaching 100% confluence, the cells were maintained for another 2 days and then exposed to a differentiation medium (MDI) consisting of DMEM supplemented with 10% FBS, 0.5 mM IBMX, 10 \u0026micro;g/ml insulin, 1 \u0026micro;M dexamethasone, and 100 \u0026micro;M indomethacin. After 2 days, the culture medium was replaced with DMEM supplemented with 10% FBS and insulin, and changed every 2 days thereafter (from day 2 to day 8).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTransfection\u003c/h2\u003e \u003cp\u003eLentivirus-expressing small interfering RNAs (siRNAs) against TGFBI (Cat. no. 4659409) and lentiviral scrambled siRNA (Cat. no. LV015-G) were purchased from ABM. Then, 293FT cells were transfected with a lentiviral vector plasmid together with each of the packaging plasmids. After transfection, the viruses were harvested and passed through a 0.4\u003cb\u003e-\u003c/b\u003e\u0026micro;m filter for infection. 3T3-L1 cells were infected with the virus in the presence of polybrene in 6-well plates. After two days, the medium containing the virus was removed and replaced with fresh medium. Subsequently, the cells were selected continuously using puromycin (1\u0026ndash;2 \u0026micro;g/ml).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of BM-MSC and BM-DM\u003c/h2\u003e \u003cp\u003eBM-MSC and bone marrow-derived macrophages (BM-DM) were isolated from the hind legs of 6\u0026ndash;8-week-old WT and TGFBI KO mice as previously described \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, with some modifications (BM isolation and BM-DM isolation). For BM-MSC differentiation into beige adipocytes, the isolated cells were treated with an adipogenic cocktail (0.5 mM IBMX, 170 nM insulin, 5 \u0026micro;M dexamethasone, 125 \u0026micro;M indomethacin, 2 nM T3, and 1 \u0026micro;M rosiglitazone). After two days, the differentiation medium was replaced with DMEM supplemented with 170 nM insulin, 2 nM T3, and 1 \u0026micro;M rosiglitazone for 10 days. Isolated BM-DM were cultured in DMEM containing 30% L929 conditioned medium and 20% FBS. A week after isolation, the BM-DM culture medium was collected and centrifuged at 500 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min at 4\u0026deg;C to remove cell debris. The resulting macrophage conditioned medium (M-CM) was used at a 1:1 ratio with a growth medium containing an adipogenic cocktail. Mature adipocytes from BM-MSCs cultured without M-CM were used as controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eBM-DM cells were isolated from the hind legs of WT and TGFBI KO mice, as described above, and then treated with Fc-block and stained with CD11b antibody for 2 h at RT. Cells were washed with PBS and analyzed using an Attune FACS system (Life Technologies, Darmstadt, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eNotch binding assay\u003c/h2\u003e \u003cp\u003eRecombinant Notch-1 protein (2 g/ml) in PBS was coated onto 96-well polystyrene microplates at room temperature (RT) overnight. The plates were washed and blocked with 1% bovine serum albumin (BSA) in PBS for 1 h at RT. The blocking solution was removed and binding proteins Jagged-1 or TGFBI were added at concentrations ranging from 0 to 2000 ng/ml. After incubation, biotinylated antibodies were added to the plate and incubated for 1 h at RT. The plates were washed, and then streptavidin-horseradish peroxidase (HRP) was added for 20\u0026ndash;30 min at RT. The plates were washed again, the substrate was added, and the OD at 450 nm was measured. Kd (nM) was calculated using the following equation: ED50 (ng/mL)/molecular weight of binding protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eOxygen consumption rate assay\u003c/h2\u003e \u003cp\u003e3T3-L1 adipocytes were seeded into XF96-well plates (Agilent Technologies, Santa Clara, CA, USA) coated with 0.1% gelatin (3 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells per well). After three days, the medium was replaced with Seahorse XF assay medium (Agilent) containing 1 mM pyruvate, 1 mM glutamine, and 0.25 mM glucose. The oxygen consumption rate (OCR) was measured under basal conditions and in response to 1 \u0026micro;M oligomycin, 2 \u0026micro;M FCCP (carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone), and 1 \u0026micro;M antimycin and rotenone exposure using an XF96 Extracellular Flux Analyzer (Agilent).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAdhesion assay\u003c/h2\u003e \u003cp\u003eBSA (20 \u0026micro;g/ml) or TGFBI was coated onto 96-well plates and incubated for 2 h at 37\u0026deg;C. 3T3-L1 preadipocytes were pretreated with Notch-1 antibody (5 \u0026micro;g/ml) or control IgG for 15 min at 37\u0026deg;C and then plated at a density of 3,000 cells per well. After incubation for 30 min at 37\u0026deg;C, the cells were washed and fixed with 4% paraformaldehyde in PBS. Adherent cells were imaged under a microscope and counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eRecombinant TGFBI or galectin-3 was coated onto 6-well chamber slides, which were maintained for 24 h at 4\u0026deg;C. Next, 3T3-L1 preadipocytes were plated into precoated wells and incubated. The following day, the cells were fixed in 4% PFA, permeabilized with 25% Triton X-100, and blocked with blocking buffer containing 1% BSA in PBS. The cells were incubated with a Notch-1 specific primary antibody (ab52627) for 2 h at RT and stained with a secondary antibody (Alexa Fluor\u0026reg; 488) under the same conditions. The samples were then mounted using DAPI-containing mounting solution (Vector Laboratories, Burlingame, CA, USA) and imaged using a Leica DM IL LED microscope (Leica, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real time polymerase chain reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003eRNA extraction, complementary DNA (cDNA) synthesis, and quantitative real-time PCR (qPCR) were performed as previously described \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. PCR was performed on an iCycler iQ\u0026trade; Real-Time PCR Detection System (Bio-Rad Laboratories, USA) using SYBR Green Master Mix (TOYOBO, Japan). The relative gene expression levels were determined using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method and expressed as the fold-change increase compared with that of wild-type mice. The primer sequences are listed in the Supplementary Table; β-actin was used as the control gene.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eProtein extraction and western blot analyses were performed as previously described \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Briefly, total protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes. The following primary antibodies were used: anti-GLUT-2 (Cat. sc-9117, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PGC-1α (Cat. ab54481; Abcam, Cambridge, UK), anti-PPARγ (cat. ab19481; Abcam), anti-UCP-1 (Cat. UCP11-A; Alpha Diagnostics, San Antonio, TX, USA), anti-cleaved Notch-1 (Cat. D3B8; Cell Signaling Technology (CST), Danvers, MA, USA), anti-Notch-1 (Cat. ab52627; Abcam), anti-Hes-1 (Cat.D6P2U; CST), C/EBPα (Cat. ab15048; Abcam), anti-tubulin (Cat. 2148;CST), anti-lamin B1 (cat. 12586;CST), and anti-β-actin (Cat. sc-47778; Santa Cruz Biotechnology) antibodies. The signals were quantified using a Fusion Solo Detector (Vilber Lourmat, Marne La Vallee, France).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eImmunoprecipitation\u003c/h2\u003e \u003cp\u003e3T3-L1 preadipocytes were treated with recombinant TGFBI (20 \u0026micro;g/ml) for 24 h, and total protein was extracted in RIPA buffer containing 10 mM nethylmaleimide (EMD Millipore, Darmstadt, Germany). Immunoprecipitation was carried out overnight at 4\u0026deg;C with primary antibody, followed by the addition of Protein G agarose beads, and incubation for 1 h. Beads were collected by pulse centrifugation and washed with lysis buffer. The precipitated proteins were analyzed by western blotting as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eInguinal white adipose tissue (iWAT) and liver tissue were fixed with 4% paraformaldehyde, processed, and embedded in paraffin. Tissue sections (5\u0026ndash;7 \u0026micro;m thick) were stained with hematoxylin and eosin (H\u0026amp;E). The size of the adipocytes in three mice per genotype was measured using ImageJ software (NIH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses for all experiments were performed using SPSS ver.20.0 (SPSS Inc., Chicago, IL, USA). Statistical differences between groups were analyzed using two-sided t-test, and statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eTGFBI KO mice exhibit resistance to high-fat diet-induced obesity\u003c/h2\u003e \u003cp\u003eA genetically modified animal model was used to determine whether TGFBI affected diet-induced obesity. The PCR analysis confirmed the deletion of the TGFBI gene, and the 376-bp product was compared with the 1099-bp product for the WT mice (Supplementary Fig.\u0026nbsp;1B). The deletion of TGFBI in the plasma of WT and KO mice was also confirmed by ELISA. The whole body weight of 11-week-old HFD-fed KO mice was significantly lower than that of WT mice of the same age (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The 20-week-old HFD-fed KO mice showed a dramatic decrease in body weight gain compared with the WT mice. These differences were also observed between the ND-fed WT and KO mice, although a smaller difference was observed compared with that for the HFD-fed groups. A significant decrease was observed in the inguinal WAT (iWAT), retroperitoneal WAT (rWAT), epididymal WAT (eWAT), and liver mass of the HFD-fed KO mice, whereas the brown adipose tissue (BAT) and subcutaneous WAT (sWAT) mass were unaltered compared with those of the HFD-fed WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C). The masses of the heart, lungs, kidneys, and spleen tissue were comparable between the WT and TGFBI KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). As expected, with suppressed adipose expansion, the size of the iWAT adipocytes decreased in the HFD-fed KO mice compared with that of the HFD-fed WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). These results suggest that TGFBI KO mice exhibit resistance to HFD-induced body weight gain and adipose tissue expansion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHFD-induced ECM remodeling was apparent not only in adipose tissue but also in the liver, and HFD-fed mice exhibited increased expression of collagen type I and III in the liver \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. This remodeling process was also closely associated with insulin resistance \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Therefore, we determined whether the lack of TGFBI contributed to improved homeostasis and liver function in HFD-fed mice. HFD-fed KO mice exhibited a marked reduction in lipid composition, triacylglycerol (TG) content, and free fatty acid (FFA) content in the liver compared with that in the HFD-fed WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-G). HFD-fed KO mice showed increased expression of gluconeogenesis-related genes, including those encoding GLUT-2, PGC-1α, and PPARα (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Glucose tolerance and insulin sensitivity improved in the HFD-fed KO mice compared with those in the HFD-fed WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI-J). To determine whether differences occurred between sexes, we evaluated the phenotype of HFD-fed female KO mice. Similar to that of the HFD-fed male KO mice, the female KO mice also exhibited lower body weight and iWAT mass and improved glucose homeostasis than the female WT mice (Supplementary Fig.\u0026nbsp;2A-F). Taken together, these results suggest that TGFBI KO mice had improved capacity for obesity-induced metabolic changes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTGFBI KO induces expression of proteins involved in the PPAR\u003c/b\u003eγ \u003cb\u003esignaling pathway\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo explore the mechanisms underlying the protective effects of TGFBI during obesity, we examined the PPARγ signaling pathway, which is closely involved in adipocyte differentiation, in iWAT from ND- and HFD-fed WT and TGFBI KO mice. We observed that the protein and mRNA expression levels of PPARγ, C/EBPα, and adiponectin did not change in the ND-fed WT and KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). Interestingly, the protein and mRNA expression levels of PPARγ and C/EBPα were significantly lower in the HFD-fed TGFBI KO mice than the WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D). In addition, the serum leptin levels were decreased in the TGFBI KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Notably, the TGFBI KO mice showed improved food efficiency, although their food intake was comparable with that of the WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These results indicated that the obesity resistance of TGFBI KO mice was not affected by the reduction of fat in the diet. Taken together, these results demonstrate that the lack of TGFBI may lead to the suppression of excess adipose expansion by inhibiting the PPARγ signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of TGFBI inhibits adipogenic differentiation\u003c/h2\u003e \u003cp\u003eWe determined whether TGFBI plays a role in preadipocyte differentiation into mature adipocytes. We confirmed the adipogenic capacity of TGFBI-knockdown cells and compared it with that of the control cells. TGFBI knockdown cells exhibited reduced Oil Red O staining and intracellular TG accumulation, which suggest reduced differentiation capacity compared with the control siRNA cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). The knockdown efficacy was confirmed by ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). TGFBI knockdown cells showed suppressed expression of adipogenesis-related genes, including those encoding PPARγ and C/EBPα (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). These proteins also showed reduced expression in TGFBI knockdown cells compared with that in the control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-G). Furthermore, maximum oxygen consumption was significantly higher in the TGFBI knockdown cells than the control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH-I). In contrast, no differences were observed in the basal state.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eTGFBI KO mice exhibit over-secretion of adipsin and higher macrophage populations\u003c/h2\u003e \u003cp\u003eTo completely understand the mechanism through which metabolism benefits HFD-fed TGFBI KO mice, we performed an RNA sequence analysis of iWAT from these mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Among the gene processes that were modulated in TGFBI KO mice, we focused on the significantly enriched complement and coagulation cascades. Adipsin is a regulatory protein of the complement cascade whose expression is downregulated in mice with acquired metabolic diseases \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Consistently, the expression of adipsin was reduced in HFD-fed obese mice compared with ND-fed normal mice (Supplementary Fig.\u0026nbsp;3A-B). We observed that the mRNA, protein, and secretory levels of adipsin were upregulated in the HFD-fed TGFBI KO mice, whereas no difference was observed between the ND-fed WT and KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we hypothesized that primary adipocyte-derived BM-MSCs could alter adipsin secretion in an autocrine manner. However, the adipsin levels of differentiated BM-MSCs were comparable between those isolated from TGFBI KO mice or WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Previous studies have indicated that fat-produced adipsin regulates inflammation and adipose tissue macrophage properties in adipsin KO mice \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. To determine the contribution of immune cells, such as macrophages, to the processes observed in the present study, we measured the macrophage population in the adipose tissues. We detected CD11b-positive macrophages in the adipose tissue and found a higher population of CD11b\u003csup\u003e+\u003c/sup\u003e macrophages in the TGFBI KO mice compared with the WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-F). In addition, the levels of the pro-inflammatory cytokines IL-6 and TNF-α were decreased in the TGFBI KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Moreover, significant differences were not observed in the expression of CD206 and CD86 between the WT and TGFBI KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). These data suggest that TGFBI facilitates the differentiation of distinct populations of macrophages and modulates the inflammatory status of obese adipose tissue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eAdipose macrophages regulate the browning and adipsin secretion of adipocytes\u003c/h2\u003e \u003cp\u003eBecause increased numbers of macrophages were present in TGFBI KO mice, we sought to determine whether adipsin expression was regulated by macrophages under indirect coculture conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We found that adipocytes cultured with TGFBI KO macrophages contained brown-like adipocytes that featured multiple smaller lipid droplets in their cytoplasm (Supplementary Fig.\u0026nbsp;4A). Consistent with these morphological differences, adipsin, UCP-1, and PGC-1α were mainly detected in adipocytes cocultured with macrophages isolated from TGFBI KO mice compared with those isolated from WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-D and Supplementary Fig.\u0026nbsp;4B). Next, we confirmed the effects of TGFBI on adipsin expression under macrophage coculture conditions. TGFBI treatment ameliorated adipsin expression in adipocytes cultured with TGFBI KO macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-G). Interestingly, we observed that TGFBI was primarily secreted by the small vascular fraction containing macrophages rather than by adipocytes. In contrast, adipsin levels were significantly higher in adipocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH-I). This result suggests that these macrophages play important roles in the regulation of TGFBI-mediated adipsin secretion from adipocytes. Additionally, TGFBI is largely derived from macrophages and may influence adipsin expression in adipocytes in a paracrine manner, and which closely associated with adipocyte characteristics and browning.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTGFBI induces Notch-1 signaling activation in adipocytes.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNotch-1-induced transcriptional regulator Hes-1 downregulates adipsin expression in adipocytes \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e and Notch-1 signaling promotes obesity progression and ameliorates adipose browning \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. However, the mechanisms underlying the activation of these processes and whether these processes are related to ECM remodeling in HFD-induced obese mice have not been clarified. HFD-feeding induced the overexpression of Notch-1 and Hes-1 in WAT obtained from WT mice compared with that from lean mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). Interestingly, Notch-1 protein expression was significantly reduced in TGFBI KO mice, whereas differences at the mRNA level relative to that in WT mice were not observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). In contrast, Hes-1 mRNA and protein levels decreased by 1.5- to 2.0-fold in TGFBI KO mice. To determine whether exogenous treatment with TGFBI affected Notch-1 activation, we measured the expression of Notch-1 and Hes-1 in TGFBI-treated adipocytes. The TGFBI treatment increased the protein expression of Hes-1 and Notch-1 in 3T3-L1 adipocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Consistent with the in vivo data, downregulation of Hes-1 mRNA expression was observed, while changes in Notch-1 expression were not observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). These data suggest that TGFBI controls Notch-1 abundance at the post-transcriptional level.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we postulated that TGFBI may facilitate the nuclear translocation of Notch-1. The results showed that the TGFBI treatment induced nuclear translocation of Notch-1, which was similar to that observed for the positive control galectin-3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Furthermore, the content of Notch-1 intracellular domain increased in the nuclear fractions of TGFBI-treated 3T3-L1 adipocytes, whereas Notch-1 activation was blocked by a Notch-1 inhibitor in cells cotreated with TGFBI (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Furthermore, Notch-1 expression remained unchanged in the cytoplasmic fractions. These results suggest that TGFBI activates nuclear translocation of Notch-1 in adipocytes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eNotch-1 binds to TGFBI and regulates cell adhesion of adipocytes\u003c/h2\u003e \u003cp\u003eNext, we determined whether TGFBI binds to Notch-1 in 3T3-L1 adipocytes. Notch-1 was physically associated with TGFBI in 3T3-L1 adipocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). In a non-cell-based system, TGFBI exhibited binding affinity for Notch-1 (Kd\u0026thinsp;=\u0026thinsp;3.18 nM), although its affinity was weaker than that of jagged-1, which is a strong ligand for Notch-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The ECM facilitates cellular attachment and mediates cellular mobility, adhesion, proliferation, and differentiation \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. To determine whether TGFBI controls adipocyte adhesion via Notch-1, we performed a cell adhesion assay using TGFBI-coated plates (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Notch-1 inhibition markedly suppressed the adhesion capacity of 3T3-L1 adipocytes on TGFBI-coated plates, although cytotoxicity was not observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-F). These results indicated that TGFBI is functionally active and directly binds to Notch-1 in adipocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eTGFBI suppresses adipocyte browning and adipsin secretion via activating Notch-1 signaling\u003c/h2\u003e \u003cp\u003eTo establish the physiological significance of TGFBI, we examined whether it regulates adipocyte browning by activating Notch-1. The TGFBI treatment markedly reduced multilocular lipid formation and browning-related gene expression in differentiated 3T3-L1 cells compared with that in control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-B). Notably, this TGFBI-induced reduction was restored by cotreatment with a Notch-1 inhibitor, and significantly elevated the expression of UCP-1 and PGC-1α (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB-C). Accordingly, adipsin expression and secretion were significantly elevated by cotreatment of the cells with the Notch inhibitor and TGFBI compared to that under treatment with TGFBI alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC-E). Moreover, the adipsin levels were unchanged by the treatment with the Notch-1 inhibitor alone. These findings indicate that TGFBI ameliorates adipsin-mediated browning via a Notch-1 dependent mechanism. Finally, we determined whether Notch-1 regulated the expression of adipsin and browning markers. Notch-1 has intracellular domains that include RAM (Rbpj interacting domain) and NLS (nuclear localization signal) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF). Deletion of the NLS domain upregulated the mRNA and protein expression of UCP-1 and adipsin (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG-I), while deletion of RAM did not induce the expression of these proteins. The promoter activity of adipsin was repressed by TGFBI (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eJ); however, this was restored by deleting the NLS domain in the presence of TGFBI (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eK). These results indicate that deletion of the NLS domain directly enhances adipsin promoter activity and markedly increases the expression of browning-related genes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eObesity is characterized by abnormal adipose expansion and fibrosis accompanied with the overaccumulation of ECM \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. A recent study indicated that remodeling is required to maintain a healthy adipose tissue condition \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. However, limitations have been observed in studies that primarily focused on major ECM proteins, such as collagen and fibronectin. TGFBI has been implicated in the pathogenesis of cancer and diabetic retinopathy \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Moreover, previous studies have detected TGFBI in adipose tissue and associated TGFBI polymorphisms with insulin levels and body mass \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. However, the gain- and loss-of-function mechanisms of TGFBI are not completely understood. In the present study, we demonstrated that the ECM component TGFBI plays a role in HFD-induced obesity and improves several physiological conditions, such as glucose/insulin resistance, adipose expansion, liver steatosis, and adipocyte differentiation.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIndeed, weight loss resulting from TGFBI deficiency occurs not only in HFD-fed mice but also in ND-fed mice. A previous study demonstrated that TGFBI regulates periosteal bone formation, including the bone mass, size, and strength \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. TGFBI KO mice show reduced bone mass and 8\u0026ndash;20% lower body weight than WT mice during postnatal development. However, why TGFBI KO mice exhibit a slight decline in body weight gain due to ND-feeding remains elusive. In the present study, we found that HFD-fed TGFBI KO mice exhibited a greater reduction in adipose and liver mass and lipid accumulation. Future studies are warranted to address the tissue-specific and direct targeted effects of TGFBI using engineered mice.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eInconsistent with our data, a previous study reported that TGFBI knockdown accelerated human preadipocyte adipogenesis \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e; however, the use of human preadipocytes isolated from subcutaneous adipose tissue or visceral adipose tissue likely led to the observed discrepancy in the results. Adipose-derived stem cells undergo multilineage changes and then differentiate into different type of adipocyte, such as white, beige, and brown adipocytes \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. These adipocytes have characteristic differences in their anatomical location and function. The 3T3-L1 mouse cell line used in our study shows characteristics typical of white adipocytes \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Based on these points, we assumed that the role of TGFBI in each type of adipocyte may differ, which may have caused the discrepancy in the results between the previous study and our study. In fact, in our results based on BM-MSC-derived adipocytes, TGFBI did not affect adipogenic capacity, but significantly promoted adipose browning.\u003c/p\u003e \u003cp\u003eTGFBI overexpression is detected in fibroblasts, peritoneal cells, macrophages, and T cells \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, among which, macrophages are predominant producers of TGFBI. In the present study, we found that TGFBI was largely produced by small vascular fractions containing various immune cells and also secreted from adipocytes. Indeed, significant changes were not observed in the upregulated adipsin expression between the BM-MSC-derived adipocytes cultured alone and those from the WT mice following TGFBI KO. This suggests that additional metabolically stimulatory conditions or factors may contribute to adipsin secretion from adipocytes. Therefore, we propose that TGFBI acts on obesity and adipose tissue in both autocrine and autocrine manners.\u003c/p\u003e \u003cp\u003eMacrophages are the primary inflammatory cells found in inflamed adipose tissue that control adipose homeostasis and energy expenditure \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Several reports have indicated the involvement of cytokines secreted from adipose tissue macrophages in beige adipocyte formation and WAT browning \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Consistent with these findings, our results provided several lines of evidence supporting that adipose-associated macrophages regulate the expression of inflammatory cytokines and browning markers in adipose tissues obtained from TGFBI KO mice. We speculate that the different phenotypes detected in our study occurred in a distinct population of CD11b\u003csup\u003e+\u003c/sup\u003e M1-like macrophages in TGFBI KO mice compared to those in WT mice.\u003c/p\u003e \u003cp\u003eInterestingly, adipsin levels were strongly elevated in adipose tissue and adipocytes cocultured with macrophages isolated from TGFBI KO mice. We confirmed that adipsin expression was positively correlated with the expression of adipose browning markers UCP-1 and PGC-1α. In TGFBI KO mice, adipsin was secreted in response to macrophages; thus, we concluded that increased adipsin expression is closely associated with adipose browning and other metabolic benefits in TGFBI KO mice.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe Notch-1 signaling pathway has been implicated in various biological processes, such as tumorigenesis, apoptosis, and proliferation \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Recently, Notch signaling was also found to be important in adipogenesis \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Inhibition of Notch-1 and its signaling mediators results in adipose browning and regulates adipsin expression \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, which is consistent with our observation that the pharmacological effects and genetic mutations of Notch-1 resulted in elevated adipose browning. Furthermore, we discovered that Notch-1 represses adipsin expression and the Notch-1 intracellular domain NLS is required for the inhibition of adipsin transcription. Notably, we discovered that TGFBI acts as a new ligand for Notch-1, and this signaling activation is required for suppression of adipsin secretion and adipose browning.\u003c/p\u003e\u003cp\u003eIn summary, the present study revealed that the deletion of the ECM protein TGFBI plays a protective role against HFD-induced metabolic disorders. Mechanistically, TGFBI activates Notch-1 signaling and subsequently regulates adipsin expression, which may explain the upregulation of adipose browning related-gene expression and the beneficial metabolic effects observed in TGFBI KO mice. Taken together, our findings offer new insights for the development of new therapeutic approaches for obesity and related disorders via TGFBI regulation.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Research Foundation of Korea (NRF) funded by the Korean government (NRF-2017R1A2B4011003, NRF-2019R1A2C1090619, 2021R1A6A3A01087252, and NRF-2019R1A2C2005921).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.G.L., T.K.K., and J.-O.N. designed the project; S.G.L., J.C., S.M.W., and S.U.S. performed the experiments; H.-J.K., H.-S.P., and D.D.S. provided consultation and guidance; I.-S.K. provided the transgenic mice and guidance; T.-K.K. and J.-O.N. supervised the project; and S.G.L., T.-K.K., and J.-O.N. wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKaila, B. \u0026amp; Raman, M. Obesity: a review of pathogenesis and management strategies. Canadian Journal of Gastroenterology and Hepatology \u003cb\u003e22\u003c/b\u003e, 61\u0026ndash;68 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeirson, L. \u003cem\u003eet al.\u003c/em\u003e Treatment for overweight and obesity in adult populations: a systematic review and meta-analysis. CMAJ open \u003cb\u003e2\u003c/b\u003e, E306 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan, T.S. \u0026amp; Lean, M.E. A clinical perspective of obesity, metabolic syndrome and cardiovascular disease. JRSM cardiovascular disease \u003cb\u003e5\u003c/b\u003e, 2048004016633371 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuilherme, A., Virbasius, J.V., Puri, V. \u0026amp; Czech, M.P. Adipocyte dysfunctions linking obesity to insulin resistance and type 2 diabetes. Nature reviews Molecular cell biology \u003cb\u003e9\u003c/b\u003e, 367 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScherer, P.E. Adipose tissue: from lipid storage compartment to endocrine organ. Diabetes \u003cb\u003e55\u003c/b\u003e, 1537\u0026ndash;1545 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShabalina, I.G. \u003cem\u003eet al.\u003c/em\u003e UCP1 in brite/beige adipose tissue mitochondria is functionally thermogenic. Cell reports \u003cb\u003e5\u003c/b\u003e, 1196\u0026ndash;1203 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, J. \u003cem\u003eet al.\u003c/em\u003e Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human. Cell \u003cb\u003e150\u003c/b\u003e, 366\u0026ndash;376 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVaicik, M.K. \u003cem\u003eet al.\u003c/em\u003e Laminin alpha4 deficient mice exhibit decreased capacity for adipose tissue expansion and weight gain. PloS one \u003cb\u003e9\u003c/b\u003e, e109854 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun, K., Kusminski, C.M. \u0026amp; Scherer, P.E. Adipose tissue remodeling and obesity. The Journal of clinical investigation \u003cb\u003e121\u003c/b\u003e, 2094\u0026ndash;2101 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, M.H. \u003cem\u003eet al.\u003c/em\u003e ECM microenvironment unlocks brown adipogenic potential of adult human bone marrow-derived MSCs. Scientific reports \u003cb\u003e6\u003c/b\u003e, 21173 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, S.G. \u003cem\u003eet al.\u003c/em\u003e Endothelial angiogenic activity and adipose angiogenesis is controlled by extracellular matrix protein TGFBI. Scientific reports \u003cb\u003e11\u003c/b\u003e, 9644 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGealekman, O. \u003cem\u003eet al.\u003c/em\u003e Depot-specific differences and insufficient subcutaneous adipose tissue angiogenesis in human obesity. Circulation \u003cb\u003e123\u003c/b\u003e, 186\u0026ndash;194 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZafar, M.I. \u003cem\u003eet al.\u003c/em\u003e Association between the expression of vascular endothelial growth factors and metabolic syndrome or its components: a systematic review and meta-analysis. Diabetology \u0026amp; metabolic syndrome \u003cb\u003e10\u003c/b\u003e, 62 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNijhawans, P., Behl, T. \u0026amp; Bhardwaj, S. Angiogenesis in obesity. Biomedicine \u0026amp; pharmacotherapy = Biomedecine \u0026amp; pharmacotherapie \u003cb\u003e126\u003c/b\u003e, 110103 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamaguchi, K. \u003cem\u003eet al.\u003c/em\u003e Notch1 haploinsufficiency in mice accelerates adipogenesis. Scientific reports \u003cb\u003e11\u003c/b\u003e, 1\u0026ndash;14 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarc\u0026eacute;s, C. \u003cem\u003eet al.\u003c/em\u003e Notch-1 controls the expression of fatty acid-activated transcription factors and is required for adipogenesis. The Journal of biological chemistry \u003cb\u003e272\u003c/b\u003e, 29729\u0026ndash;29734 (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBae, J.S. \u003cem\u003eet al.\u003c/em\u003e Transforming growth factor β-induced protein promotes severe vascular inflammatory responses. American journal of respiratory and critical care medicine \u003cb\u003e189\u003c/b\u003e, 779\u0026ndash;786 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, J.E. \u003cem\u003eet al.\u003c/em\u003e A TGF-beta-inducible cell adhesion molecule, betaig-h3, is downregulated in melorheostosis and involved in osteogenesis. Journal of cellular biochemistry \u003cb\u003e77\u003c/b\u003e, 169\u0026ndash;178 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoleimani, M. \u0026amp; Nadri, S. A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. Nature protocols \u003cb\u003e4\u003c/b\u003e, 102\u0026ndash;106 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToda, G., Yamauchi, T., Kadowaki, T. \u0026amp; Ueki, K. Preparation and culture of bone marrow-derived macrophages from mice for functional analysis. STAR protocols \u003cb\u003e2\u003c/b\u003e, 100246 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, J.S. \u003cem\u003eet al.\u003c/em\u003e Eupatilin inhibits adipogenesis through suppression of PPARgamma activity in 3T3-L1 cells. Biomedicine \u0026amp; pharmacotherapy = Biomedecine \u0026amp; pharmacotherapie \u003cb\u003e103\u003c/b\u003e, 135\u0026ndash;139 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, S.G., Kim, J.S., Min, K., Kwon, T.K. \u0026amp; Nam, J.O. Hispidulin inhibits adipogenesis in 3T3-L1 adipocytes through PPARgamma pathway. Chemico-biological interactions \u003cb\u003e293\u003c/b\u003e, 89\u0026ndash;93 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams, A.S. \u003cem\u003eet al.\u003c/em\u003e Integrin alpha1-null mice exhibit improved fatty liver when fed a high fat diet despite severe hepatic insulin resistance. The Journal of biological chemistry \u003cb\u003e290\u003c/b\u003e, 6546\u0026ndash;6557 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonner, J.S. \u003cem\u003eet al.\u003c/em\u003e Relaxin treatment reverses insulin resistance in mice fed a high-fat diet. Diabetes \u003cb\u003e62\u003c/b\u003e, 3251\u0026ndash;3260 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRyu, K.Y. \u0026amp; Jeon, E.J. Regulation of Adipsin Expression by Endoplasmic Reticulum Stress in Adipocytes. \u003cb\u003e10\u003c/b\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLo, J.C. \u003cem\u003eet al.\u003c/em\u003e Adipsin is an adipokine that improves β cell function in diabetes. Cell \u003cb\u003e158\u003c/b\u003e, 41\u0026ndash;53 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, Y. \u003cem\u003eet al.\u003c/em\u003e Fat-Produced Adipsin Regulates Inflammatory Arthritis. Cell reports \u003cb\u003e27\u003c/b\u003e, 2809\u0026ndash;2816.e2803 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSearfoss, G.H. \u003cem\u003eet al.\u003c/em\u003e Adipsin, a biomarker of gastrointestinal toxicity mediated by a functional gamma-secretase inhibitor. The Journal of biological chemistry \u003cb\u003e278\u003c/b\u003e, 46107\u0026ndash;46116 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBi, P. \u003cem\u003eet al.\u003c/em\u003e Inhibition of Notch signaling promotes browning of white adipose tissue and ameliorates obesity. \u003cb\u003e20\u003c/b\u003e, 911\u0026ndash;918 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoh, K.H. \u003cem\u003eet al.\u003c/em\u003e Nonimmune cell-derived ICOS ligand functions as a renoprotective αvβ3 integrin-selective antagonist. The Journal of clinical investigation \u003cb\u003e129\u003c/b\u003e, 1713\u0026ndash;1726 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpencer, M. \u003cem\u003eet al.\u003c/em\u003e Adipose Tissue Extracellular Matrix and Vascular Abnormalities in Obesity and Insulin Resistance. \u003cem\u003eThe Journal of Clinical Endocrinology \u0026amp; Metabolism\u003c/em\u003e \u003cb\u003e96\u003c/b\u003e, E1990-E1998 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuiz-Ojeda, F.J., M\u0026eacute;ndez-Guti\u0026eacute;rrez, A., Aguilera, C.M. \u0026amp; Plaza-D\u0026iacute;az, J. Extracellular Matrix Remodeling of Adipose Tissue in Obesity and Metabolic Diseases. International journal of molecular sciences \u003cb\u003e20\u003c/b\u003e, 4888 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOzawa, D. \u003cem\u003eet al.\u003c/em\u003e TGFBI Expression in Cancer Stromal Cells is Associated with Poor Prognosis and Hematogenous Recurrence in Esophageal Squamous Cell Carcinoma. Annals of surgical oncology \u003cb\u003e23\u003c/b\u003e, 282\u0026ndash;289 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBetts-Obregon, B.S. \u003cem\u003eet al.\u003c/em\u003e TGFβ induces BIGH3 expression and human retinal pericyte apoptosis: a novel pathway of diabetic retinopathy. Eye (London, England) \u003cb\u003e30\u003c/b\u003e, 1639\u0026ndash;1647 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark, K.S. \u003cem\u003eet al.\u003c/em\u003e Genetic polymorphisms in the transforming growth factor beta-induced gene associated with BMI. Human mutation \u003cb\u003e25\u003c/b\u003e, 322 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu, H., Wergedal, J.E., Zhao, Y. \u0026amp; Mohan, S. Targeted disruption of TGFBI in mice reveals its role in regulating bone mass and bone size through periosteal bone formation. Calcified tissue international \u003cb\u003e91\u003c/b\u003e, 81\u0026ndash;87 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHirata, K. \u003cem\u003eet al.\u003c/em\u003e Role of leukotriene B(4) receptor signaling in human preadipocyte differentiation. Biochemical and biophysical research communications \u003cb\u003e429\u003c/b\u003e, 197\u0026ndash;203 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng, B., Cao, B., Li, G. \u0026amp; Huard, J. Mouse adipose-derived stem cells undergo multilineage differentiation in vitro but primarily osteogenic and chondrogenic differentiation in vivo. Tissue engineering \u003cb\u003e12\u003c/b\u003e, 1891\u0026ndash;1901 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark, A., Kim, W.K. \u0026amp; Bae, K.H. Distinction of white, beige and brown adipocytes derived from mesenchymal stem cells. World journal of stem cells \u003cb\u003e6\u003c/b\u003e, 33\u0026ndash;42 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorrison, S. \u0026amp; McGee, S.L. 3T3-L1 adipocytes display phenotypic characteristics of multiple adipocyte lineages. Adipocyte \u003cb\u003e4\u003c/b\u003e, 295\u0026ndash;302 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLecker, L.S. \u003cem\u003eet al.\u003c/em\u003e TGFBI production by macrophages contributes to an immunosuppressive microenvironment in ovarian cancer. Cancer research \u003cb\u003e81\u003c/b\u003e, 5706\u0026ndash;5719 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSteitz, A.M. \u003cem\u003eet al.\u003c/em\u003e Tumor-associated macrophages promote ovarian cancer cell migration by secreting transforming growth factor beta induced (TGFBI) and tenascin C. Cell death \u0026amp; disease \u003cb\u003e11\u003c/b\u003e, 1\u0026ndash;15 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWolf, Y. \u003cem\u003eet al.\u003c/em\u003e Brown-adipose-tissue macrophages control tissue innervation and homeostatic energy expenditure. Nature immunology 18, 665\u0026ndash;674 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKotzbeck, P. \u003cem\u003eet al.\u003c/em\u003e Brown adipose tissue whitening leads to brown adipocyte death and adipose tissue inflammation. J Lipid Res 59, 784\u0026ndash;794 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeuper, M. On the role of macrophages in the control of adipocyte energy metabolism. Endocrine connections 8, R105-r121 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, D., Masiero, M., Banham, A.H. \u0026amp; Harris, A.L. The notch ligand JAGGED1 as a target for anti-tumor therapy. Frontiers in oncology 4, 254 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShan, T., Liu, J., Wu, W., Xu, Z. \u0026amp; Wang, Y. Roles of Notch Signaling in Adipocyte Progenitor Cells and Mature Adipocytes. Journal of cellular physiology 232, 1258\u0026ndash;1261 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBi, P. \u003cem\u003eet al.\u003c/em\u003e Inhibition of Notch signaling promotes browning of white adipose tissue and ameliorates obesity. Nature medicine 20, 911\u0026ndash;918 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSearfoss, G.H. \u003cem\u003eet al.\u003c/em\u003e Adipsin, a biomarker of gastrointestinal toxicity mediated by a functional γ-secretase inhibitor. Journal of Biological Chemistry 278, 46107\u0026ndash;46116 (2003).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"adipocyte browning, extracellular matrix, macrophage, obesity, TGFBI, Notch-1","lastPublishedDoi":"10.21203/rs.3.rs-1739900/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1739900/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExtracellular matrix proteins are associated with metabolically healthy adipose tissue and regulate inflammation, fibrosis, angiogenesis, and subsequent metabolic deterioration. In this study, we demonstrated that transforming growth factor-beta (TGFBI), an extracellular matrix (ECM) component, plays an important role in adipose metabolism and browning during high-fat diet-induced obesity. TGFBI KO mice were resistant to adipose tissue hypertrophy, liver steatosis, and insulin resistance. Furthermore, adipose tissue from TGFBI KO mice presented a large population of CD11b\u003csup\u003e+\u003c/sup\u003e macrophages, which control adipokine secretion through paracrine mechanisms. Mechanistically, we showed that inhibiting TGFBI-stimulated release of adipsin by Notch-1-dependent signaling resulted in adipocytes browning. TGFBI was physiologically bound to Notch-1 and stimulated its activation in adipocytes. Our findings revealed a novel protective effect of TGFBI deficiency in obesity that is realized via the activation of the Notch-1 signaling pathway.\u003c/p\u003e","manuscriptTitle":"TGFBI remodels adipose metabolism by regulating the Notch-1 signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-09-30 19:04:38","doi":"10.21203/rs.3.rs-1739900/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2022-07-18T00:19:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-06-14T05:13:43+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2022-06-13T05:08:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental \u0026 Molecular Medicine","date":"2022-06-09T01:47:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-06-09T01:47:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e63f203f-5b20-4f22-b8e7-3056e9103662","owner":[],"postedDate":"September 30th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-03-01T08:07:31+00:00","versionOfRecord":{"articleIdentity":"rs-1739900","link":"https://doi.org/10.1038/s12276-023-00947-9","journal":{"identity":"experimental-and-molecular-medicine","isVorOnly":false,"title":"Experimental \u0026 Molecular Medicine"},"publishedOn":"2023-03-01 05:00:00","publishedOnDateReadable":"March 1st, 2023"},"versionCreatedAt":"2022-09-30 19:04:38","video":"","vorDoi":"10.1038/s12276-023-00947-9","vorDoiUrl":"https://doi.org/10.1038/s12276-023-00947-9","workflowStages":[]},"version":"v1","identity":"rs-1739900","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1739900","identity":"rs-1739900","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}