GALNT15, induced during adipogenesis of human SGBS cells but not in mouse 3T3-L1 cells, regulates adipocyte differentiation

GALNT15, induced during adipogenesis of human SGBS cells but not in mouse 3T3-L1 cells, regulates adipocyte differentiation | 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 GALNT15, induced during adipogenesis of human SGBS cells but not in mouse 3T3-L1 cells, regulates adipocyte differentiation Asuka Takahashi, Ryo Koike, Shota Watanabe, Kyoko Kuribayashi, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4244309/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Aug, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Adipogenesis involves intricate molecular mechanisms regulated by various transcription factors and signaling pathways. In this study, we aimed to identify factors specifically induced during adipogenesis in the human preadipocyte cell line, SGBS, but not in the mouse preadipocyte cell line, 3T3-L1. Microarray analysis revealed distinct gene expression profiles, with 1460 genes induced in SGBS cells and 1297 genes induced in 3T3-L1 cells during adipogenesis, with only 297 genes commonly induced. Among the genes uniquely induced in SGBS cells, we focused on GALNT15 , which encodes polypeptide N-acetylgalactosaminyl transferase-15. Its expression increased transiently during adipogenesis in SGBS cells but remained low in 3T3-L1 cells. Overexpression of GALNT15 increased mRNA levels of CCAAT-enhancer binding protein (C/EBPα) and leptin but had no significant impact on adipogenesis in SGBS cells. Conversely, knockdown of GALNT15 suppressed mRNA expression of adipocyte marker genes, reduced lipid accumulation, and decreased the percentage of cells with oil droplets. The induction of C/EBPα and peroxisome proliferator-activated receptor γ during adipogenesis was promoted or suppressed in SGBS cells subjected to overexpression or knockdown of GALNT1 5, respectively. These data suggest that GALNT15 is a novel regulatory molecule that enhances adipogenesis in SGBS cells. Biological sciences/Developmental biology Health sciences/Endocrinology/Endocrine system and metabolic diseases/Obesity adipogenesis polypeptide N-acetylgalactosaminyl transferase GALNT15 preadipocytes adipocytes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Obesity is a condition characterized by the excessive accumulation of adipose tissue in the body, which, along with its common complications such as, type 2 diabetes, dyslipidemia, and hypertension, increases the risk of vascular diseases related to arteriosclerosis 1 . Adipose tissue comprises mature adipocytes with oil droplets that store triglycerides, and stromal vascular fractions containing preadipocytes, which are precursor cells of adipocytes. Excessive accumulation of adipose tissue is thought to result from the hypertrophy of individual adipocytes due to increased triglyceride accumulation, and an increased number of adipocytes due to the accelerated differentiation of preadipocytes into mature adipocytes 2 . During adipogenesis, preadipocytes acquire the machinery for lipid transport and synthesis, insulin sensitivity, and secretion of adipocyte-specific proteins 2 . During this process, the induction and activation of transcription factors, specifically the CCAAT-enhancer binding protein (C/EBP) family and peroxisome proliferator-activated receptor γ (PPARγ), are crucial 2 , 3 . C/EBPβ and C/EBPδ are transiently induced early in adipogenesis 4 and they induce expressions of C/EBPα and PPARγ, which function to promote transcription of many types of genes involved in adipocyte phenotype and function 2 , 3 . In addition to these key transcriptional regulators, various other transcriptional regulators of adipogenesis and factors affecting adipogenesis have been reported. While many of these findings regarding adipogenesis have been obtained using in vitro mouse models utilizing cell lines, such as 3T3-L1 or 3T3-F442A, the mechanisms of adipogenesis in humans and mice are not entirely identical. For instance, 3T3-L1 and 3T3-F442A cells undergo an increase in cell number known as mitotic clonal expansion prior to differentiation, which is thought to be necessary for subsequently differentiation. However, human primary preadipocytes or SGBS cells, a human preadipocyte cell line established from the subcutaneous adipose tissue of an infant suspected of having Simpson-Golabi-Behmel Syndrome 5 , do not necessarily undergo mitotic clonal expansion during adipogenesis 6 , 7 . Furthermore, LIM domain only 3 ( LMO3 ), which positively regulates adipogenesis, is upregulated during adipogenesis in humans but not in mice 8 . Additionally, D-dopachrome tautomerase, an adipokine secreted by adipocytes, suppresses adipogenesis in SGBS cells but not in 3T3-L1 cells 9 . Thus, identifying the factors that affect adipogenesis in a human-specific manner may lead to the identification of novel target molecules for the development of anti-obesity drugs. In the present study, we conducted a comparative analysis of mRNA expression induced during adipogenesis in SGBS cells and 3T3-L1 cells using microarray to identify genes whose expression is induced in SGBS but not in 3T3-L1 cells. GALNT15 , encoding polypeptide N-acetylgalactosaminyl transferase (GalNAc-T)-15, was identified as one of the candidate genes. GALNT15 , a member of the GALNT family comprising 20 species in humans, is expressed in most human tissues 10 . Its translation product catalyzes the initiation of mucin-type O -linked glycosylation by adding N -acetylgalactosamine to the serine or threonine residues of the polypeptide 10 . Mucin-type O -glycosylation is initiated and regulated by the GalNAc-T family that catalyzes the first step in the biosynthesis forming the GalNAcα1- O -serine/threonine linkage in O-glycoproteins. O -linked glycosylation, the most diverse form of post-translational modifications, affects various aspects of protein function, therefore, many GalNAc-Ts are considered to have potentials for differential regulation in cells and tissues 11 . Aberrant O -glycosylation by some GalNAc-Ts has been observed in many types of cancer and is associated with noncancerous developmental and metabolic disorders 12 , 13 ; however, the involvement of GALNT15 in these diseases has not been reported, and its physiological function remains largely unknown. Therefore, we focused on GALNT15 and investigated its effects on adipogenesis in SGBS cells. Results GALNT15 expression was induced during adipogenesis in SGBS but not in 3T3-L1 cells The mRNA expression profiles of SGBS and 3T3-L1 cells on days 0, 1, 3, and 7 after adipogenic induction were analyzed using microarray analysis. Genes whose expression was upregulated by more than 2-fold compared with that in cells before adipogenic induction (day 0) were extracted. In SGBS cells, 565, 880, and 955 genes were upregulated on days 1, 3, and 7, respectively, after adipogenic induction (Fig. 1 A). Excluding overlapping genes, 1460 genes were identified as induced during adipogenesis in SGBS cells. Similarly, in 3T3-L1 cells, 807, 662, and 903 genes were induced on days 1, 3, and 7 after adipogenic induction, respectively (Fig. 1 B), resulting in 1297 induced genes during adipogenesis. In both cell types, only 297 genes were found to be commonly induced (Fig. 1 C), which included well-known adipocyte marker genes, such as ADIPOQ (adiponectin), CEBPA (C/EBPα), DGAT2 (diacylglycerol O -acyltransferase 2), FABP4 (fatty acid binding protein 4), and PPARG (PPARγ) (Table 1 ). Among the genes induced exclusively in SGBS cells, 61 exhibited more than 20-fold induction, including LMO3 , whose expression is induced during adipogenesis in humans but not in mice 8 . Among of these, we focused on GALNT15 , whose function in adipocytes remains largely unknown. Microarray analysis revealed that the signal intensity of GALNT15 in SGBS cells increased 63.73-, 37.05-, and 1.30-fold on days 1, 3, and 7 after adipogenic induction, respectively (Table 1 ). By contrast, the signal intensity of Galnt15 in 3T3-L1 cells showed minor fluctuations, with fold changes of 1.02, -1.07, and 1.09, respectively, at the same time points following adipogenic induction. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) validated these findings, highlighting a transient increase in GALNT15 mRNA during adipogenesis in SGBS but not in 3T3-L1 cells (Fig. 2 A). Western blotting also confirmed a similar pattern, with GalNAc-T15 protein levels transiently increased in SGBS cells but barely detected throughout adipogenesis in 3T3-L1 cells (Fig. 2 B). Table 1 Fold changes of the microarray probeset signals for representative genes induced at day 1, 3, and 7 after adipogenic induction. Gene symbol (human/mouse) SGBS cells 3T3-L1 cells day 1 day 3 day 7 day 1 day 3 day 7 ADIPOQ/Adipoq -1.10 164.85 425.87 1.09 27.14 299.76 AGT/Agt 1.16 3.29 22.47 13.49 17.38 35.45 CEBPA 1.20 24.03 67.72 1.56 1.81 3.43 CIDEC 4.16 48.59 111.01 2.62 40.81 111.06 DGAT2 1.22 8.23 23.62 -1.27 3.36 7.59 FABP4 3.11 637.63 1053.8 54.42 407.06 597.63 GALNT15/Galnt15 63.73 37.05 1.30 1.02 -1.07 1.09 LIPE/Lipe 1.08 3.28 14.74 3.64 4.82 7.04 LMO3/Lmo3 22.42 44.06 6.62 1.06 1.10 -1.01 LPL/Lpl 1.10 128.67 362.19 1.31 6.27 8.18 PPARG/Pparg2 5.84 17.43 19.45 2.24 2.97 5.36 GALNT15 overexpression enhances CEBPA and LEP mRNA expression in SGBS cells To investigate the impact of GALNT15 on adipogenesis in SGBS cells, we first constructed an adenovirus overexpressing the GalNAc-T15-FLAG fusion protein and assessed adipogenesis in SGBS cells transfected with this virus compared with that in cells transfected with a control virus (vehicle). Western blotting demonstrated the ectopic expression of the GalNAc-T15-FLAG fusion protein in SGBS cells (Fig. 3 A). GALNT15 overexpression significantly increased the mRNA expressions of CEBPA and LEP , but not that of ADIPOQ, FABP4 , and PPARG , in SGBS cells 7 days after adipogenic induction (Fig. 3 B). The amount of triglyceride accumulation and the percentage of cells with oil droplets were comparable between GALNT15 -overexpressing and control cells (Fig. 3 C, D). GALNT15 knockdown inhibits adipogenesis in SGBS cells Subsequently, we constructed adenoviruses expressing a short hairpin RNA (shRNA) against GALNT15 mRNA (shGALNT15) and a non-targeting control shRNA (shNC) and assessed adipogenesis in transfected SGBS cells. The induction of GALNT15 mRNA and protein expressions during adipogenesis was inhibited in SGBS cells transfected with an adenovirus expressing shGALNT15 (Fig. 4 A, B). GALNT15 knockdown suppressed the mRNA expression of all tested adipocyte marker genes (Fig. 4 B), triglyceride accumulation (Fig. 4 D, E), and the percentage of cells with oil droplets (Fig. 4 F, G), indicating that GALNT15 knockdown inhibited adipogenesis in SGBS cells. GALNT15 enhances the induction of PPARG and CEBPA during adipogenesis To elucidate the molecular mechanisms by which GALNT15 participates in adipogenesis, we examined the mRNA levels of two key adipogenic transcriptional regulatoroy genes, PPARG and CEBPA , during adipogenesis. GALNT15 overexpression significantly enhanced the induction levels of both PPARG and CEBPA mRNA during adipogenesis in SGBS cells (Fig. 5 A), whereas GALNT15 knockdown inhibited the induction of PPARG and CEBPA mRNA 4 days after adipogenic induction compared with that in control cells (Fig. 5 B). These results indicate that GALNT15 is involved in adipogenesis by enhancing the expression of PPARG and CEBPA during adipogenesis in SGBS cells. Furthermore, the effect of GALNT15 on the mRNA expressions of CEBPB , a transcription factor upstream of PPARG and CEBPA , in the early stages of adipogenesis was investigated. Neither overexpression nor knockdown of GALNT15 altered the expression of CEBPB mRNA in SGBS cells at 1 day after adipogenic induction (Fig. 5 C, D), suggesting that mechanisms other than inducing the expression of CEBPB are involved in the regulation of PPARG and CEBPA by GALNT15 . Discussion Microarray analysis revealed distinct expression patterns of many genes during adipogenesis in human SGBS cells and mouse 3T3-L1 cells. This may be attributed to species differences between humans and mice; however, there may also be other differences in their properties as preadipocytes. For instance, the profile of genes expressed by differentiated adipocytes derived from 3T3-L1 cells is markedly different from that expressed by mature adipocytes in mouse 15 . By contrast, the mRNA expression profile of adipocytes derived from SGBS cells is similar to that of primary human white subcutaneous adipocytes 16 – 18 . Nevertheless, numerous molecules implicated in adipogenesis have been identified and validated using 3T3-L1 cells. Thus, identifying genes induced during the adipogenic process in SGBS cells, but not in 3T3-L1 cells in this study, could serve as a viable strategy for identifying adipogenic factors as yet undiscovered in humans. In fact, among the 61 genes exhibiting more than 20-fold induction during the SGBS adipogenic process, but not in 3T3-L1 cells, some genes, except for GALNT15 , whose involvement in adipogenesis is unknown to our knowledge, were included. Notably, with LMO3 , a human-specific adipogenic gene 8 , being one of these 61 genes, there is a possibility that unidentified adipogenic factors may also be present within this group. In this study, GALNT15 is identified as a gene induced during the adipogenesis of SGBS cells, consistent with reports indicating that GALNT15 is listed as one of upregulated during adipocyte differentiation from human adipose-derived stem cells 19 . Although the mRNA expression of Galnt15 was not induced, and protein expression was not detected during adipogenesis of 3T3-L1 cells, it cannot be ruled out that the induction was undetectable due to extremely low expression levels compared with those in SGBS cells. The regulation of Galnt15 gene expression is influenced by corticosterone and the stress response in the mouse hippocampus 20 , and the medium used to induce adipogenesis in 3T3-L1 cells also contains dexamethasone (DEX), a synthetic glucocorticoids. This study focused on the identification of novel human adipogenesis-related factors, therefore, the investigation in mice was limited to confirming Galnt15 expression in 3T3-L1 cells. However, it is necessary to carefully consider whether an transient increase in the expression of Galnt15 is observed during mouse adipogenesis or whether Galnt15 is also involved in mouse adipogenesis. Although inhibition of GALNT15 induction clearly impeded adipogenesis in SGBS cells, overexpression of GALNT15 did not affect the accumulation of triglycerides or the proportion of cells containing lipid droplets, despite affecting the induction of PPARG mRNA 4 days after adipogenic induction in SGBS cells. This suggests that the induced expression levels of PPARG during the adipogenic process in control SGBS cells may be sufficient to affect adipogenesis under our experimental conditions, with further overexpression potentially having no additional effect on these aspects. Furthermore, overexpression of GALNT15 enhanced only the mRNA expression of CEBPA and its direct target gene LEP 21 , among the tested adipogenic marker genes. The role of CEBPA in adipogenesis is limited to the induction and maintenance of PPARG expression and the establishment of insulin sensitivity 22 . This suggests that the lack of adipogenic promotion in SGBS cells overexpressing GALNT15 could be attribute to sufficient levels of PPARG expression. Conversely, GALNT15 knockdown may affect adipogenesis by suppressing CEBPA expression, resulting in insufficient PPARG expression levels for adipogenic differentiation. Abnormal O -GalNAc-glycosylation catalyzed by the GALNT family is associated with various human diseases, with particular attention focused on the link between GALNT2 and metabolic disorders, such as obesity, type 2 diabetes, and lipid abnormalities 23 . In vitro analysis has shown that a reduction in GALNT2 expression in HepG2 cells, a human hepatocarcinoma cell line, impairs insulin signaling and action 24 . Conversely, GALNT2 overexpression stimulates adipocyte maturation and enlargement in 3T3-L1 cells 25 . However, our microarray data revealed that, except for GALNT15 , other GALNT family members did not exhibit a remarkable increase during the adipogenic process in SGBS cells (Supplementary table 2 ). This may suggest a more profound role for GALNT15 than for GALNT2 in human adipogenesis. GALNT15 does not show a significant relation with other GALNT family member 11 , and to our knowledge, its physiological function has not been thoroughly investigated. Our findings, combined with the fact that GALNT15 also serves as a marker gene candidate during osteocyte differentiation from canine adipose derived stem cells 26 , suggest that GALNT15 may play an important role in the differentiation of mesenchymal stem cells. In conclusion, we have demonstrated that GALNT15 contributes to adipogenesis in SGBS cells by upregulating CEBPA and PPARG . However, the specific molecular mechanisms driving GALNT15 -induced adipogenesis, including the potential involvement of unidentified substrates or non-enzymatic functions of GalNAc-T15, remain unclear and require further investigation for comprehensive elucidation. Our findings suggest that GLANT15 is an attractive drug target for the treatment of obesity. Methods Cell culture and adipogenic induction SGBS cells provided by our co-author, Dr. Martin Wabitsch, Ulm University Medical Center, Germany, were cultured in 6-well plates with culture medium consisting of Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium (DMEM/F12; Fujifilm, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Sigma, St Louis, MO, USA), 3 µM biotin, 17 µM pantothenic acid, and 0.5% penicillin-streptomycin-amphotericin B suspension (Fujifilm) in an incubator at 37°C with humidified air at 5% CO 2 . To induce adipogenesis, cells grown to 80–90% confluency were cultured with FBS-free medium containing 0.01 mg/ml transferrin, 0.1 µM cortisol, 200 pM triiodothyronine, 20 nM insulin, 0.25 µM DEX, 500 µM 3-isobutyl-1-methylxanthine, and 2 µM troglitazone for 4 days. Subsequently, the cells were cultured in a maintenance medium consisting of FBS-free medium containing 0.01 mg/ml transferrin, 0.1 µM cortisol, 200 pM triiodothyronine, and 20 nM insulin. The maintenance medium was changed every 3 days. 3T3-L1 cells were generously provide by Oral Bioscience Laboratory, Tokushima University. Japan. The cells were cultured in 6-well plates with culture medium consisting of Dulbecco’s modified Eagle’s medium (Fujifilm) supplemented with 10% FBS and 0.5% penicillin-streptomycin-amphotericin B suspension. To induce adipogenesis, cells grown to 100% confluency were cultured for an additional 2 days and then cultured in medium containing 10 µM insulin, 1 µM DEX, 500 µM 3-isobutyl-1-methylxanthine, and 2 µM troglitazone for 3 days. Subsequently, the cells were cultured in maintenance medium consisting of medium containing 10 µM insulin. The maintenance medium was changed every 3 days. Microarray Total RNA was extracted from SGBS and 3T3-L1 cells before (day 0) and on days 1, 3, and 7 after adipogenic induction, using ISOGEN (Nippongene, Toyama, Japan). The extracted RNA was used to generate biotin-labeled cRNA using the Affymetrix GeneChip ™ 3′ IVT PLUS Reagent Kit (Thermo Fisher Scientific, Waltham, MA, USA). The biotin-labeled RNA was then hybridized to either an Affymetrix Human Genome U-219 Array plate (Thermo Fisher Scientific) or a mouse genome MG-430 PM array plate (Thermo Fisher Scientific) following the manufacturer’s instructions. After washing and staining the array strips, the signals were developed and scanned using the Affymetrix Gene Atlas system (Thermo Fisher Scientific), and the data were analyzed using Transcriptome Analysis Console software (Thermo Fisher Scientific). Average hybridization signal intensities were used for data analysis, and genes with a mean signal intensity greater than 5 (log base 2 scale) in either of the adipogenic-induced samples were considered detectable. Genes with a signal intensity more than 2-fold higher than that in each cell before adipogenic induction were considered induced genes, and comparisons were made between SGBS and 3T3-L1 cells. Genes with the same symbol in humans and mice were designated as common genes. qRT-PCR Each cDNA was synthesized from total RNA using the ReverTra Ace® qPCR RT Kit (Toyobo, Osaka, Japan) following the manufacturer's protocol. The cDNA was then subjected to qRT-PCR on a Thermal Cycler Dice® Real Time System (Takara, Shiga, Japan) using Thunderbird™ SYBR® qPCR Mix (Toyobo) and gene-specific primer sets via the following program: 30 sec at 95°C, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The specificity of each primer set was confirmed by dissociation curve analysis following amplification. The nucleotide sequences of the primer sets are listed in Supplementary table 1 . The mRNA level of each gene was normalized to that of the human and mouse glyceraldehyde 3-phosphate dehydrogenase gene ( GAPDH/Gapdh ). Western blotting The cells were lysed with lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM ethylenediamine tetraacetic acid, 1% Triton X-100, and complete mini (Roche, Basel, Switzerland)), and the lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Immobilon Transfer Membranes; Millipore, Bedford, MA, USA). After incubation in blocking solution (Blocking One; Nakalai tesque, Kyoto, Japan), the membranes were incubated with a 1:1000 dilution of mouse anti-FLAG M2 antibody (Sigma), a 1:2000 dilution of mouse anti-β actin (Sigma), a 1:500 dilution of rabbit anti-GalNAc-T15 antibody (Thermo Fisher), or a 1:1000 dilution of mouse anti-adiponectin antibody (Proteintech, Rosemont, IL, USA), and subsequently incubated with an anti-rabbit or anti-mouse IgG-horseradish peroxidase-conjugated secondary antibody (Jackson Lab, Farmington, CT, USA). The signal was detected using Immobilon Western Detection Reagent (Millipore) with a Luminograph III (Atto, Tokyo, Japan). Construction of adenoviruses Adenoviruses expressing GalNAc-T15 fused to FLAG at the C-terminus and a shGALNT15 were constructed as previously described 14 . Briefly, cDNA encoding the translational region lacking the stop codon of GALNT15 was amplified from SGBS adipocyte cDNA using PCR, and DNA with FLAG cDNA sequences added to its 3'-end was inserted into the pAxCAwtit cosmid vector (TakaRa). Recombinant adenoviral genomic DNA was excised from the cosmid and transfected into HEK293 cells to produce an adenovirus. An adenovirus produced from intact pAxCAwtit was used as a control. Cosmids for adenovirus production, which were inserted with a cDNA encoding shGALNT15 and a cDNA encoding a shNC, were purchased from Vector Builder (Chicago, IL, USA), and adenoviruses were produced in the same manner. Evaluation of adipogenesis The degree of adipocyte differentiation was evaluated based on the expression of adipocyte marker genes, triglyceride accumulation, and the percentage of cells with oil droplets. SGBS cells were infected with each adenoviruses for 24 h and subjected to adipogenic induction. Seven days after adipogenic induction, total RNA was extracted from the cells and the expression of adipocyte marker genes was measured by qRT-PCR. Ten days after adipogenic induction, SGBS cells ere fixed with 4% paraformaldehyde and stained with Oil Red O to evaluate triglyceride accumulation or 4,6-diamidine-2-phenylindole dihydrochloride and Sudan III to count cells with oil droplets. To measure the amount of triglycerides, stained Oil Red O was eluted with isopropanol, and the absorbance was measured at 500 nm using a spectrophotometer (Ultrospec 6300 pro; GE Healthcare, Chicago, IL, USA). To assess the percentage of cells with oil droplets, the ratio of Sudan III-positive cells to 4,6-diamidine-2-phenylindole dihydrochloride-stained cells was determined in 3 randomly selected low-power fields (x100). Data analysis Each experiment in Fig. 2 – 5 was repeated several times, and representative results are shown. Each bar on the graph is expressed as the mean ± SE. Statistical analyses were performed using Student’s t -test for the comparison of two groups and Dunnett's test for the comparison of three or more groups versus the control. Differences were considered significant when the P -value was less than 0.05. Declarations Competing interests The authors declare no competing interests. Author Contribution Conceptualization; T.I., Data collection; T.I., A.T., R.K., S.W., and K.K., Funding acquisition; T.I., A.K., M.S., M.N. and A.I., Project administration; T.I. and K.Y., Methodology and Resources; T.I., M.M., and M.W., Writing the original draft; T.I. and A.T.. Acknowledgement We thank Dr. M. Fukuhara and T. Yamaguchi (Department of Microbiology, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Medical and Life Sciences) for providing measurement equipment. Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. References Rosen, E. D. & Spiegelman, B. M. Adipocytes as Regulators of Energy Balance and Glucose Homeostasis. Nature 444, 847–853. https://doi.org/10.1038/nature05483 (2006). Hausman D. B., DiGirolamo M., Bartness T. J., Hausman G. J. & Martin R. J. The biology of white adipocyte proliferation. Obes. Rev. 2, 239–254. https://doi.org/10.1046/j.1467-789X.2001.00042.x (2001). Rosen, E. D. & MacDougald, O. A Adipocyte differentiation from the inside out. Nat. Rev. Mol. Cell Biol. 7, 885–986. doi: 10.1038/nrm2066 . (2006). Tang, Q. Q. & Lane, M. D. Activation and centromeric localization of CCAAT/enhancer-binding proteins during the mitotic clonal expansion of adipocyte differentiation. Genes Dev. 13, 2231–2241. doi: 10.1101/gad.13.17.2231 . (1999). Wabitsch, M. et al. Characterization of a human preadipocyte cell strain with high capacity for adipose differentiation. Int. J. Obe.s Relat. Metab. Disord. 25, 8–15. doi: 10.1038/sj.ijo.0801520 . (2001). Entenmann, G. & Hauner, H. Relationship between replication and differentiation in cultured human adipocyte precursor cells. Am. J. Physiol. 270, C1011-C1016. doi: 10.1152/ajpcell.1996.270.4.C1011 . (1996). Newell, F. S. et al. Characterization of the transcriptional and functional effects of fibroblast growth factor-1 on human preadipocyte differentiation. FASEB J. 20, 2615–2617. doi: 10.1096/fj.05-5710fje . (2006). Lindroos, J. et al. Human but not mouse adipogenesis is critically dependent on LMO3. Cell Metab. 18, 62–74. doi: 10.1016/j.cmet.2013.05.020 . (2013). Ishimoto, K. et al. D-dopachrome tautomerase promotes IL-6 expression and inhibits adipogenesis in preadipocytes. Cytokine 60, 772 – 727. doi: 10.1016/j.cyto.2012.07.037 . (2012). Cheng, L. et al . Characterization of a novel human UDP-GalNAc transferase, pp-GalNAc-T15. FEBS Lett. 566, 17–24. doi: 10.1016/j.febslet.2004.03.108 . (2004). Bennett, E. P. et al. Control of mucin-type O-glycosylation: A classification of the polypeptide GalNAc-transferase gene family. Glycobiology 22, 736–756. doi: 10.1093/glycob/cwr182 . (2012). Hussain, M. R. M., Hoessli, D. C. & Fang, M. N-acetylgalactosaminyltransferases in cancer. Oncotarget 7, 54067–54081. doi: 10.18632/oncotarget.10042 . (2016). Kato, K., Hansen, L. & Clausen, H. Polypeptide N -acetylgalactosaminyltransferase-Associated Phenotypes in Mammals. Molecules 26, 5504. https://doi.org/10.3390/molecules26185504 . (2021). Iwata, T. et al. The action of D-dopachrome tautomerase as an adipokine in adipocyte lipid metabolism. PLoS One 7, e33402. doi: 10.1371/journal.pone.0033402 (2012). Soukas, A. et al. Distinct transcriptional profiles of adipogenesis in vivo and in vitro. J. Biol. Chem. 276, 34167–34174. DOI 10.1074/jbc.M104421200 (2001). Yao, C. R. et al. SGBS cells as a model of human adipocyte browning: A comprehensive comparative study with primary human white subcutaneous adipocytes. Sci. Rep. 7, 4031. DOI: 10.1038/s41598-017-04369-2 . (2017). Kalkhof, S. et al. In depth quantitative proteomic and transcriptomic characterization of human adipocyte differentiation using the SGBS cell line. Proteomics 8, e1900405. doi: 10.1002/pmic.201900405 . (2020). Tews, D. et al. 20 Years with SGBS cells - a versatile in vitro model of human adipocyte biology. Int. J. Obes. 46, 1939–1947. doi: 10.1038/s41366-022-01199-9 .(2022). Chen, K., Xie, S. & Jin, W. Crucial lncRNAs associated with adipocyte differentiation from human adipose- derived stem cells based on co-expression and ceRNA network analyses. PeerJ 7, e7544. doi: 10.7717/peerj.7544 . (2019). Jaszczyk, A. et al. Overnight corticosterone and gene expression in mouse hippocampus: Time course during resting period. Int. J. Mol. Sci. 24, 2828. https://doi.org/10.3390/ijms24032828 . (2023). Miller, S. G. et al. The adipocyte specific transcription factor C/EBPalpha modulates human ob gene expression. Proc. Natl. Acad. Sci. U.S.A. 93, 5507–5511. doi: 10.1073/pnas.93.11.5507 . (1996). Rosen, E. D. et al. C/EBPalpha induces adipogenesis through PPARgamma: a unified pathway. Genes Dev. 16, 22–26. doi: 10.1101/gad.948702 . (2002). Antonucci, A., Marucci, A., Trischitta, V., & Di Paola, R. Role of GALNT2 on Insulin Sensitivity, Lipid Metabolism and Fat Homeostasis. Int. J. Mol. Sci. 23, 929. doi: 10.3390/ijms23020929 . (2022). Marucci, A. et al. Role of GALNT2 in the modulation of ENPP1 expression, and insulin signaling and action: GALNT2: A novel modulator of insulin signaling. Biochim. Biophys. Acta 1833, 1388–1395 doi: 10.1016/j.bbamcr.2013.02.032. (2013). Marucci, A. et al. GALNT2 as a novel modulator of adipogenesis and adipocyte insulin signaling. Int. J. Obes. 43, 2448–2457. doi: 10.1038/s41366-019-0367-3 . (2019). Jankowski, M. et al. Expression profile of new marker genes involved in differentiation of canine adipose-derived stem cells into osteoblasts. Int. J. Mol. Sci. 22, 6663. doi: 10.3390/ijms22136663 . (2021). Additional Declarations No competing interests reported. Supplementary Files supplementaryTable1.docx supplementaryTable2.docx Cite Share Download PDF Status: Published Journal Publication published 29 Aug, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 26 May, 2024 Reviews received at journal 25 May, 2024 Reviewers agreed at journal 11 May, 2024 Reviews received at journal 07 May, 2024 Reviewers agreed at journal 29 Apr, 2024 Reviewers invited by journal 21 Apr, 2024 Editor assigned by journal 21 Apr, 2024 Editor invited by journal 18 Apr, 2024 Submission checks completed at journal 18 Apr, 2024 First submitted to journal 09 Apr, 2024 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-4244309","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":294276835,"identity":"1427aa84-c56f-4eea-8590-a7c164c47fd8","order_by":0,"name":"Asuka Takahashi","email":"","orcid":"","institution":"Niigata University of Pharmacy and Medical and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Asuka","middleName":"","lastName":"Takahashi","suffix":""},{"id":294276837,"identity":"f3397302-9c3e-4862-858f-1a7bf70d8aa2","order_by":1,"name":"Ryo Koike","email":"","orcid":"","institution":"Niigata University of Pharmacy and Medical and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ryo","middleName":"","lastName":"Koike","suffix":""},{"id":294276839,"identity":"c839b689-01fe-4623-bce8-7560d9774a99","order_by":2,"name":"Shota Watanabe","email":"","orcid":"","institution":"Niigata University of Pharmacy and Medical and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shota","middleName":"","lastName":"Watanabe","suffix":""},{"id":294276840,"identity":"c6964143-9f98-4795-9b7b-8e1671f2777e","order_by":3,"name":"Kyoko Kuribayashi","email":"","orcid":"","institution":"Ehime University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kyoko","middleName":"","lastName":"Kuribayashi","suffix":""},{"id":294276841,"identity":"f02a0908-0b2b-471e-9bdb-57551e311c92","order_by":4,"name":"Martin Wabitsch","email":"","orcid":"","institution":"Ulm University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Wabitsch","suffix":""},{"id":294276842,"identity":"9b90dea1-21e6-4620-8696-cd7637ab585d","order_by":5,"name":"Masahiko Miyamoto","email":"","orcid":"","institution":"Niigata University of Pharmacy and Medical and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Masahiko","middleName":"","lastName":"Miyamoto","suffix":""},{"id":294276843,"identity":"b90a8ce2-1ee5-4048-833a-93a2f20b8e94","order_by":6,"name":"Akihiko Komuro","email":"","orcid":"","institution":"Niigata University of Pharmacy and Medical and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Akihiko","middleName":"","lastName":"Komuro","suffix":""},{"id":294276844,"identity":"0461cdc1-ca8b-4482-8ce4-ae02e2e70d39","order_by":7,"name":"Mineaki Seki","email":"","orcid":"","institution":"Niigata University of Pharmacy and Medical and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mineaki","middleName":"","lastName":"Seki","suffix":""},{"id":294276845,"identity":"d6c7c80b-31c0-4799-97a3-b36cc048b39e","order_by":8,"name":"Masayuki Nashimoto","email":"","orcid":"","institution":"Niigata University of Pharmacy and Medical and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Masayuki","middleName":"","lastName":"Nashimoto","suffix":""},{"id":294276846,"identity":"6ee50c55-9712-47de-b4be-9c13d8ffc362","order_by":9,"name":"Akiko Ibuka","email":"","orcid":"","institution":"Kanagawa University","correspondingAuthor":false,"prefix":"","firstName":"Akiko","middleName":"","lastName":"Ibuka","suffix":""},{"id":294276847,"identity":"b2f84de2-03a7-4bc5-8bc0-7360edae9ed5","order_by":10,"name":"Kikuji Yamashita","email":"","orcid":"","institution":"Niigata University of Pharmacy and Medical and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Kikuji","middleName":"","lastName":"Yamashita","suffix":""},{"id":294276848,"identity":"00b5fe5b-15bc-4266-be71-7128949f52cd","order_by":11,"name":"Takeo Iwata","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYBACNjBZASKYG8BsxgbCWoBqziCpJagFrIaxjVi1IMAnkfz8wc95dxK3sx9s/sBQY8fAPJuAVjaJNMPG3m3PEnf2JLZJMBxLZmCcc4CAFukEwwbebYcTNxxIBDqP7QAD44wEQlrSPzb+nQPUcv4h0GH/iNKSY9jM2wDUciOxQYKxjRgt8m8KZ8scO2y8c8bDNonEvmQegn6R7zm+4eObmsOy2/mTD3/48M1OzpBQiMGBAYgAOonHcAaROiBawPZKEKtlFIyCUTAKRgoAABkESQ13rV9HAAAAAElFTkSuQmCC","orcid":"","institution":"Niigata University of Pharmacy and Medical and Life Sciences","correspondingAuthor":true,"prefix":"","firstName":"Takeo","middleName":"","lastName":"Iwata","suffix":""}],"badges":[],"createdAt":"2024-04-10 00:44:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4244309/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4244309/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-70930-5","type":"published","date":"2024-08-29T15:58:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55192477,"identity":"aa2115bf-f4a3-4002-8308-3c0a207906a3","added_by":"auto","created_at":"2024-04-23 20:31:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":144714,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of genes with increased expression during adipogenesis in SGBS and 3T3-L1 cells using microarray. The numbers represent the count of genes that increased more than 2-fold after adipogenic induction at day 1, 3, and 7, compared with that before induction (day 0) in SGBS cells (A) and in 3T3-L1 cells (B). (C) A Venn diagram illustrating the overlap between number of genes upregulated more than 2-fold after adipogenic induction at either day 1, 3, or 7 compared with that before induction in SGBS cells and in 3T3-L1 cells.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4244309/v1/dd10c3aa93f727766780cfbd.png"},{"id":55192911,"identity":"780c8a5d-1608-449f-889a-67f817f984ff","added_by":"auto","created_at":"2024-04-23 20:39:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":87263,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eGALNT15/Galnt15\u003c/em\u003eexpression during adipogenesis in SGBS and 3T3-L1 cells. (A) \u003cem\u003eGALNT15/Galnt15.\u003c/em\u003emRNA expression after adipogenic induction at 0, 1, 3, and 7 days in SGBS cells (black columns) and 3T3-L1 cells (white columns). Data represents relative values compared with those before adipogenesis in each cell type. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 (n=3). (B) Representative western blotting images using lysates from SGBS and 3T3-L1 cells subjected to adipogenic induction for the indicated days, probed with an anti-GalNAc-T15 antibody. Anti-adiponectin antibody and anti-β-actin antibodies were used as positive controls for adipogenesis and internal controls, respectively.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4244309/v1/77e776905b3bdf4c341c0185.png"},{"id":55192480,"identity":"daa40202-8512-41ce-926b-6ee16fb2625d","added_by":"auto","created_at":"2024-04-23 20:31:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":419465,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eGALNT15\u003c/em\u003eoverexpression on adipogenesis in SGBS cells. SGBS cells were infected with adenovirus overexpressing GalNAc-T15-FLAG fusion protein (GALNT15; black columns) or adenovirus without insertion (vehicle; white columns) for 24 h, followed by adipogenic induction. Adipogenesis was evaluated on day 7 (B) or day 10 (C-F) after adipogenic induction. (A) Exogenous GalNAc-T15 expression in SGBS cells subjected to adipogenic induction for 7 days confirmed by western blotting with an anti-FLAG antibody. An anti-β-actin antibody was used as an internal control. (B) mRNA expression of adipocyte marker genes in SGBS cells subjected to adipogenic induction. The graph represents relative values compared with those in control cells. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 (n=3). (C, D)\u003cem\u003e \u003c/em\u003eLipid accumulation in SGBS cells subjected to adipogenic induction. Representative images (C) and the colorimetric quantification (D) in SGBS cells stained with Oil Red O are shown. n=3. (E, F) Percentage of cells with oil droplets in SGBS cells subjected to adipogenic induction. Representative images (E) and percentage (F) in SGBS cells stained both by 4,6-diamidine-2-phenylindole dihydrochloride(DAPI) and Sudan III are presented. \u0026nbsp;n=3.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4244309/v1/63606b7f08bf044bab42b362.png"},{"id":55192910,"identity":"4a22efaf-b265-4b37-a4bd-79e3766644c8","added_by":"auto","created_at":"2024-04-23 20:39:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":414181,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eGALNT15\u003c/em\u003e knockdown on adipogenesis in SGBS cells. SGBS cells were infected with adenovirus expressing shRNA against \u003cem\u003eGALNT15\u003c/em\u003e mRNA (shGALNT15; gray columns) or control adenovirus expressing negative control shRNA (shNC; white columns) for 24 h, followed by adipogenic induction. Adipogenesis was evaluated on day 7 (C) or day 10 (D-G) after adipogenic induction. (A, B) Suppression of \u003cem\u003eGALNT15\u003c/em\u003e mRNA (A) and GalNAc-T15 (B) expressions by shGALNT15 during adipogensis in SGBS cells was confirmed by quantitative reverse transcription polymerase chain reaction (A) and western blotting (B), respectively. \u0026nbsp;The graph represents relative values compared with those before adipogenesis in control cells. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 (n=3). (C) mRNA expression of adipocyte marker genes in SGBS cells subjected to adipogenic induction. The graph represents relative values compared with those in control cells. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 (n=3). *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 (n=3). (D, E)\u003cem\u003e \u003c/em\u003eThe effect on lipid accumulation in SGBS cells subjected to adipogenic induction was assessed by Oil Red O staining. Representative images (D) and the colorimetric quantification (E) of SGBS cells stained by oil red O are shown. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 (n=3). (F, G) The effect on the percentage of cells with oil droplets in SGBS cells subjected to adipogenic induction. \u0026nbsp;Representative images (F) and the percentage (G) of SGBS cells stained with both 4,6-diamidine-2-phenylindole dihydrochloride (DAPI) and Sudan III are presented. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 (n=3).\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4244309/v1/3224e420572714c73b3ed879.png"},{"id":55192479,"identity":"d04e3466-85c5-4141-a8ff-83befc7fd00f","added_by":"auto","created_at":"2024-04-23 20:31:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":101413,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eGALNT15\u003c/em\u003e overexpression or knockdown on the induction of PPARG, CEBPA, and CEBPB mRNA expression during SGBS adipogenesis. SGBS cells were infected with adenovirus overexpressing GalNAc-T15-FLAG fusion protein (black columns in A and C), adenovirus expressing shGALNT15 (gray columns in B and D), or corresponding control adenovirus (white columns) for 24 h, followed by adipogenic induction. mRNA expression was measured on day 4 for \u003cem\u003ePPARG or CEBPA \u003c/em\u003e(A, B) and on day 1 for \u003cem\u003eCEBPB\u003c/em\u003e(C, D) after adipogenic induction and compared with that of cells before adipogenic induction. \u0026nbsp;The data represents relative values compared with those before adipogenesis in each control cell. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 (n=3).\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4244309/v1/b1455b9b13542b29d860f0dd.png"},{"id":63821108,"identity":"60f9020a-886c-4b7c-9af5-d0d04e33ac7f","added_by":"auto","created_at":"2024-09-02 16:11:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1632161,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4244309/v1/e24d0ef7-b5db-467e-b436-3dd0807812f0.pdf"},{"id":55192482,"identity":"a8a33847-df3e-4ea7-a99f-a420fa651d36","added_by":"auto","created_at":"2024-04-23 20:31:29","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":14820,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4244309/v1/133d270a7b60c64e0f24d405.docx"},{"id":55192483,"identity":"93610602-8486-44ab-af71-1a24379922f1","added_by":"auto","created_at":"2024-04-23 20:31:30","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":20483,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryTable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4244309/v1/f0f055ea84ba07bcc71bc735.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"GALNT15, induced during adipogenesis of human SGBS cells but not in mouse 3T3-L1 cells, regulates adipocyte differentiation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eObesity is a condition characterized by the excessive accumulation of adipose tissue in the body, which, along with its common complications such as, type 2 diabetes, dyslipidemia, and hypertension, increases the risk of vascular diseases related to arteriosclerosis\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Adipose tissue comprises mature adipocytes with oil droplets that store triglycerides, and stromal vascular fractions containing preadipocytes, which are precursor cells of adipocytes. Excessive accumulation of adipose tissue is thought to result from the hypertrophy of individual adipocytes due to increased triglyceride accumulation, and an increased number of adipocytes due to the accelerated differentiation of preadipocytes into mature adipocytes\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDuring adipogenesis, preadipocytes acquire the machinery for lipid transport and synthesis, insulin sensitivity, and secretion of adipocyte-specific proteins\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. During this process, the induction and activation of transcription factors, specifically the CCAAT-enhancer binding protein (C/EBP) family and peroxisome proliferator-activated receptor γ (PPARγ), are crucial\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. C/EBPβ and C/EBPδ are transiently induced early in adipogenesis\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e and they induce expressions of C/EBPα and PPARγ, which function to promote transcription of many types of genes involved in adipocyte phenotype and function\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In addition to these key transcriptional regulators, various other transcriptional regulators of adipogenesis and factors affecting adipogenesis have been reported. While many of these findings regarding adipogenesis have been obtained using \u003cem\u003ein vitro\u003c/em\u003e mouse models utilizing cell lines, such as 3T3-L1 or 3T3-F442A, the mechanisms of adipogenesis in humans and mice are not entirely identical. For instance, 3T3-L1 and 3T3-F442A cells undergo an increase in cell number known as mitotic clonal expansion prior to differentiation, which is thought to be necessary for subsequently differentiation. However, human primary preadipocytes or SGBS cells, a human preadipocyte cell line established from the subcutaneous adipose tissue of an infant suspected of having Simpson-Golabi-Behmel Syndrome\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, do not necessarily undergo mitotic clonal expansion during adipogenesis\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Furthermore, LIM domain only 3 (\u003cem\u003eLMO3\u003c/em\u003e), which positively regulates adipogenesis, is upregulated during adipogenesis in humans but not in mice\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Additionally, D-dopachrome tautomerase, an adipokine secreted by adipocytes, suppresses adipogenesis in SGBS cells but not in 3T3-L1 cells\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Thus, identifying the factors that affect adipogenesis in a human-specific manner may lead to the identification of novel target molecules for the development of anti-obesity drugs.\u003c/p\u003e \u003cp\u003eIn the present study, we conducted a comparative analysis of mRNA expression induced during adipogenesis in SGBS cells and 3T3-L1 cells using microarray to identify genes whose expression is induced in SGBS but not in 3T3-L1 cells. \u003cem\u003eGALNT15\u003c/em\u003e, encoding polypeptide N-acetylgalactosaminyl transferase (GalNAc-T)-15, was identified as one of the candidate genes. \u003cem\u003eGALNT15\u003c/em\u003e, a member of the \u003cem\u003eGALNT\u003c/em\u003e family comprising 20 species in humans, is expressed in most human tissues\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Its translation product catalyzes the initiation of mucin-type \u003cem\u003eO\u003c/em\u003e-linked glycosylation by adding \u003cem\u003eN\u003c/em\u003e-acetylgalactosamine to the serine or threonine residues of the polypeptide\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Mucin-type \u003cem\u003eO\u003c/em\u003e-glycosylation is initiated and regulated by the GalNAc-T family that catalyzes the first step in the biosynthesis forming the GalNAcα1-\u003cem\u003eO\u003c/em\u003e-serine/threonine linkage in O-glycoproteins. \u003cem\u003eO\u003c/em\u003e-linked glycosylation, the most diverse form of post-translational modifications, affects various aspects of protein function, therefore, many GalNAc-Ts are considered to have potentials for differential regulation in cells and tissues\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Aberrant \u003cem\u003eO\u003c/em\u003e-glycosylation by some GalNAc-Ts has been observed in many types of cancer and is associated with noncancerous developmental and metabolic disorders\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e; however, the involvement of \u003cem\u003eGALNT15\u003c/em\u003e in these diseases has not been reported, and its physiological function remains largely unknown. Therefore, we focused on \u003cem\u003eGALNT15\u003c/em\u003e and investigated its effects on adipogenesis in SGBS cells.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eGALNT15 expression was induced during adipogenesis in SGBS but not in 3T3-L1 cells\u003c/h2\u003e\n \u003cp\u003eThe mRNA expression profiles of SGBS and 3T3-L1 cells on days 0, 1, 3, and 7 after adipogenic induction were analyzed using microarray analysis. Genes whose expression was upregulated by more than 2-fold compared with that in cells before adipogenic induction (day 0) were extracted. In SGBS cells, 565, 880, and 955 genes were upregulated on days 1, 3, and 7, respectively, after adipogenic induction (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Excluding overlapping genes, 1460 genes were identified as induced during adipogenesis in SGBS cells. Similarly, in 3T3-L1 cells, 807, 662, and 903 genes were induced on days 1, 3, and 7 after adipogenic induction, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB), resulting in 1297 induced genes during adipogenesis. In both cell types, only 297 genes were found to be commonly induced (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC), which included well-known adipocyte marker genes, such as \u003cem\u003eADIPOQ\u003c/em\u003e (adiponectin), \u003cem\u003eCEBPA\u003c/em\u003e (C/EBP\u0026alpha;), \u003cem\u003eDGAT2\u003c/em\u003e (diacylglycerol \u003cem\u003eO\u003c/em\u003e-acyltransferase 2), \u003cem\u003eFABP4\u003c/em\u003e (fatty acid binding protein 4), and \u003cem\u003ePPARG\u003c/em\u003e (PPAR\u0026gamma;) (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Among the genes induced exclusively in SGBS cells, 61 exhibited more than 20-fold induction, including \u003cem\u003eLMO3\u003c/em\u003e, whose expression is induced during adipogenesis in humans but not in mice\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Among of these, we focused on \u003cem\u003eGALNT15\u003c/em\u003e, whose function in adipocytes remains largely unknown. Microarray analysis revealed that the signal intensity of \u003cem\u003eGALNT15\u003c/em\u003e in SGBS cells increased 63.73-, 37.05-, and 1.30-fold on days 1, 3, and 7 after adipogenic induction, respectively (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). By contrast, the signal intensity of \u003cem\u003eGalnt15\u003c/em\u003e in 3T3-L1 cells showed minor fluctuations, with fold changes of 1.02, -1.07, and 1.09, respectively, at the same time points following adipogenic induction. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) validated these findings, highlighting a transient increase in \u003cem\u003eGALNT15\u003c/em\u003e mRNA during adipogenesis in SGBS but not in 3T3-L1 cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Western blotting also confirmed a similar pattern, with GalNAc-T15 protein levels transiently increased in SGBS cells but barely detected throughout adipogenesis in 3T3-L1 cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eFold changes of the microarray probeset signals for representative genes induced at day 1, 3, and 7 after adipogenic induction.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eGene symbol\u003c/p\u003e\n \u003cp\u003e(human/mouse)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eSGBS cells\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e3T3-L1 cells\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eday 1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eday 3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eday 7\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eday 1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eday 3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eday 7\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eADIPOQ/Adipoq\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e164.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e425.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e299.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAGT/Agt\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEBPA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e24.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e67.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.43\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCIDEC\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e48.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e111.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e40.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e111.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eDGAT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eFABP4\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e637.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1053.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e54.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e407.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e597.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eGALNT15/Galnt15\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e63.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e37.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLIPE/Lipe\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLMO3/Lmo3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e44.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLPL/Lpl\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e128.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e362.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ePPARG/Pparg2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eGALNT15 overexpression enhances CEBPA and LEP mRNA expression in SGBS cells\u003c/h2\u003e\n \u003cp\u003eTo investigate the impact of \u003cem\u003eGALNT15\u003c/em\u003e on adipogenesis in SGBS cells, we first constructed an adenovirus overexpressing the GalNAc-T15-FLAG fusion protein and assessed adipogenesis in SGBS cells transfected with this virus compared with that in cells transfected with a control virus (vehicle). Western blotting demonstrated the ectopic expression of the GalNAc-T15-FLAG fusion protein in SGBS cells (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). \u003cem\u003eGALNT15\u003c/em\u003e overexpression significantly increased the mRNA expressions of \u003cem\u003eCEBPA\u003c/em\u003e and \u003cem\u003eLEP\u003c/em\u003e, but not that of \u003cem\u003eADIPOQ, FABP4\u003c/em\u003e, and \u003cem\u003ePPARG\u003c/em\u003e, in SGBS cells 7 days after adipogenic induction (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). The amount of triglyceride accumulation and the percentage of cells with oil droplets were comparable between \u003cem\u003eGALNT15\u003c/em\u003e-overexpressing and control cells (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC, D).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eGALNT15 knockdown inhibits adipogenesis in SGBS cells\u003c/h2\u003e\n \u003cp\u003eSubsequently, we constructed adenoviruses expressing a short hairpin RNA (shRNA) against \u003cem\u003eGALNT15\u003c/em\u003e mRNA (shGALNT15) and a non-targeting control shRNA (shNC) and assessed adipogenesis in transfected SGBS cells. The induction of \u003cem\u003eGALNT15\u003c/em\u003e mRNA and protein expressions during adipogenesis was inhibited in SGBS cells transfected with an adenovirus expressing shGALNT15 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). \u003cem\u003eGALNT15\u003c/em\u003e knockdown suppressed the mRNA expression of all tested adipocyte marker genes (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB), triglyceride accumulation (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD, E), and the percentage of cells with oil droplets (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF, G), indicating that \u003cem\u003eGALNT15\u003c/em\u003e knockdown inhibited adipogenesis in SGBS cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eGALNT15 enhances the induction of PPARG and CEBPA during adipogenesis\u003c/h2\u003e\n \u003cp\u003eTo elucidate the molecular mechanisms by which \u003cem\u003eGALNT15\u003c/em\u003e participates in adipogenesis, we examined the mRNA levels of two key adipogenic transcriptional regulatoroy genes, \u003cem\u003ePPARG\u003c/em\u003e and \u003cem\u003eCEBPA\u003c/em\u003e, during adipogenesis. \u003cem\u003eGALNT15\u003c/em\u003e overexpression significantly enhanced the induction levels of both \u003cem\u003ePPARG\u003c/em\u003e and \u003cem\u003eCEBPA\u003c/em\u003e mRNA during adipogenesis in SGBS cells (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA), whereas \u003cem\u003eGALNT15\u003c/em\u003e knockdown inhibited the induction of \u003cem\u003ePPARG\u003c/em\u003e and \u003cem\u003eCEBPA\u003c/em\u003e mRNA 4 days after adipogenic induction compared with that in control cells (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). These results indicate that \u003cem\u003eGALNT15\u003c/em\u003e is involved in adipogenesis by enhancing the expression of \u003cem\u003ePPARG\u003c/em\u003e and \u003cem\u003eCEBPA\u003c/em\u003e during adipogenesis in SGBS cells. Furthermore, the effect of \u003cem\u003eGALNT15\u003c/em\u003e on the mRNA expressions of \u003cem\u003eCEBPB\u003c/em\u003e, a transcription factor upstream of \u003cem\u003ePPARG\u003c/em\u003e and \u003cem\u003eCEBPA\u003c/em\u003e, in the early stages of adipogenesis was investigated. Neither overexpression nor knockdown of \u003cem\u003eGALNT15\u003c/em\u003e altered the expression of \u003cem\u003eCEBPB\u003c/em\u003e mRNA in SGBS cells at 1 day after adipogenic induction (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC, D), suggesting that mechanisms other than inducing the expression of \u003cem\u003eCEBPB\u003c/em\u003e are involved in the regulation of \u003cem\u003ePPARG\u003c/em\u003e and \u003cem\u003eCEBPA\u003c/em\u003e by \u003cem\u003eGALNT15\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMicroarray analysis revealed distinct expression patterns of many genes during adipogenesis in human SGBS cells and mouse 3T3-L1 cells. This may be attributed to species differences between humans and mice; however, there may also be other differences in their properties as preadipocytes. For instance, the profile of genes expressed by differentiated adipocytes derived from 3T3-L1 cells is markedly different from that expressed by mature adipocytes in mouse\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. By contrast, the mRNA expression profile of adipocytes derived from SGBS cells is similar to that of primary human white subcutaneous adipocytes\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Nevertheless, numerous molecules implicated in adipogenesis have been identified and validated using 3T3-L1 cells. Thus, identifying genes induced during the adipogenic process in SGBS cells, but not in 3T3-L1 cells in this study, could serve as a viable strategy for identifying adipogenic factors as yet undiscovered in humans. In fact, among the 61 genes exhibiting more than 20-fold induction during the SGBS adipogenic process, but not in 3T3-L1 cells, some genes, except for \u003cem\u003eGALNT15\u003c/em\u003e, whose involvement in adipogenesis is unknown to our knowledge, were included. Notably, with \u003cem\u003eLMO3\u003c/em\u003e, a human-specific adipogenic gene\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, being one of these 61 genes, there is a possibility that unidentified adipogenic factors may also be present within this group.\u003c/p\u003e \u003cp\u003eIn this study, \u003cem\u003eGALNT15\u003c/em\u003e is identified as a gene induced during the adipogenesis of SGBS cells, consistent with reports indicating that \u003cem\u003eGALNT15\u003c/em\u003e is listed as one of upregulated during adipocyte differentiation from human adipose-derived stem cells\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Although the mRNA expression of \u003cem\u003eGalnt15\u003c/em\u003e was not induced, and protein expression was not detected during adipogenesis of 3T3-L1 cells, it cannot be ruled out that the induction was undetectable due to extremely low expression levels compared with those in SGBS cells. The regulation of \u003cem\u003eGalnt15\u003c/em\u003e gene expression is influenced by corticosterone and the stress response in the mouse hippocampus\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, and the medium used to induce adipogenesis in 3T3-L1 cells also contains dexamethasone (DEX), a synthetic glucocorticoids. This study focused on the identification of novel human adipogenesis-related factors, therefore, the investigation in mice was limited to confirming \u003cem\u003eGalnt15\u003c/em\u003e expression in 3T3-L1 cells. However, it is necessary to carefully consider whether an transient increase in the expression of \u003cem\u003eGalnt15\u003c/em\u003e is observed during mouse adipogenesis or whether \u003cem\u003eGalnt15\u003c/em\u003e is also involved in mouse adipogenesis.\u003c/p\u003e \u003cp\u003eAlthough inhibition of \u003cem\u003eGALNT15\u003c/em\u003e induction clearly impeded adipogenesis in SGBS cells, overexpression of \u003cem\u003eGALNT15\u003c/em\u003e did not affect the accumulation of triglycerides or the proportion of cells containing lipid droplets, despite affecting the induction of \u003cem\u003ePPARG\u003c/em\u003e mRNA 4 days after adipogenic induction in SGBS cells. This suggests that the induced expression levels of \u003cem\u003ePPARG\u003c/em\u003e during the adipogenic process in control SGBS cells may be sufficient to affect adipogenesis under our experimental conditions, with further overexpression potentially having no additional effect on these aspects. Furthermore, overexpression of \u003cem\u003eGALNT15\u003c/em\u003e enhanced only the mRNA expression of \u003cem\u003eCEBPA\u003c/em\u003e and its direct target gene \u003cem\u003eLEP\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, among the tested adipogenic marker genes. The role of \u003cem\u003eCEBPA\u003c/em\u003e in adipogenesis is limited to the induction and maintenance of \u003cem\u003ePPARG\u003c/em\u003e expression and the establishment of insulin sensitivity\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. This suggests that the lack of adipogenic promotion in SGBS cells overexpressing \u003cem\u003eGALNT15\u003c/em\u003e could be attribute to sufficient levels of \u003cem\u003ePPARG\u003c/em\u003e expression. Conversely, \u003cem\u003eGALNT15\u003c/em\u003e knockdown may affect adipogenesis by suppressing \u003cem\u003eCEBPA\u003c/em\u003e expression, resulting in insufficient \u003cem\u003ePPARG\u003c/em\u003e expression levels for adipogenic differentiation.\u003c/p\u003e \u003cp\u003eAbnormal \u003cem\u003eO\u003c/em\u003e-GalNAc-glycosylation catalyzed by the \u003cem\u003eGALNT\u003c/em\u003e family is associated with various human diseases, with particular attention focused on the link between \u003cem\u003eGALNT2\u003c/em\u003e and metabolic disorders, such as obesity, type 2 diabetes, and lipid abnormalities\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eIn vitro\u003c/em\u003e analysis has shown that a reduction in \u003cem\u003eGALNT2\u003c/em\u003e expression in HepG2 cells, a human hepatocarcinoma cell line, impairs insulin signaling and action\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Conversely, \u003cem\u003eGALNT2\u003c/em\u003e overexpression stimulates adipocyte maturation and enlargement in 3T3-L1 cells\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. However, our microarray data revealed that, except for \u003cem\u003eGALNT15\u003c/em\u003e, other \u003cem\u003eGALNT\u003c/em\u003e family members did not exhibit a remarkable increase during the adipogenic process in SGBS cells (Supplementary table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This may suggest a more profound role for \u003cem\u003eGALNT15\u003c/em\u003e than for \u003cem\u003eGALNT2\u003c/em\u003e in human adipogenesis. \u003cem\u003eGALNT15\u003c/em\u003e does not show a significant relation with other \u003cem\u003eGALNT\u003c/em\u003e family member\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, and to our knowledge, its physiological function has not been thoroughly investigated. Our findings, combined with the fact that \u003cem\u003eGALNT15\u003c/em\u003e also serves as a marker gene candidate during osteocyte differentiation from canine adipose derived stem cells\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, suggest that \u003cem\u003eGALNT15\u003c/em\u003e may play an important role in the differentiation of mesenchymal stem cells.\u003c/p\u003e \u003cp\u003eIn conclusion, we have demonstrated that \u003cem\u003eGALNT15\u003c/em\u003e contributes to adipogenesis in SGBS cells by upregulating \u003cem\u003eCEBPA\u003c/em\u003e and \u003cem\u003ePPARG\u003c/em\u003e. However, the specific molecular mechanisms driving \u003cem\u003eGALNT15\u003c/em\u003e -induced adipogenesis, including the potential involvement of unidentified substrates or non-enzymatic functions of GalNAc-T15, remain unclear and require further investigation for comprehensive elucidation. Our findings suggest that GLANT15 is an attractive drug target for the treatment of obesity.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and adipogenic induction\u003c/h2\u003e \u003cp\u003eSGBS cells provided by our co-author, Dr. Martin Wabitsch, Ulm University Medical Center, Germany, were cultured in 6-well plates with culture medium consisting of Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium/Ham\u0026rsquo;s F-12 medium (DMEM/F12; Fujifilm, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Sigma, St Louis, MO, USA), 3 \u0026micro;M biotin, 17 \u0026micro;M pantothenic acid, and 0.5% penicillin-streptomycin-amphotericin B suspension (Fujifilm) in an incubator at 37\u0026deg;C with humidified air at 5% CO\u003csub\u003e2\u003c/sub\u003e. To induce adipogenesis, cells grown to 80\u0026ndash;90% confluency were cultured with FBS-free medium containing 0.01 mg/ml transferrin, 0.1 \u0026micro;M cortisol, 200 pM triiodothyronine, 20 nM insulin, 0.25 \u0026micro;M DEX, 500 \u0026micro;M 3-isobutyl-1-methylxanthine, and 2 \u0026micro;M troglitazone for 4 days. Subsequently, the cells were cultured in a maintenance medium consisting of FBS-free medium containing 0.01 mg/ml transferrin, 0.1 \u0026micro;M cortisol, 200 pM triiodothyronine, and 20 nM insulin. The maintenance medium was changed every 3 days.\u003c/p\u003e \u003cp\u003e3T3-L1 cells were generously provide by Oral Bioscience Laboratory, Tokushima University. Japan. The cells were cultured in 6-well plates with culture medium consisting of Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (Fujifilm) supplemented with 10% FBS and 0.5% penicillin-streptomycin-amphotericin B suspension. To induce adipogenesis, cells grown to 100% confluency were cultured for an additional 2 days and then cultured in medium containing 10 \u0026micro;M insulin, 1 \u0026micro;M DEX, 500 \u0026micro;M 3-isobutyl-1-methylxanthine, and 2 \u0026micro;M troglitazone for 3 days. Subsequently, the cells were cultured in maintenance medium consisting of medium containing 10 \u0026micro;M insulin. The maintenance medium was changed every 3 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMicroarray\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTotal RNA was extracted from SGBS and 3T3-L1 cells before (day 0) and on days 1, 3, and 7 after adipogenic induction, using ISOGEN (Nippongene, Toyama, Japan). The extracted RNA was used to generate biotin-labeled cRNA using the Affymetrix GeneChip\u003csup\u003e\u0026trade;\u003c/sup\u003e 3\u0026prime; IVT PLUS Reagent Kit (Thermo Fisher Scientific, Waltham, MA, USA). The biotin-labeled RNA was then hybridized to either an Affymetrix Human Genome U-219 Array plate (Thermo Fisher Scientific) or a mouse genome MG-430 PM array plate (Thermo Fisher Scientific) following the manufacturer\u0026rsquo;s instructions. After washing and staining the array strips, the signals were developed and scanned using the Affymetrix Gene Atlas system (Thermo Fisher Scientific), and the data were analyzed using Transcriptome Analysis Console software (Thermo Fisher Scientific). Average hybridization signal intensities were used for data analysis, and genes with a mean signal intensity greater than 5 (log base 2 scale) in either of the adipogenic-induced samples were considered detectable. Genes with a signal intensity more than 2-fold higher than that in each cell before adipogenic induction were considered induced genes, and comparisons were made between SGBS and 3T3-L1 cells. Genes with the same symbol in humans and mice were designated as common genes.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eqRT-PCR\u003c/h2\u003e \u003cp\u003eEach cDNA was synthesized from total RNA using the ReverTra Ace\u0026reg; qPCR RT Kit (Toyobo, Osaka, Japan) following the manufacturer's protocol. The cDNA was then subjected to qRT-PCR on a Thermal Cycler Dice\u0026reg; Real Time System (Takara, Shiga, Japan) using Thunderbird\u0026trade; SYBR\u0026reg; qPCR Mix (Toyobo) and gene-specific primer sets via the following program: 30 sec at 95\u0026deg;C, followed by 40 cycles of 95\u0026deg;C for 15 sec and 60\u0026deg;C for 1 min. The specificity of each primer set was confirmed by dissociation curve analysis following amplification. The nucleotide sequences of the primer sets are listed in Supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The mRNA level of each gene was normalized to that of the human and mouse glyceraldehyde 3-phosphate dehydrogenase gene (\u003cem\u003eGAPDH/Gapdh\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eThe cells were lysed with lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM ethylenediamine tetraacetic acid, 1% Triton X-100, and complete mini (Roche, Basel, Switzerland)), and the lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Immobilon Transfer Membranes; Millipore, Bedford, MA, USA). After incubation in blocking solution (Blocking One; Nakalai tesque, Kyoto, Japan), the membranes were incubated with a 1:1000 dilution of mouse anti-FLAG M2 antibody (Sigma), a 1:2000 dilution of mouse anti-β actin (Sigma), a 1:500 dilution of rabbit anti-GalNAc-T15 antibody (Thermo Fisher), or a 1:1000 dilution of mouse anti-adiponectin antibody (Proteintech, Rosemont, IL, USA), and subsequently incubated with an anti-rabbit or anti-mouse IgG-horseradish peroxidase-conjugated secondary antibody (Jackson Lab, Farmington, CT, USA). The signal was detected using Immobilon Western Detection Reagent (Millipore) with a Luminograph III (Atto, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of adenoviruses\u003c/h2\u003e \u003cp\u003eAdenoviruses expressing GalNAc-T15 fused to FLAG at the C-terminus and a shGALNT15 were constructed as previously described\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Briefly, cDNA encoding the translational region lacking the stop codon of \u003cem\u003eGALNT15\u003c/em\u003e was amplified from SGBS adipocyte cDNA using PCR, and DNA with FLAG cDNA sequences added to its 3'-end was inserted into the pAxCAwtit cosmid vector (TakaRa). Recombinant adenoviral genomic DNA was excised from the cosmid and transfected into HEK293 cells to produce an adenovirus. An adenovirus produced from intact pAxCAwtit was used as a control. Cosmids for adenovirus production, which were inserted with a cDNA encoding shGALNT15 and a cDNA encoding a shNC, were purchased from Vector Builder (Chicago, IL, USA), and adenoviruses were produced in the same manner.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of adipogenesis\u003c/h2\u003e \u003cp\u003eThe degree of adipocyte differentiation was evaluated based on the expression of adipocyte marker genes, triglyceride accumulation, and the percentage of cells with oil droplets. SGBS cells were infected with each adenoviruses for 24 h and subjected to adipogenic induction. Seven days after adipogenic induction, total RNA was extracted from the cells and the expression of adipocyte marker genes was measured by qRT-PCR. Ten days after adipogenic induction, SGBS cells ere fixed with 4% paraformaldehyde and stained with Oil Red O to evaluate triglyceride accumulation or 4,6-diamidine-2-phenylindole dihydrochloride and Sudan III to count cells with oil droplets. To measure the amount of triglycerides, stained Oil Red O was eluted with isopropanol, and the absorbance was measured at 500 nm using a spectrophotometer (Ultrospec 6300 pro; GE Healthcare, Chicago, IL, USA). To assess the percentage of cells with oil droplets, the ratio of Sudan III-positive cells to 4,6-diamidine-2-phenylindole dihydrochloride-stained cells was determined in 3 randomly selected low-power fields (x100).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eEach experiment in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e was repeated several times, and representative results are shown. Each bar on the graph is expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE. Statistical analyses were performed using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test for the comparison of two groups and Dunnett's test for the comparison of three or more groups versus the control. Differences were considered significant when the \u003cem\u003eP\u003c/em\u003e-value was less than 0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization; T.I., Data collection; T.I., A.T., R.K., S.W., and K.K., Funding acquisition; T.I., A.K., M.S., M.N. and A.I., Project administration; T.I. and K.Y., Methodology and Resources; T.I., M.M., and M.W., Writing the original draft; T.I. and A.T..\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Dr. M. Fukuhara and T. Yamaguchi (Department of Microbiology, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Medical and Life Sciences) for providing measurement equipment.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRosen, E. D. \u0026amp; Spiegelman, B. M. Adipocytes as Regulators of Energy Balance and Glucose Homeostasis. Nature 444, 847\u0026ndash;853. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nature05483\u003c/span\u003e\u003cspan address=\"10.1038/nature05483\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHausman D. B., DiGirolamo M., Bartness T. J., Hausman G. J. \u0026amp; Martin R. J. The biology of white adipocyte proliferation. Obes. Rev. 2, 239\u0026ndash;254. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1046/j.1467-789X.2001.00042.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1467-789X.2001.00042.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosen, E. D. \u0026amp; MacDougald, O. A Adipocyte differentiation from the inside out. Nat. Rev. Mol. Cell Biol. 7, 885\u0026ndash;986. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nrm2066\u003c/span\u003e\u003cspan address=\"10.1038/nrm2066\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang, Q. Q. \u0026amp; Lane, M. D. Activation and centromeric localization of CCAAT/enhancer-binding proteins during the mitotic clonal expansion of adipocyte differentiation. Genes Dev. 13, 2231\u0026ndash;2241. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/gad.13.17.2231\u003c/span\u003e\u003cspan address=\"10.1101/gad.13.17.2231\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWabitsch, M. \u003cem\u003eet al.\u003c/em\u003e Characterization of a human preadipocyte cell strain with high capacity for adipose differentiation. Int. J. Obe.s Relat. Metab. Disord. 25, 8\u0026ndash;15. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/sj.ijo.0801520\u003c/span\u003e\u003cspan address=\"10.1038/sj.ijo.0801520\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEntenmann, G. \u0026amp; Hauner, H. Relationship between replication and differentiation in cultured human adipocyte precursor cells. Am. J. Physiol. 270, C1011-C1016. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1152/ajpcell.1996.270.4.C1011\u003c/span\u003e\u003cspan address=\"10.1152/ajpcell.1996.270.4.C1011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (1996).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNewell, F. S. \u003cem\u003eet al.\u003c/em\u003e Characterization of the transcriptional and functional effects of fibroblast growth factor-1 on human preadipocyte differentiation. FASEB J. 20, 2615\u0026ndash;2617. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1096/fj.05-5710fje\u003c/span\u003e\u003cspan address=\"10.1096/fj.05-5710fje\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLindroos, J. \u003cem\u003eet al.\u003c/em\u003e Human but not mouse adipogenesis is critically dependent on LMO3. Cell Metab. 18, 62\u0026ndash;74. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cmet.2013.05.020\u003c/span\u003e\u003cspan address=\"10.1016/j.cmet.2013.05.020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIshimoto, K. \u003cem\u003eet al.\u003c/em\u003e D-dopachrome tautomerase promotes IL-6 expression and inhibits adipogenesis in preadipocytes. \u003cem\u003eCytokine\u003c/em\u003e 60, 772\u0026thinsp;\u0026ndash;\u0026thinsp;727. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cyto.2012.07.037\u003c/span\u003e\u003cspan address=\"10.1016/j.cyto.2012.07.037\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng, L. \u003cem\u003eet al\u003c/em\u003e. Characterization of a novel human UDP-GalNAc transferase, pp-GalNAc-T15. FEBS Lett. 566, 17\u0026ndash;24. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.febslet.2004.03.108\u003c/span\u003e\u003cspan address=\"10.1016/j.febslet.2004.03.108\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBennett, E. P. \u003cem\u003eet al.\u003c/em\u003e Control of mucin-type O-glycosylation: A classification of the polypeptide GalNAc-transferase gene family. Glycobiology 22, 736\u0026ndash;756. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/glycob/cwr182\u003c/span\u003e\u003cspan address=\"10.1093/glycob/cwr182\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHussain, M. R. M., Hoessli, D. C. \u0026amp; Fang, M. N-acetylgalactosaminyltransferases in cancer. Oncotarget 7, 54067\u0026ndash;54081. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18632/oncotarget.10042\u003c/span\u003e\u003cspan address=\"10.18632/oncotarget.10042\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKato, K., Hansen, L. \u0026amp; Clausen, H. Polypeptide \u003cem\u003eN\u003c/em\u003e-acetylgalactosaminyltransferase-Associated Phenotypes in Mammals. Molecules 26, 5504. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules26185504\u003c/span\u003e\u003cspan address=\"10.3390/molecules26185504\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIwata, T. \u003cem\u003eet al.\u003c/em\u003e The action of D-dopachrome tautomerase as an adipokine in adipocyte lipid metabolism. PLoS One 7, e33402. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0033402\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0033402\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoukas, A. \u003cem\u003eet al.\u003c/em\u003e Distinct transcriptional profiles of adipogenesis in vivo and in vitro. J. Biol. Chem. 276, 34167\u0026ndash;34174. DOI \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1074/jbc.M104421200\u003c/span\u003e\u003cspan address=\"10.1074/jbc.M104421200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao, C. R. \u003cem\u003eet al.\u003c/em\u003e SGBS cells as a model of human adipocyte browning: A comprehensive comparative study with primary human white subcutaneous adipocytes. Sci. Rep. 7, 4031. DOI:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-017-04369-2\u003c/span\u003e\u003cspan address=\"10.1038/s41598-017-04369-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalkhof, S. \u003cem\u003eet al.\u003c/em\u003e In depth quantitative proteomic and transcriptomic characterization of human adipocyte differentiation using the SGBS cell line. Proteomics 8, e1900405. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/pmic.201900405\u003c/span\u003e\u003cspan address=\"10.1002/pmic.201900405\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTews, D. \u003cem\u003eet al.\u003c/em\u003e 20 Years with SGBS cells - a versatile in vitro model of human adipocyte biology. Int. J. Obes. 46, 1939\u0026ndash;1947. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41366-022-01199-9\u003c/span\u003e\u003cspan address=\"10.1038/s41366-022-01199-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.(2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, K., Xie, S. \u0026amp; Jin, W. Crucial lncRNAs associated with adipocyte differentiation from human adipose- derived stem cells based on co-expression and ceRNA network analyses. PeerJ 7, e7544. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7717/peerj.7544\u003c/span\u003e\u003cspan address=\"10.7717/peerj.7544\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJaszczyk, A. \u003cem\u003eet al.\u003c/em\u003e Overnight corticosterone and gene expression in mouse hippocampus: Time course during resting period. Int. J. Mol. Sci. 24, 2828. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms24032828\u003c/span\u003e\u003cspan address=\"10.3390/ijms24032828\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiller, S. G. \u003cem\u003eet al.\u003c/em\u003e The adipocyte specific transcription factor C/EBPalpha modulates human ob gene expression. \u003cem\u003eProc. Natl. Acad. Sci. U.S.A.\u003c/em\u003e 93, 5507\u0026ndash;5511. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.93.11.5507\u003c/span\u003e\u003cspan address=\"10.1073/pnas.93.11.5507\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (1996).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosen, E. D. \u003cem\u003eet al.\u003c/em\u003e C/EBPalpha induces adipogenesis through PPARgamma: a unified pathway. Genes Dev. 16, 22\u0026ndash;26. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/gad.948702\u003c/span\u003e\u003cspan address=\"10.1101/gad.948702\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAntonucci, A., Marucci, A., Trischitta, V., \u0026amp; Di Paola, R. Role of GALNT2 on Insulin Sensitivity, Lipid Metabolism and Fat Homeostasis. Int. J. Mol. Sci. 23, 929. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms23020929\u003c/span\u003e\u003cspan address=\"10.3390/ijms23020929\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarucci, A. \u003cem\u003eet al.\u003c/em\u003e Role of GALNT2 in the modulation of ENPP1 expression, and insulin signaling and action: GALNT2: A novel modulator of insulin signaling. Biochim. Biophys. Acta 1833, 1388\u0026ndash;1395 doi: 10.1016/j.bbamcr.2013.02.032. (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarucci, A. \u003cem\u003eet al.\u003c/em\u003e GALNT2 as a novel modulator of adipogenesis and adipocyte insulin signaling. Int. J. Obes. 43, 2448\u0026ndash;2457. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41366-019-0367-3\u003c/span\u003e\u003cspan address=\"10.1038/s41366-019-0367-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJankowski, M. \u003cem\u003eet al.\u003c/em\u003e Expression profile of new marker genes involved in differentiation of canine adipose-derived stem cells into osteoblasts. Int. J. Mol. Sci. 22, 6663. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms22136663\u003c/span\u003e\u003cspan address=\"10.3390/ijms22136663\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (2021).\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"adipogenesis, polypeptide N-acetylgalactosaminyl transferase, GALNT15, preadipocytes, adipocytes","lastPublishedDoi":"10.21203/rs.3.rs-4244309/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4244309/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAdipogenesis involves intricate molecular mechanisms regulated by various transcription factors and signaling pathways. In this study, we aimed to identify factors specifically induced during adipogenesis in the human preadipocyte cell line, SGBS, but not in the mouse preadipocyte cell line, 3T3-L1. Microarray analysis revealed distinct gene expression profiles, with 1460 genes induced in SGBS cells and 1297 genes induced in 3T3-L1 cells during adipogenesis, with only 297 genes commonly induced. Among the genes uniquely induced in SGBS cells, we focused on \u003cem\u003eGALNT15\u003c/em\u003e, which encodes polypeptide N-acetylgalactosaminyl transferase-15. Its expression increased transiently during adipogenesis in SGBS cells but remained low in 3T3-L1 cells. Overexpression of \u003cem\u003eGALNT15\u003c/em\u003e increased mRNA levels of CCAAT-enhancer binding protein (C/EBPα) and leptin but had no significant impact on adipogenesis in SGBS cells. Conversely, knockdown of \u003cem\u003eGALNT15\u003c/em\u003e suppressed mRNA expression of adipocyte marker genes, reduced lipid accumulation, and decreased the percentage of cells with oil droplets. The induction of C/EBPα and peroxisome proliferator-activated receptor γ during adipogenesis was promoted or suppressed in SGBS cells subjected to overexpression or knockdown of \u003cem\u003eGALNT1\u003c/em\u003e5, respectively. These data suggest that \u003cem\u003eGALNT15\u003c/em\u003e is a novel regulatory molecule that enhances adipogenesis in SGBS cells.\u003c/p\u003e","manuscriptTitle":"GALNT15, induced during adipogenesis of human SGBS cells but not in mouse 3T3-L1 cells, regulates adipocyte differentiation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-23 20:31:24","doi":"10.21203/rs.3.rs-4244309/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-26T12:07:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-25T16:12:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"53368807303719226551126293778763484488","date":"2024-05-11T13:11:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-08T00:07:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"23a4d1e5-3f11-4d39-ba17-96935f13c8f6","date":"2024-04-29T07:15:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-21T22:33:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-21T22:18:29+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-18T17:07:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-18T17:00:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-04-10T00:37:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"08f227b1-34df-4a61-a1dc-7261a1f69370","owner":[],"postedDate":"April 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":31020419,"name":"Biological sciences/Developmental biology"},{"id":31020420,"name":"Health sciences/Endocrinology/Endocrine system and metabolic diseases/Obesity"}],"tags":[],"updatedAt":"2024-09-02T16:04:51+00:00","versionOfRecord":{"articleIdentity":"rs-4244309","link":"https://doi.org/10.1038/s41598-024-70930-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-08-29 15:58:05","publishedOnDateReadable":"August 29th, 2024"},"versionCreatedAt":"2024-04-23 20:31:24","video":"","vorDoi":"10.1038/s41598-024-70930-5","vorDoiUrl":"https://doi.org/10.1038/s41598-024-70930-5","workflowStages":[]},"version":"v1","identity":"rs-4244309","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4244309","identity":"rs-4244309","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}