Adipose-specific BMP and activin membrane-bound inhibitor (BAMBI) deletion promotes adipogenesis by accelerating ROS production

Abstract

With the improvement of people's living standards, the number of obese patients has also grown rapidly. It is reported that the level of oxidative stress in obese patients has significantly increased, mainly caused by the increase in reactive oxygen species (ROS) levels in adipose tissue. Studies have shown that the use of siRNA to interfere with bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) expression could promote adipocyte differentiation, and under hypoxic conditions, BAMBI could act as a regulator of HIF1α to regulate the polarity damage of epithelial cells. In view of these results, we speculated that BAMBI may regulate adipogenesis by regulating the level of ROS. In this study, we generated adipose-specific BAMBI knockout mice (BAMBI AKO) and found that compared with control mice, BAMBI AKO mice showed obesity when fed with high-fat diet, accompanied by insulin resistance, glucose intolerance, hypercholesterolemia, and increased inflammation in adipose tissue. Interestingly, adipose-specific deficiency of BAMBI could cause an increase in the expression level of Nox4, thereby promoting ROS production in cytoplasm and mitochondria and the DNA-binding activity of C/EBPβ and ultimately promoting adipogenesis. Consistently, our findings indicated that BAMBI may be a reactive oxygen regulator to affect adipogenesis, thereby controlling obesity and metabolic syndrome. With the improvement of people's living standards, the number of obese patients has also grown rapidly. It is reported that the level of oxidative stress in obese patients has significantly increased, mainly caused by the increase in reactive oxygen species (ROS) levels in adipose tissue. Studies have shown that the use of siRNA to interfere with bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) expression could promote adipocyte differentiation, and under hypoxic conditions, BAMBI could act as a regulator of HIF1α to regulate the polarity damage of epithelial cells. In view of these results, we speculated that BAMBI may regulate adipogenesis by regulating the level of ROS. In this study, we generated adipose-specific BAMBI knockout mice (BAMBI AKO) and found that compared with control mice, BAMBI AKO mice showed obesity when fed with high-fat diet, accompanied by insulin resistance, glucose intolerance, hypercholesterolemia, and increased inflammation in adipose tissue. Interestingly, adipose-specific deficiency of BAMBI could cause an increase in the expression level of Nox4, thereby promoting ROS production in cytoplasm and mitochondria and the DNA-binding activity of C/EBPβ and ultimately promoting adipogenesis. Consistently, our findings indicated that BAMBI may be a reactive oxygen regulator to affect adipogenesis, thereby controlling obesity and metabolic syndrome. In recent years, obesity has prevailed worldwide, and 300 million adults have been classified as obese (BMI >30) (1Reilly J.J. El-Hamdouchi A. Diouf A. Monyeki A. Somda S.A. Determining the worldwide prevalence of obesity.Lancet. 2018; 391: 1773-1774Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Obesity is closely related to many pathological conditions and diseases, including hypertension, type 2 diabetes, dyslipidemia, kidney disease, heart disease, certain types of cancer, acute respiratory distress syndrome, and osteoarthritis (2Ayer J. Charakida M. Deanfield J.E. Celermajer D.S. Lifetime risk: childhood obesity and cardiovascular risk.Eur. Heart J. 2015; 36: 1371-1376Crossref PubMed Scopus (90) Google Scholar, 3Abuyassin B. Laher I. Obesity-linked diabetes in the Arab world: a review.East Mediterr. Health J. 2015; 21: 420-439Crossref PubMed Google Scholar, 4Ghaben A.L. Scherer P.E. Adipogenesis and metabolic health.Nat. Rev. Mol. Cell Biol. 2019; 20: 242-258Crossref PubMed Scopus (247) Google Scholar). Generally, obesity is characterized by excessive accumulation of white adipose tissue, which is produced by the increased size (hypertrophy) or number (hyperplasia) of adipocytes or the combination of hypertrophy and hyperplasia. Therefore, the study of adipocytes differentiation is of great value in alleviating various diseases caused by obesity. Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) is a transmembrane glycoprotein that is highly conserved from human to zebrafish development in vertebrates (5Grotewold L. Plum M. Dildrop R. Peters T. Rüther U. Bambi is coexpressed with Bmp-4 during mouse embryogenesis.Mech. Dev. 2001; 100: 327-330Crossref PubMed Scopus (94) Google Scholar, 6Onichtchouk D. Chen Y.-G. Dosch R. Gawantka V. Delius H. Massagué J. Niehrs C. Silencing of TGF-β signalling by the pseudoreceptor BAMBI.Nature. 1999; 401: 480-485Crossref PubMed Scopus (529) Google Scholar, 7Tsang M. Kim R. de Caestecker M.P. Kudoh T. Roberts A.B. Dawid I.B. Zebrafish nma is involved in TGFβ family signaling.Genesis. 2000; 28: 47-57Crossref PubMed Scopus (57) Google Scholar). Because it is highly homologous to the transforming growth factor (TGF) beta superfamily type I receptor, BAMBI is considered to be a pseudoreceptor of the TGFβ-related signaling pathway and acts as a negative regulator of the TGFβ signaling pathway. It has been reported that BAMBI was involved in many aspects of biological processes, such as organ development and disease (8Luo X. Hutley L.J. Webster J.A. Kim Y.H. Liu D.F. Newell F.S. Widberg C.H. Bachmann A. Turner N. Schmitz-Peiffer C. Prins J.B. Yang G.S. Whitehead J.P. Identification of BMP and activin membrane-bound inhibitor (BAMBI) as a potent negative regulator of adipogenesis and modulator of autocrine/paracrine adipogenic factors.Diabetes. 2012; 61: 124-136Crossref PubMed Scopus (49) Google Scholar). On the one hand, in vitro studies have shown that BAMBI could promote skeletal muscle myogenic differentiation and transformation of myofiber type in C2C12 cells by activating Wnt/β-catenin signaling (9Zhang Q. Shi X.E. Song C. Sun S. Yang G. Li X. BAMBI promotes C2C12 myogenic differentiation by enhancing Wnt/beta-catenin signaling.Int. J. Mol. Sci. 2015; 16: 17734-17745Crossref PubMed Scopus (19) Google Scholar, 10Yao X. Yu T. Xi F. Xu Y. Ma L. Pan X. Chen S. Han M. Yin Y. Dai X. Xu G. Zhang H. Yang G. Xie L. BAMBI shuttling between cytosol and membrane is required for skeletal muscle development and regeneration.Biochem. Biophys. Res. Commun. 2019; 509: 125-132Crossref PubMed Scopus (5) Google Scholar), whereas in myocardium, BAMBI played a critical role in resisting cardiac hypertrophy by inhibiting TGF-β signaling (11Villar A.V. Garcia R. Llano M. Cobo M. Merino D. Lantero A. Tramullas M. Hurle J.M. Hurle M.A. Nistal J.F. BAMBI (BMP and activin membrane-bound inhibitor) protects the murine heart from pressure-overload biomechanical stress by restraining TGF-beta signaling.Biochim. Biophys. Acta. 2013; 1832: 323-335Crossref PubMed Scopus (48) Google Scholar). On the other hand, BAMBI also inhibits TGFβ-induced downregulation of estradiol and progesterone, indicating the potential role of BAMBI in reproduction (12Bai L. Chu G. Wang W. Xiang A. Yang G. BAMBI promotes porcine granulosa cell steroidogenesis involving TGF-beta signaling.Theriogenology. 2017; 100: 24-31Crossref PubMed Scopus (8) Google Scholar). In addition, BAMBI has also been reported to play a key role in the development process of adipose tissue. The downregulated expression of BAMBI by siRNA could inhibit promotion of FGF-1 on lipogenesis (8Luo X. Hutley L.J. Webster J.A. Kim Y.H. Liu D.F. Newell F.S. Widberg C.H. Bachmann A. Turner N. Schmitz-Peiffer C. Prins J.B. Yang G.S. Whitehead J.P. Identification of BMP and activin membrane-bound inhibitor (BAMBI) as a potent negative regulator of adipogenesis and modulator of autocrine/paracrine adipogenic factors.Diabetes. 2012; 61: 124-136Crossref PubMed Scopus (49) Google Scholar). Meanwhile, BAMBI was also confirmed to be able to inhibit adipogenesis by a feedback loop, which forms with canonical Wnt/β-catenin signaling (13Mai Y. Zhang Z. Yang H. Dong P. Chu G. Yang G. Sun S. BMP and activin membrane-bound inhibitor (BAMBI) inhibits the adipogenesis of porcine preadipocytes through Wnt/beta-catenin signaling pathway.Biochem. Cell Biol. 2014; 92: 172-182Crossref PubMed Scopus (16) Google Scholar). However, at present, research on BAMBI is focused on the cellular level, and in vivo tests are rarely reported, especially in terms of adipose tissue development. Reactive oxygen species (ROS) can significantly regulate the process of adipocyte differentiation and then affect the occurrence and development of obesity and related diseases. In cells, ROS is mainly produced under the action of the mitochondrial respiratory system, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, endoplasmic reticulum stress, certain metabolism, and detoxifying enzymes (14Wang X. Hai C.X. Novel insights into redox system and the mechanism of redox regulation.Mol. Biol. Rep. 2016; 43: 607-628Crossref PubMed Scopus (38) Google Scholar). In the past few years, increasing evidence has supported that ROS is an essential factor for adipocyte differentiation and is considered an important medium for adipogenic hormone-induced C/EBPβ production and adipogenesis (15Rhee Y.H. Ahn J.C. Melatonin attenuated adipogenesis through reduction of the CCAAT/enhancer binding protein beta by regulating the glycogen synthase 3 beta in human mesenchymal stem cells.J. Physiol. Biochem. 2016; 72: 145-155Crossref PubMed Scopus (20) Google Scholar, 16Wang W.M. Zhang Y. Lu W.Y. Liu K.Y. Mitochondrial reactive oxygen species regulate adipocyte differentiation of mesenchymal stem cells in hematopoietic stress induced by arabinosylcytosine.PLoS One. 2015; 10e0120629PubMed Google Scholar). Studies have shown that ROS can further promote the DNA binding activity of C/EBPβ by affecting the mitotic clonal expansion (MCE) phase of differentiation and ultimately promote the adipogenic process (17Lee H. Lee Y.J. Choi H. Ko E.H. Kim J.W. Reactive oxygen species facilitate adipocyte differentiation by accelerating mitotic clonal expansion.J. Biol. Chem. 2009; 284: 10601-10609Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). NADPH oxidase (NOX) is an important enzyme that produces ROS in cells (18Kim Y.M. Cho M. Activation of NADPH oxidase subunit NCF4 induces ROS-mediated EMT signaling in HeLa cells.Cell Signal. 2014; 26: 784-796Crossref PubMed Scopus (34) Google Scholar). In the NOX family, only NOX4 and p47phox isoforms are involved in adipocyte differentiation (19Seo M.J. Seo Y.J. Pan C.H. Lee O.H. Kim K.J. Lee B.Y. Fucoxanthin accumulation and ROS production during differentiation in Res. 2016; PubMed Scopus Google Scholar, F. A. The role of reactive oxygen species in mesenchymal stem cell adipogenic and a Dev. 2015; PubMed Scopus Google Scholar). In mesenchymal stem cells, that differentiation can ROS and and of NOX4 can the level of ROS and can the differentiation of In the study, we BAMBI NOX4 activity levels are during the development of adipose tissue in mice with high-fat we a mouse in which BAMBI has been in adipocytes to the role of BAMBI in an obesity that BAMBI deficiency in adipocyte caused hypertrophy and of adipocytes and an ROS level during the development of obesity. mice with adipocytes in BAMBI an increased of insulin with adipose tissue these that BAMBI may play a role in adipose tissue development. of the between and adipocyte differentiation could for the of the that the and of BAMBI the it was to BAMBI knockout mice to the of Therefore, we BAMBI mice from The and mice from and then generated adipose-specific BAMBI knockout (BAMBI AKO) mice by these types of mice the of a few these mouse and and we ultimately adipose-specific BAMBI knockout mice, BAMBI AKO mice the we the of BAMBI on adipogenesis under we found that in tissue adipose tissue adipose tissue adipocyte and adipogenesis between the of mice Therefore, we the of BAMBI on accumulation with of the knockout of BAMBI in and The showed that BAMBI was in and the of the mice, BAMBI AKO mice showed under conditions, with in the mice and the of BAMBI knockout on tissue found that the and adipose tissue of the knockout mice of the control mice In addition, levels of and and levels of in BAMBI AKO mice The that the adipose-specific knockout of BAMBI can cause obesity and under In the increased size of adipocytes (hypertrophy) an increase in adipose tissue the mechanism of increased in BAMBI AKO mice, adipocyte size was in the adipose tissue of control and BAMBI AKO and indicated that adipocytes in and of BAMBI AKO compared with control mice and adipocyte supported the of increased adipocyte size in BAMBI AKO adipose tissue and we the expression levels of adipogenic and in or of BAMBI AKO mice at the and protein and the with by thereby that increased in BAMBI AKO mice was caused by adipocyte hypertrophy and also the of BAMBI in adipose tissue on metabolic BAMBI AKO mice showed significantly oxygen and production compared with respiratory indicating that the metabolic may be the cause of obesity in BAMBI AKO and insulin are closely related to obesity Obesity and insulin 2000; PubMed Scopus Google Scholar, obesity to insulin and type 2 PubMed Scopus Google Scholar, C.H. insulin and of the adipose a 2019; PubMed Scopus Google Scholar). In the study, adipose BAMBI expression caused glucose and insulin in mice, in mice we the of adipose-specific BAMBI on glucose and insulin in the BAMBI AKO mice glucose and insulin compared with mice in the signaling pathway are to the insulin signaling pathway is the damage to the insulin signaling pathway BAMBI we the expression levels of in and muscle of was attenuated in the adipose tissue of BAMBI AKO mice, that BAMBI knockout could the insulin that BAMBI knockout could insulin in adipose tissue. to further the in vivo and to a cell for we preadipocytes from and of control and BAMBI AKO with in vivo the preadipocytes from and of mice showed differentiation In addition, we in lipogenesis and inflammation in BAMBI knockout preadipocytes The confirmed the of adipocyte differentiation in expansion in the adipose of the preadipocytes from of mice into adipocytes with expression levels of lipogenesis and expression levels of The of and also the at the and protein that this cell could be for studies that significantly in from BAMBI AKO mice to BAMBI mice these involved in adipogenesis, and redox including the signaling the signaling and oxidative a of BAMBI in adipocyte Interestingly, our that of BAMBI AKO mice is characterized by in involved in redox such as and The of and in adipocytes and of BAMBI AKO mice confirmed by with these the protein levels of mitochondrial oxidative increased in adipocytes and of BAMBI AKO mice compared with BAMBI mice, that BAMBI may regulate the adipogenesis process by increasing the ROS this we the mitochondrial and ROS levels in adipocytes of and In the mitochondrial and ROS level significantly increased in of the that knockout of BAMBI in adipocytes could the mitochondrial and ROS level of we cell during are by ROS preadipocytes the cell in the of cells by of Interestingly, BAMBI in preadipocytes caused an increase in the during adipocyte differentiation have shown that of C/EBPβ was with ROS and it was only to on DNA cells the phase at in a in shown in the of C/EBPβ was at the in BAMBI AKO C/EBPβ in the control the could cause the The of showed that the use of the differentiation of indicated that BAMBI knockout and the of cell was confirmed by the expression levels of and as shown in that BAMBI is important for the of during adipocyte further our we the to ROS production in preadipocytes at the of The involved the in to the I and then the I to the cell for the of the of was significantly indicating that the ROS produced by mitochondria on the ROS results, the of the in the adipocyte cells was significantly indicating that the ROS produced in the cells shown in the of showed that the of phase in BAMBI AKO preadipocytes indicating that the phase of the of C/EBPβ showed a compared with when was indicating that the of could the DNA-binding of C/EBPβ with The of also showed that the was significantly and the value of at was also significantly indicating that the adipogenic differentiation of preadipocytes that is closely with obesity and insulin resistance, we the of adipose BAMBI on mice showed accumulation in the mice and accumulation BAMBI AKO mice under an The levels of significantly increased in the of BAMBI AKO mice, and expression levels only and levels significantly in BAMBI AKO mice to these that of BAMBI in adipocyte in to increased and In addition, the levels of protein factor and in BAMBI AKO of increased number of in the of BAMBI AKO mice adipose-specific BAMBI deficiency may cause increased inflammation in to the excessive accumulation of in cells to various are many of mainly related to metabolic diabetes, In our study, the of and the expression of binding protein and such as synthase in the BAMBI knockout mice significantly of control the expression of has that the increased and in are important for the of tissue in BAMBI AKO mice On the other hand, the expression level of and in AKO mice was significantly that of control that the of may cause the increase of in tissue of BAMBI AKO mice the of showed that the expression of and in BAMBI knockout mice was significantly that of control mice, indicated that BAMBI AKO mice is on the results, we that deficiency of BAMBI may cause by promoting the of from adipose tissue, regulating the of the adipogenesis in In the study, we BAMBI adipose-specific knockout mice through the this showed highly with with obesity. BAMBI AKO mice white adipose expansion with de lipogenesis and accompanied by increased and insulin Consistently, the was found in we that BAMBI could the adipogenesis process by increased ROS knockout of BAMBI could cause an increase in the expression level of Nox4, thereby promoting the ROS production in the cytoplasm and mitochondria and the DNA-binding activity of ultimately promoting adipogenesis. In other our findings indicated that BAMBI could as a reactive oxygen regulator to affect adipogenesis. In our recent study, BAMBI protein is highly in skeletal muscle and is only in the of In addition, BAMBI are in in with in BAMBI protein is on the cell membrane during muscle growth and that important by BAMBI may be an important of muscle growth and X. Yu T. Xi F. Xu Y. Ma L. Pan X. Chen S. Han M. Yin Y. Dai X. Xu G. Zhang H. Yang G. Xie L. BAMBI shuttling between cytosol and membrane is required for skeletal muscle development and regeneration.Biochem. Biophys. Res. Commun. 2019; 509: 125-132Crossref PubMed Scopus (5) Google Scholar). It can be from the that BAMBI an important role in many and pathological of the including at present, the of BAMBI on adipogenesis has been in research that BAMBI can promote the of to through the signaling and under hypoxic conditions, BAMBI could as a regulator of HIF1α to promote TGFβ signaling and then induced of epithelial cells, indicating that BAMBI may affect adipogenesis through inflammation or S. Chen L. M. J. Z. Han H. S. C. X. BAMBI the differentiation of through the TGF-β pathway in Res. 2019; 20: PubMed Scopus Google Scholar, I. F. A. J. BAMBI is a modulator of of cell polarity during Cell Sci. 2018; PubMed Scopus Google Scholar). we generated BAMBI knockout mice and found that BAMBI knockout can to obesity and insulin resistance, thereby our of BAMBI is in the the role of ROS on adipogenesis, for the ROS is considered to promote adipogenesis X. Hai C. of adipocyte of and insights into of adipogenesis and Biol. 2015; PubMed Scopus (34) Google Scholar). we is that as as was evidence that ROS was during the process of adipogenesis H. H. of of the and growth factor on adipose and in J. PubMed Scopus Google Scholar), and increasing of ROS as a factor for adipocyte differentiation W.M. Zhang Y. Lu W.Y. Liu K.Y. Mitochondrial reactive oxygen species regulate adipocyte differentiation of mesenchymal stem cells in hematopoietic stress induced by arabinosylcytosine.PLoS One. 2015; 10e0120629PubMed Google Scholar). have found that ROS levels increase adipogenic and with or of could inhibit adipocyte differentiation in mesenchymal stem cells D.F. an in vitro for of and Health PubMed Scopus Google Scholar). ROS can also be involved in adipocyte C. P. induced protein promotes adipogenesis oxidative stress, endoplasmic reticulum stress and Physiol. Biochem. 2012; PubMed Scopus Google showed that of protein could increase levels and of ROS production attenuated adipogenesis in In addition, important is that C/EBPβ and are the in the ROS-mediated adipogenesis when is to ROS can C/EBPβ and by thereby enhancing DNA-binding activity from stem cell to Rev. Biochem. 2012; PubMed Scopus Google Scholar). when cells with differentiation with increasing expression (17Lee H. Lee Y.J. Choi H. Ko E.H. Kim J.W. Reactive oxygen species facilitate adipocyte differentiation by accelerating mitotic clonal expansion.J. Biol. Chem. 2009; 284: 10601-10609Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). In this study, we have that BAMBI adipose-specific knockout can promote the production of ROS in the cytoplasm and thereby promoting C/EBPβ to and ultimately promoting adipogenesis, which may the potential of BAMBI during adipogenesis. ROS are produced during the adipogenesis and the production of ROS is for adipogenesis. Therefore, inhibiting the production of ROS may inhibit adipogenesis, thereby the expansion of adipose tissue. Studies have reported that ROS is mainly produced by the mitochondrial respiratory system, NADPH oxidase and other in cells. On the one hand, the preadipocytes are induced to the differentiation the and of mitochondrial and the adipocytes to this and to an essential or increased mitochondrial activity and could to ROS On the other hand, NADPH oxidase (NOX) is an important enzyme that produces ROS in cells N. J.J. in inflammation and metabolic Rev. PubMed Scopus Google Scholar), of which are in In the NOX family, only the NOX4 and p47phox are involved in adipocyte It has been that the increase in ROS is accompanied by an increase in NOX4 activity in adipose tissue S. T. M. M. Y. Y. M. M. I. oxidative stress in obesity and on metabolic 2017; Scopus Google Scholar), and the increased NOX4 activity was closely related to the increase in expression L. L. B. B. A. L. W. NOX4 activity is by levels and a of ROS J. PubMed Scopus Google Scholar), which is with our Therefore, the increased expression of NOX4 to BAMBI knockout may promote the obese in BAMBI AKO However, at this we is between BAMBI and NOX4 and the further study on this evidence has shown that of adipocytes could to the of cells and the development and of insulin in the metabolic of obese In our study, BAMBI deficiency insulin and in adipose tissue. the obese BAMBI AKO mice also which is characterized by excessive accumulation of In we that BAMBI knockout ROS in in adipose tissue of BAMBI adipose and glucose and insulin BAMBI may be a potential for obesity and metabolic mice from and mice from the J. Y. R. The TGF-beta pseudoreceptor Bambi is for mouse development and PubMed Scopus Google Scholar). mice generated by mice with mice as at of mice fed an of or and was on a and to and by the of at and from or of mice as J. J. H. Zhang X. Y. Li X. B. Yang G. Shi X. promotes white adipocyte by inhibiting the signaling 2019; Google Scholar). 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