RESEARCH ARTICLE

Downregulated brain and muscle aryl hydrocarbon receptornuclear translocator-like protein-1 inhibits osteogenesis of BMSCsthrough p53 in type 2 diabetes mellitusXiaofei Mao1, Xiaoguang Li2, Wei Hu1, Siwei Hao1, Yifang Yuan1, Lian Guan1 and Bin Guo1,*

ABSTRACTThe bone marrow mesenchymal stem cells (BMSCs)-mediatedabnormal bone metabolism can delay and impair the boneremodeling process in type 2 diabetes mellitus (T2DM). Our previousstudy demonstrated that the downregulation of brain and muscle arylhydrocarbon receptor nuclear translocator-like protein 1 (BMAL1), acircadian clock protein, inhibited the Wnt/β-catenin pathway viaenhanced GSK-3β in diabetic BMSCs. In this article, we confirmedthat the downregulated BMAL1 in T2DM played an inhibitory role inosteogenic differentiation of BMSCs. Upregulation of BMAL1 in thediabetic BMSCs significantly recovered the expression pattern ofosteogenic marker genes and alkaline phosphatase (Alp) activity. Wealso observed an activation of the p53 signaling pathways, exhibited byincreased p53 and p21 in diabetic BMSCs. Downregulation of p53resulting from overexpression of BMAL1 was detected, and when weapplied p53 gene silencing (shRNA) and the p53 inhibitor, pifithrin-α(PFT-α), the impaired osteogenic differentiation ability of diabeticBMSCs was greatly restored. However, there was no change in thelevel of expression of BMAL1. Taken together, our results first revealedthat BMAL1 regulated osteogenesis of BMSCs through p53 in T2DM,providing a novel direction for further exploration of the mechanismunderlying osteoporosis in diabetes.

KEY WORDS: Brain and muscle aryl hydrocarbon receptor nucleartranslocator-like protein-1 (BMAL1), p53, Type 2 diabetes mellitus(T2DM), Bone marrow mesenchymal stem cells (BMSCs),Osteogenic differentiation

INTRODUCTIONAs a chronic metabolic disorder disease, diabetes mellitus (DM) hasaffected millions of people, and its prevalence has been increasingsignificantly worldwide (Guariguata et al., 2014). More than 90% ofthese patients are diagnosed with type 2 diabetes mellitus (T2DM)(McGurnaghan et al., 2019). Most of them are suffering fromosteoporosis and following fractures. Bone marrow mesenchymalstem cells (BMSCs) are vital for bone regeneration, due to itsmultidirectional differentiation potential and the differentiation ability

into osteoblasts. Previous studies revealed that the proliferation andmultipotency of BMSCs might change in diabetic pathologicalmicroenvironment (Zhou et al., 2016). BMSCs-mediated imbalanceof bone formation and bone resorption would occur in T2DM (Brownet al., 2014). However, there have been few articles on the molecularmechanisms of diabetic osteoporosis until now.

The core cellular circadian pacemaker inmammals drives circadianrhythms of behaviors, which is coupled to the environmental lightcycle (Honma, 2018). Brain and muscle aryl hydrocarbon receptornuclear translocator-like protein 1 (BMAL1) is a core component ofthe circadian clock, which has been reported to express universally inboth suprachiasmatic nucleus and peripheral tissues and regulatevarious cellular metabolic processes (Zhang et al., 2014). It combineswith CLOCK to form heterodimers and activates transcriptionalactivity of downstream genes, affecting numerous physiological andbiochemical reactions (Gaucher et al., 2018). Researchers have foundthe involvement of BMAL1 in regulating the rhythmic secretion ofpancreatic islet B cells, and its deletion will lead to the occurrence ofT2DM (Albrecht, 2017). The expression pattern of BMAL1 wasreported to be disrupted in the microenvironment of T2DM (Wegeret al., 2017). Moreover, decreased osteogenesis ability of BMSCswas observed in BMAL1 deletion or inhibition mice (Miyamotoet al., 2010). Our previous study preliminarily verified that BMAL1regulated the osteogenesis of diabetic BMSCs by modulatingGSK-3β (Li et al., 2017). However, more work remains to be doneto ascertain the mechanism between BMAL1 and osteogenicdifferentiation of diabetic BMSCs.

Recently, classical tumor suppressor p53 has been reported toregulate glucose metabolism and osteogenic differentiation ofBMSCs (Labuschagne et al., 2018). The p53 mutant mice showedsymptoms of premature aging, and increased bone density andbone formation ability (He et al., 2015). p53 was illustrated tosuppress the expression of runt-related transcription factor 2(Runx2) and osterix (Osx), which were identified as mastertranscription factors that control the differentiation of BMSCs intoosteoblasts, and modulate the expression of several osteogenicdifferentiation-related microRNAs (He et al., 2015). Studies havealso confirmed that BMAL1 performed as a putative regulator ofp53 in cancer (Mullenders et al., 2009). However, the associationbetween BMAL1 and p53 in BMSCs is still uncertain, let alone inT2DM.

In this study, we investigated the inhibitory effect of BMAL1 onosteogenic differentiation of BMSCs in type 2 diabetic rat models.And, with the help of p53 gene silencing (shRNA) and the p53inhibitor, pifithrin-α (PFT-α), we identified the key molecule p53,which could mediate the regulatory function of BMAL1 onosteogenic differentiation of BMSCs. Our results indicate that thedecreased expression of BMAL1 inhibits osteogenesis of BMSCsthrough p53 in T2DM.Received 11 February 2020; Accepted 27 May 2020

1Department of Stomatology, Chinese PLA General Hospital, Beijing 100853,China. 2Department of Stomatology, Shandong Provincial Hospital Affiliated toShandong University, Jinan, Shandong 250021, China.

*Author for correspondence (guobin0408@126.com)

X.M., 0000-0003-2020-0520; X.L., 0000-0002-6243-9452; B.G., 0000-0002-8103-1815

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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RESULTSCharacteristics of BMSCsFlow cytometric analysis of phenotypic expression showed thatCD34 and CD45 were negatively expressed, while CD29, CD44 andCD90 were positively expressed in cells from both groups (Fig. 1A).These cells isolated from rats’ femurs were able to differentiate intoosteoblasts or adipocytes after induction of osteogenic or adipogenicdifferentiation, as demonstrated by Alizarin Red staining and Oil RedO staining (Fig. 1B). The results conformed with previous reports(Degen et al., 2016; Kang et al., 2015). Therefore, cells separatedfrom Wistar rats and Goto-Kakizaki (GK) rats showed the typicalcharacteristics of BMSCs.

Overexpression of BMAL1 rescued the impaired osteogenicdifferentiation of BMSCs in T2DMOur previous research has demonstrated that expression of BMAL1was clearly downregulated in diabetic BMSCs (Li et al., 2017).However, the role of BMAL1 in the regulation of osteogenesis shouldbe assayed in a more rigorous and well-structured way. In this study,we reconfirmed that BMAL1 expression was evidently decreased in

diabetic BMSCs, after stable overexpression of BMAL1 in diabeticGK BMSCs with lentiviral infection. When BMAL1 expression wassignificantly upregulated at both mRNA and protein levels, successfulosteogenic differentiation was indicated by the upregulation of theosteogenesis related markers Runx2, Osx and alkaline phosphatase(Alp) (Fig. 2A,B). But, the expression level of osteocalcin (Ocn), amarker of terminal osteogenic differentiation, showed no statisticaldifference between diabetic GK BMSCs (GK) and BMAL1overexpressed diabetic GK BMSCs (GK-BMAL1) (Fig. 2B). Whatis more, BMAL1 overexpression led to a significant upregulation ofosteogenesis related markers Runx2, Alp and Osx at both mRNA andprotein levels, after osteogenic differentiation for 7 days (Fig. 2A,B).After 7 days of osteogenic induction, the Alp activity was significantlylower in diabetic GK BMSCs (GK), but higher in BMAL1overexpressed diabetic GK BMSCs (GK-BMAL1), compared withthe control wild-type (WT) Wistar BMSCs (Fig. 2C). Based on thedata above, it is concluded that BMAL1 has an important effect on theregulation of BMSCs osteogenic differentiation in T2DM. BMAL1overexpression helps rescue the impaired osteogenic differentiationability of diabetic GK BMSCs.

Fig. 1. Characteristics of BMSCs. (A) Flow cytometry analysis of the typical surface markers of BMSCs. (B) BMSCs could differentiate into osteoblasts oradipocytes after osteogenic or adipogenic induction, indicated by Alizarin Red staining and Oil Red O staining. Scale bars: 500 μm.

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Decreased BMAL1 level inhibited proliferation of BMSCsin T2DMSufficient cells are the basis of normal differentiation of BMSCs, soit is plausible that the proliferation and differentiation of BMSCs arecorrelative (Liu and Li, 2010). Results of CCK-8 assay revealed thatdiabetic GK BMSCs had a decreased cell growth rate comparedwith WT Wistar rats. Overexpressing BMAL1 in GK BMSCshelped recover its proliferation, and its proliferation rate was evenhigher than that of WT group (Fig. 3A). Cell apoptosis wasdetermined by the Annexin V-FITC/PI apoptosis detection kit. Asshown in Fig. 3B, overexpression of BMAL1 by lentiviral infectionin diabetic GK BMSCs greatly reduced the percentage of apoptoticcells. Similarly, according to the Alp staining results, GK-BMAL1BMSCs showed the highest Alp activity, while GK BMSCs had thelowest Alp activity (Fig. 3C). Even up to passage 6, when Alpactivity had an age-dependent decrease during osteogenesis, theosteogenic differentiation ability of BMSCs in GK-BMAL1 groupwas still the highest among the three groups (Fig. 3C). These resultsindicate that osteogenic differentiation potential of BMSCs inT2DM is affected by BMAL1 expression pattern, which is alsorelated to the proliferation of BMSCs.

Downregulated BMAL1 promoted p53 expression in T2DMPreviously, BMAL1 was reported to perform as a putative regulatorof p53 in pancreatic cancer (Jiang et al., 2016). Recently, p53 hasbeen reported to inhibit osteogenic differentiation of BMSCs bysuppressing the expression of several osteogenic transcriptionfactors, such as Runx2 and Osx (Oren, 2019). Therefore, we

wondered whether therewas a correlation between BMAL1 and p53in the regulation of osteogenic differentiation of diabetic BMSCs.

Our results validated that the expression patterns of p53 and p21,two p53 signaling pathway-related proteins, were significantlyupregulated at the protein level in diabetic GK BMSCs (Fig. 4A). Inaddition, after BMAL1 was overexpressed in GK BMSCs, theexpressions of p53 and p21 were clearly decreased (Fig. 4A). Samechanges were observed at mRNA level (Fig. 4B). At the same time, thedownregulation of p53 expression level resulted from BMAL1upregulation was also confirmed by immunofluorescence (Fig. 4C).Results above enlighten us that the process of BMAL1 regulatingosteogenic differentiation of diabetic BMSCsmay bemediated by p53.

Inhibition of osteogenic differentiation of BMSCs by BMAL1downregulation in T2DM was mediated by p53To investigate whether the recovery of osteogenic differentiationcapability of BMAL1 overexpressed diabetic BMSCs was mediatedby p53, pifithrin-α (PFT-α), a well-known p53 inhibitor, was used inthe osteogenic differentiation process of GKBMSCs andGK-BMAL1BMSCs. Treatment with PFT-α in both GKBMSCs and GK-BMAL1BMSCs resulted in an increased expression of osteogenesis relatedmarkers Runx2, Osx and Alp at both protein and mRNA levels(Fig. 5A,B). The GK-BMAL1 BMSCs with PFT-α showed a furtherupregulation of osteogenesis markers than GK-BMAL1 BMSCs(Fig. 5A,B). Meanwhile, Alp activity was largely recovered in GK andGK-BMAL1 BMSCs after treatment with PFT-α (Fig. 5C). Besides,no statistically significant changes in BMAL1 expression level wereobserved at mRNA or protein level (Fig. 5A,B). However, reports

Fig. 2. BMAL1 overexpression rescued the impaired osteogenic differentiation of BMSCs in T2DM. (A) Expression of the genes BMAL1, Runx2, Alp,Ocn and Osx, determined by qRT-PCR assay, in WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs after 1 week of osteogenic differentiation.(B) Western blot analysis of BMAL1, Runx2, Alp, Ocn and Osx expression in WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs afterosteogenic differentiation for 7 days. (C) Alkaline phosphatase activity staining of WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs after1 week of osteogenic differentiation. All data are mean±s.e.m. and representative of three independent experiments. β-actin was used as a loading control.n=3. Statistical significance was defined as *P<0.05; **P<0.01. n.s: not significant.

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reminded us of the potential off-target effects of PFT-α (Kanno et al.,2015). In this regard, the same conclusions abovewere reconfirmed byp53 gene silencing (shRNA). As shown in Fig. 6A–C, upregulatedosteogenic markers (Runx2, Osx and Alp) and higher Alp activitywere observed in GK BMSCs and GK-BMAL1 BMSCs, when p53gene expression was silenced. Application of p53 gene silencing alsohad no influence on the BMAL1 expression pattern (Fig. 6A–C). Insummary, the relationship between BMAL1 and p53 was effectivelydemonstrated. Impaired osteogenic differentiation ability of diabetic

GKBMSCs could be partly rescued by p53 shutdown. Interferences ofp53 expression had no effect on BMAL1 expression level at neithermRNA nor protein level. Collectively, these data suggest that the roleof BMAL1 in osteogenic differentiation of BMSCs in T2DM ismediated by p53.

DISCUSSIONLong-term hyperglycemia will lead to the disruption of bonehomeostasis, resulting in impaired bone formation and increased

Fig. 3. Decreased BMAL1 level inhibited proliferation of BMSCs in T2DM. (A) Proliferation rate of WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs as determined by CCK-8 assay, respectively. (B) Cell apoptosis analysis of WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCsby flow cytometry. (C) WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs from passage 4 and passage 6 were cultured in osteogenic inductionmedium for 7 days before being stained for alkaline phosphatase activity. All data are mean±s.e.m. and representative of three independent experiments.n=3. Statistical significance was defined as *P<0.05; #P<0.03.

Fig. 4. Downregulated BMAL1 promoted p53 expression in T2DM. (A) Protein expression of BMAL1, p53 and p21 in WT Wistar BMSCs, diabetic GKBMSCs and GK-BMAL1 BMSCs as determined by western blot. (B) qRT-PCR analysis of BMAL1, p53 and p21 in WT Wistar BMSCs, diabetic GK BMSCsand GK-BMAL1 BMSCs. (C) Immunofluorescence staining of p53 in WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs. Scale bars: 100 µm.All data are mean±s.e.m. and representative of three independent experiments. n=3. β-actin was used as a loading control. Statistical significance wasdefined as *P<0.05; **P<0.01.

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bone resorption (Karner and Long, 2018). Research has shown thatbone metabolic disorders are primarily caused by the defect in boneformation in T2DM (Farr and Khosla, 2016). At present, there arefew studies on the molecular mechanism of osteoporosis in diabetes.Bone homeostasis is mediated by mesenchymal stem cell-derivedosteoblasts and hematopoietic-derived osteoclasts, respectively(Raggatt and Partridge, 2010). BMSCs are multipotentprogenitors capable of forming multiple tissue types and centralmediators of osteogenesis (Bianco et al., 2008). The multipotentialdifferentiation ability of BMSCs was suppressed in T2DM (Aikawaet al., 2016). Therefore, BMSCs could be the ideal target forresearches of osteoporosis in T2DM.It was in the 1960s that circadian clock components such as

BMAL1 were first discovered to impair the mitochondrial functionin pancreatic B cells (Jarrett and Keen, 1969). When circadian clockcomponents such as BMAL1 are disrupted, hypoinsulinism anddiabetes occur (Marcheva et al., 2010). The BMAL1-knockout miceshowed a lack of rhythm in insulin action and disrupted insulinresponsiveness, which were characteristics of T2DM (Sadacca et al.,2011). Decreased BMSC osteogenesis ability was also observed inBMAL1 inhibition mice (Kondratov et al., 2006). The correlationbetween BMAL1 and osteogenic differentiation of BMSCs inT2DM could be an interesting area. Previously, we have reported themechanism underlying the regulation of BMAL1 on osteogenesis ina preliminary way. We verified that decreased BMAL1 upregulatedGSK-3β and inhibited Wnt/β-catenin pathway in type 2 diabetic rat

models, leading to the decreased osteogenic ability of diabetic GKBMSCs (Li et al., 2017). However, it may contain several flaws inreasoning. Here, our results demonstrated again in a relativelyscientific way that upregulated BMAL1 restored osteogenicdifferentiation of BMSCs in T2DM (Fig. 2).

It is commonly known that the osteogenic differentiation of stemcells is controlled by a variety of lineage-specific transcriptionfactors, including Runx2 and Osx (Liu et al., 2016). Several non-coding RNAs and proteins have been reported to regulate theexpression of these transcription factors (Farr and Khosla, 2019).Our results demonstrated that upregulated BMAL1 expressioninfluenced the expression of Runx2, Osx and Alp (Fig. 2). Analysisof data showed that BMAL1 expression had no significant effect onthe expression level of Ocn (Fig. 2A). It is understandable that Ocnis a marker of terminal stages osteogenic differentiation, whichsignifies the conversion of osteoblast to osteocyte and alwaysexpresses after 21 days of osteogenic differentiation inductionin vitro (Tataria et al., 2006). These results were the same as theprevious report that BMAL1−/− mice had a low bone massphenotype (Samsa et al., 2016).

Stem cells would eventually differentiate into specific cells, andweexampled the results of osteogenic differentiation with Alp activitystaining in this study. The differentiation of stem cells must involvedifferences between cell proliferation and apoptosis. According to ourresults, the variations of BMAL1 expression did change the states ofBMSCs’ proliferation and apoptosis (Fig. 3A,B). The effect that

Fig. 5. Inhibition of osteogenic differentiation of BMSCs by BMAL1 downregulation in T2DM was mediated by p53. (A) Western blot analysis ofBMAL1, Runx2, Alp, Osx and p53 expression in WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs with or without 20 μM PFT-α treatmentafter 7 days of osteogenic differentiation. (B) The mRNA expression of Runx2, Osx and Alp in WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1BMSCs were determined by qRT-PCR, following 7 days of osteogenic differentiation, with or without 20 μM PFT-α treatment. (C) Osteogenic differentiationability of WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs with or without 20 μM PFT-α treatment as determined by alkaline phosphataseactivity after osteogenic differentiation for 7 days. Data are from five independent experiments and are expressed as mean±s.e.m.. n=5. Statisticalsignificance was defined as *P<0.05; #P<0.03. n.s: not significant.

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upregulated BMAL1 promoted the osteogenic differentiation ofBMSCs existed continuously until the sixth passage (Fig. 3). Somemechanisms could be explored among this phenomenon other thanGSK-3β pathway, which we have published about previously(Li et al., 2017). In T2DM, p53 modulates blood glucose byinterfering with glycolysis, oxidative phosphorylation and pentosephosphate (Halim et al., 2019). The p53 signaling pathways havebeen widely reported due to its negative regulation of bone formation(Kastenhuber and Lowe, 2017). Moreover, p53 has also beensubstantiated to suppress the expression of Runx2 and Osx in vitrodifferentiation models (Tataria et al., 2006). We wondered whetherBMAL1 could regulate osteogenic differentiation of BMSCs bymodulating the p53 expression in T2DM. As shown in Fig. 4, theupregulated p53 expression was observed in diabetic GK BMSCs,while the expression of p53 significantly decreased at both proteinand mRNA levels after BMAL1 overexpression using lentiviralinfection in diabetic GK BMSCs. The conclusions above were alsoconfirmed by immunofluorescence (Fig. 4C). With the treatment ofPFT-α and p53 gene silencing, decreased p53 level and upregulatedexpressions of osteogenic markers were detected (Figs 5 and 6). Alpstaining analysis verified the same results as above (Figs 5 and 6).Moreover, inhibition of p53 expression pattern could not reduce theBMAL1 expression, which was significantly increased by lentiviralinfection (Figs 5 and 6). All these results demonstrate thatdownregulated BMAL1 inhibits the osteogenic differentiationpotential of BMSCs in T2DM, in a partially p53-dependent

manner. To our knowledge, this is the first report of therelationship between BMAL1 and p53 in the regulation ofosteogenic differentiation of BMSCs in T2DM.

However, what we have done still leaves much to be desired.Firstly, although we want to examine the relationship betweenBMAL1 and p53 in T2DM perfectly, we could not find a relativelyordinary T2DM control cell line, which could be used to performexperiments about knocking-down BMAL1 expression to observethe change of p53. The particularity of the pathological environmentof T2DM always results in a significantly reduced BMAL1expression (Marcheva et al., 2010). As for WT Wistar rats, theycannot simulate the microenvironment of T2DM, and we do notknow whether upregulation of BMAL1 in their BMSCs wouldmake the cells abnormal, such as the occurrence of biorhythmdisorder. Therefore, in this study, we only referred BMSCs of WTWistar rats as a datum dimension. Secondly, considering theparticularity of p53, deeper research need to be done about thespecific molecular mechanisms between BMAL1 and p53. Reportshave already shown that the evolutionarily conserved p53 responseelement overlaps with the E-BOX element critical for BMAL1/CLOCK binding, which suggests a specific location of interactionbetween p53 and BMAL1 (Miki et al., 2013). Thirdly, we noticedthe p53 shutdown could not fully restore the impaired osteogenesisas the BMAL1 overexpression by lentiviral infection did (Figs 5 and 6).There must be some other molecules involved in the regulation ofosteogenesis of BMSCs by BMAL1.

Fig. 6. p53 gene silencing confirmed the regulation role of BMAL1 on p53 in diabetic BMSCs’ osteogenic differentiation ability. (A) Proteinexpression of BMAL1, Runx2, Alp, Osx and p53 in WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs with or without p53 gene silencing asdetermined by western blot after osteogenic differentiation for 7 days. (B) The mRNA expression of Runx2, Osx and Alp in WT Wistar BMSCs, diabetic GKBMSCs and GK-BMAL1 BMSCs were determined by qRT-PCR, following 7 days of osteogenic differentiation, with or without p53 gene silencing.(C) Osteogenic differentiation ability of WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs with or without p53 gene silencing asdetermined by alkaline phosphatase activity after 7 days of osteogenic differentiation. Data are from five independent experiments and are expressed asmean±s.e.m. n=5. Statistical significance was defined as *P<0.05; #P<0.03. n.s: not significant.

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In conclusion, our research reveals for the first time thatdownregulated BMAL1 inhibits osteogenesis of BMSCs throughp53 in T2DM. The results presented in our study suggest a potentialtherapeutic strategy in clinical practice to treat osteoporosis inT2DM.

MATERIALS AND METHODSAnimal careTen 8-week-old male GK rats, a well-known spontaneous model of T2DM(D’Souza et al., 2011), and ten age-sex-matched Wistar rats, which have thesame genetic background as GK rats, were obtained from KavensExperimental Animals Co., Ltd. (Changzhou, China). All rats were keptin the Chinese PLA General Hospital Research Institute mouse facility at22°C on a 12 h light-dark cycle with free access to water and food for1 month. All experiments were conducted in accordance with the guidelineslaid down by the National Institute of Health (NIH), USA, and approved bythe Institutional Animal Care and Use Committee of Chinese PLA GeneralHospital.

Cell cultureAfter regular detection of body weight, fasting blood glucose level, fastinginsulin level and oral glucose tolerance tests (OGTT), eight 12-week-old malestable T2DM GK rats were selected. While ten age–sex-matched Wistarrats were used as the control group. Then all rats were euthanized viaintraperitoneal injection of excess pentobarbital at the same time(100 mg kg−1). Under aseptic conditions, we isolated BMSCs from femursby cutting the ends of them and flushing the marrow with 5 ml α-minimumessential medium (α-MEM; Hyclone, UT, USA) using a syringe fitted with a25-gauge needle. All cells were cultured in α-MEM supplemented with 10%fetal bovine serum (FBS; GIBCO, NY, USA) and 100 U ml−1 penicillin-streptomycin (Hyclone) at 37°C with 5% CO2 in a completely humidifiedatmosphere. The medium was replaced every 3 days. Cells were detachedusing 0.25% trypsin and ethylenediaminetetraacetic acid (Trypsin-EDTA;Hyclone) when they reached 80–90% confluence. All BMSCs used in thisstudy were passage 3 BMSCs except special situations with correspondingdescriptions.

Identification of BMSCsBMSCs from Wistar rats and GK rats were incubated with fluoresceinisothiocyanate (FITC)-conjugated monoclonal antibodies for CD29, CD34,CD44, CD45 and CD90 (Abcam, Cambridge, UK) to investigate the clusterof differentiation (CD) phenotype of BMSCs. All cells were subjected toflow cytometric analysis using a BD FACS Calibur flow cytometry (BDBiosciences, NJ, USA).

Induction of osteogenic differentiationBefore the mediumwas replaced with osteogenic induction medium, BMSCswere cultured in the basal medium until they reached approximately 80%confluence. To induce osteogenic differentiation, BMSCs were cultured inosteogenic induction medium with the following compositions: α-MEMsupplemented with 10% FBS, 100 U ml−1 penicillin-streptomycin, 1 mMdexamethasone, 1 M β-glycerophosphate and 10 mMascorbic acid (Solarbio,Beijing, China). The osteogenic inductionmediumwas replaced every 3 days.Alp activity assay of the differentiated cells was conducted with a BCIP/NBTAlkaline Phosphatase Color Development Kit (Beyotime, Shanghai, China)according to the manufacturer’s instructions. Alizarin Red staining was usedto assay the deposition of calcium into the extracellular matrix according tostandard protocols.

Induction of adipogenic differentiationBMSCs from each group were maintained in six-well plates at a density of5×105 cells well−1. After they reached about 80% confluence, we replaced thebasal medium with adipogenic induction medium, composed of α-MEMsupplementedwith 0.5 mMmethylisobutylxanthine, 0.5 mMhydrocortisone,60 mM indomethacin, 10% FBS, and 100 U ml−1 penicillin-streptomycin.The adipogenic induction medium was replaced every 3 days. All BMSCswere incubated for 3 weeks in adipogenic induction medium, and then Oil

Red O staining was performed to assay intracellular lipid accumulationaccording to standard protocols.

Infection of BMAL1 overexpression lentiviral vectorDiabetic GK BMSCs were cultured in six-well plates, and lentivirusinfection was performed after cells reached 80% confluence. BMAL1overexpression lentiviral vector was purchased from Inovogen Tech. Co.,Ltd. (Beijing, China; Gene ID: 29657) and diabetic GK BMSCs wereinfected with lentivirus at a multiplicity of infection of 20 according to thecorresponding manufacturer’s instructions (Inovogen Tech. Co., Ltd.,Beijing, China). Diabetic GK BMSCs in the experimental group (GK-BMAL1 BMSCs) were infected with BMAL1 overexpression lentiviralvector, BMSCs in positive control group were infected using enhancedgreen fluorescent protein (EGFP)-expressing lentiviral vector, and BMSCswithout any infection were defined as negative control group.

Cell counting kit-8 (CCK-8) assayBMSCs were seeded in 96-well plates at a density of 5×104 cells well−1 andthen cultured in the incubator at 37°C with 5% CO2. 10 µl CCK-8 solution(Solarbio, Beijing, China) was added to each well at the indicated timepoints, and absorbance at 450 nm was measured by enzyme labelinginstrument after 4 h of incubation. The OD450 value was measured from thefirst day to the seventh day in each well per group. To plot the growth curve,the cultured time was considered as the abscissa and the OD450 value wasconsidered as the ordinate.

Flow cytometry analysis of cell apoptosisBMSCs fromWTWistar group, diabetic GK group and GK-BMAL1 groupwere cultured for 5 days and then the cell apoptosis assay was performedwith an Annexin V-FITC/PI Apoptosis Detection Kit (Solarbio, Beijing,China) according to the manufacturer’s instructions using a BD FACSCalibur flow cytometry.

Quantitative real-time polymerase chain reaction (qRT-PCR)Total RNA of BMSCs was extracted with TrIquick Reagent (Solarbio,Beijing, China). The concentration of total RNA was quantified using aspectrophotometer at the absorbance of 260 nm and 280 nm. A260/A280ratios of all the samples were between 1.9 to 2.0, indicating high purity.Total RNA from each sample was subjected to reverse transcription tocDNA with a PrimeScript™ RT Reagent Kit (TaKaRa, Japan). Weconducted qRT-PCR on a Bio-Rad CFX96 Touch (Bio-Rad, Hercules,California, USA) with 20 µl qRT-PCRmix, which contains 10 µl TransStartTop Green qPCR SuperMix (TransGen Biotech, Beijing, China), 0.8 µM ofeach primer, 1 µl passive reference Dye-II, 1 µl cDNA and distilled water(containing diethylpyrocarbonate). The thermal cycling conditionswere 95°Cfor 30 s, followed by 45 cycles of 95°C for 5 s, 60°C for 30 s, and incrementsof 0.5°C for 5 s from 65°C to 95°C for the melting curve. β-actin was used asan internal control. Primer sequences used for qRT-PCR are listed inTable S1.

Western blot analysisTotal proteins were extracted from 85–90% confluent BMSCs using a TotalProtein Extraction Kit (Solarbio, Beijing, China), and the proteinconcentration was determined with a BCA protein assay kit (Solarbio,Beijing, China) according to the manufacturer’s instructions. All proteinsamples were separated by 10% sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred to Polyvinylidene fluoride(PVDF) membranes (Bio-Rad). All membranes were blocked by 5% non-fatmilk for 1 h and incubated overnight at 4°C with the following primaryantibodies (all from Abcam): β-actin (#ab8227, 1:2000 dilution), BMAL1(#ab3350, 1:1500 dilution), p53 (#ab26, 1:1500 dilution), p21 (#ab80633,1:2000 dilution), Runx2 (#ab23981, 1:1000 dilution), Osx (#ab22552,1:1000 dilution), Ocn (#ab134220, 1:1000 dilution) and Alp (#ab83259,1:1000 dilution). After washing with TBST three times, the membraneswere incubated with horseradish peroxidase (HRP)-conjugated anti-rabbitIgG antibody (1:2500, Solarbio, Beijing, China) and anti-mouse IgGantibody (1:2500, Solarbio, Beijing, China) for 1 h. β-actin was used as an

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internal control. Finally, the ECLWestern Blotting Detection Kit (Solarbio,Beijing, China) and Western-Light Chemiluminescent Detection System(GE Healthcare, NY, USA) were used to acquire the protein bands.

ImmunofluorescenceBMSCs were fixed with 4% paraformaldehyde for 20 min, permeabilizedwith 0.1% Triton X-100 for 5 min, and then blocked in 5% BSA for 1 h.Samples were incubated with a p53 polyclonal antibody (#ab26, Abcam) ata dilution of 1:100 at 4°C overnight. Subsequently, they were washed forthree times with phosphate buffered solution (PBS; Solarbio, Beijing,China) and incubated with the corresponding secondary antibody in 5%BSA for 1 h. Then the nuclei were labeled with 1 mg ml−1 DAPI for 15 min.Cells were visualized by a confocal laser scanning microscope after theywere washed with PBS for three times.

Inhibition and knockdown of p53Inhibition of p53 was conducted by adding the famous p53 inhibitorpifithrin-α (PFT-α; Selleck, Houston, TX, USA) (20 μM) to the osteogenicinduction medium described above and renewing the medium every 3 days.The lentiviral vector carrying specific p53-targeting shRNA was obtainedfrom GenePharma Co., Ltd. (Shanghai, China; Gene ID: 24842). BMSCswere infected with the lentiviral vector at a multiplicity of infection of 100according to the protocols described above. The sequence of p53 shRNAwas 5′-GAG AAT ATT TCA CCC TTA A-3′ (Zheng et al., 2016).

Statistical analysisData were expressed as mean±s.e.m. and analyzed with SPSS 19.0 software(SPSS Inc., IBM, Chicago, USA). All experiments were performed at leastin triplicate. According to the study design, an unpaired Student’s t-test orANOVAwith post hoc test was used to determine the statistical significance.The threshold for statistical significance was set at *P<0.05; #P<0.03;**P<0.01.

AcknowledgementsWe thank all the members of our group for helpful discussions and assistance duringour research.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsMethodology: X.M., X.L., W.H., S.H.; Software: S.H., Y.Y.; Formal analysis: Y.Y.,L.G.; Data curation: W.H.; Writing - original draft: X.M.; Writing - review & editing:B.G.; Supervision: B.G.; Funding acquisition: B.G.

FundingThis research was funded by the National Natural Science Foundation of China[grant number 81470754] and the Natural Science Foundation of ShandongProvince [grant number ZR2019PH015].

Supplementary informationSupplementary information available online athttps://bio.biologists.org/lookup/doi/10.1242/bio.051482.supplemental

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