A Novel Role for Bcl2l13 in Promoting Beige Adipocyte Biogenesis

Biochemical and Biophysical Research Communications xxx (2018) 1e7

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Biochemical and Biophysical Research Communications

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A novel role for Bcl2l13 in promoting beige adipocyte biogenesis

Liping Ju 1, Shuqin Chen 1, Miriayi Alimujiang, Ningning Bai, Han Yan, Qichen Fang, ** * Junfeng Han, Xiaojing Ma , Ying Yang , Weiping Jia

Shanghai Diabetes Institute, Shanghai Clinical Center for Diabetes, Department of Endocrinology and Metabolism, Shanghai JiaoTong University Afﬁliated Sixth People's Hospital, Shanghai, 200233, China article info abstract

Article history: Bcl2l13 is a member of the Bcl-2 family that has been found to play a central role in regulating apoptosis. Received 30 September 2018 Recently Bcl2l13 has been reported to induce mitophagy as a functional mammalian homolog of Atg32. Accepted 5 October 2018 However, the role of Bcl2l13 in adipose tissue has not been investigated yet. In the present study, we Available online xxx found that Bcl2l13 expression was increased in white adipose tissue browning process stimulated by cold exposure or b3-adrenergic agonist CL-316,243 in vivo as well as during brown adipocytes differentiation Keywords: in vitro. Moreover, Bcl2l13 disruption dramatically inhibited the browning program of preadipocytes, Bcl2l13 evidenced by reduced Prdm16, Ucp1, Dio2 and Adrb3 expression. Our ﬁndings revealed that the inhi- Beige adipocyte Mitochondria biogenesis bition effect of Bcl2l13 disruption on browning program may be independent of altering autophagy Mitophagy activity, but through regulating mitochondrial dynamic and biogenesis, supported by decreased mitochondrial ﬁssion/fussion genes, PGC-1a and mitochondrial respiratory chain complexes expression. Taken together, our study uncovered a novel function of Bcl2l13 in adipocytes differentiation and promoting browning program. © 2018 Published by Elsevier Inc.

1. Introduction weight [2]. Mitochondria are essential organelles that produce most of the energy and have crucial roles in the thermogenic Brown adipose tissue (BAT) is characterized by breaking down function of beige/brown adipocytes [3]. Therefore, it is important to lipids to generate heat for defending against hypothermia, which is understand the mechanisms underlying the regulation of mito- mediated by uncoupling protein 1 (Ucp1), a mitochondrial protein chondrial homeostasis in brown/beige adipocytes. that uncouples electron transport from ATP production [1]. Recent Bcl2l13, also termed Bcl-rambo, is localized to outer mitochon- studies identified a distinct type of thermogenic fat cells expressing drial membrane and ubiquitously expressed in human cells [4,5]. Ucp1, called beige or brite adipocytes, in white adipose tissue Bcl2l13 is a member of the Bcl-2 family containing 4 conserved N- (WAT), which acquire thermogenic, fat-burning properties of terminal BH domains (BH1-4), and 2 WXXL/I motifs. The BH do- brown adipocytes. Inducing browning of WAT is becoming a target mains are involved in Bcl2l13-induced mitochondrial fragmenta- of obesity treatment for its close association with reduced body tion, whereas the WXXI motif, an LC3 interacting region, is important for mitophagy [6]. Bcl2l13 was previously shown induced apoptosis in various cell lines [7,8]. However, Bcl2l13 has * Corresponding author. Shanghai Key Laboratory of Diabetes, Shanghai Institute also been reported as an antiapoptotic protein in part by inhibiting for Diabetes, Shanghai Clinical Medical Centre of Diabetes, Shanghai Key Clinical proapoptotic ceramide synthases 2 and 6 activity [9]. In 2015, Centre of Metabolic Diseases, Department of Endocrinology and Metabolism, Bcl2l13 was first reported as a functional mammalian homolog of fi Shanghai JiaoTong University Af liated Sixth People's Hospital, 600 Yishan Road, Atg32. Atg32 is essential for mitophagy in yeast, and functions as a Shanghai, 200233, China. ** Corresponding author. Shanghai Key Laboratory of Diabetes, Shanghai Institute receptor of mitophagy through its interaction with Atg8 and Atg11 for Diabetes, Shanghai Clinical Medical Centre of Diabetes, Shanghai Key Clinical [10,11]. The multi-directional biological action of Bcl2l13 extending Centre of Metabolic Diseases, Department of Endocrinology and Metabolism, apoptosis to mitophagy suggests that Bcl2l13 may be a key regu- Shanghai JiaoTong University Affiliated Sixth People's Hospital, 600 Yishan Road, lator of mitochondrial remodeling in response cell conversion. Shanghai, 200233, China. Recently, mitochondrial biogenesis has been demonstrated to be E-mail addresses: [email protected] (X. Ma), [email protected] (Y. Yang). required in the process of brown/beige adipocyte differentiation 1 These authors contributed equally to this article. and thermogenesis [12], however, no reference is available https://doi.org/10.1016/j.bbrc.2018.10.034 0006-291X/© 2018 Published by Elsevier Inc.

Please cite this article in press as: L. Ju, et al., A novel role for Bcl2l13 in promoting beige adipocyte biogenesis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.10.034 2 L. Ju et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7 regarding the expression and potential function for Bcl2l13 in 2.6. Statistical analysis adipocytes. In the present study, we aimed to clarify the direct role and Data are presented as means ± standard error of mean (SEM) for possible mechanism of Bcl2l13 in the browning program of white three independent experiments. Statistical differences were adipocytes by using diverse browning models and in vitro experi- calculated by two-tailed Student's t-test or one-way ANOVA. Sig- ments. We found that Bcl2l13 disruption dramatically inhibited the niﬁcance was shown as *p < 0.05, **p < 0.01 or ***p < 0.001. browning program of preadipocytes, showing reduced Ucp1 expression and mitochondrial proteins, and the effects might be 3. Results achieved at least partially through impairing mitochondrial ﬁ biogenesis. These ndings provide more extensive information 3.1. The expression and modulation of Bcl2l13 in adipocytes regarding the role of Bcl2l13 in the browning process. It has been reported that Bcl2l13 is an outer mitochondrial 2. Methods membrane protein. However, little is known about its functions, especially its physiological roles in metabolism. Thus, the expres- 2.1. Mice sion of Bcl2l13 was measured in iWAT, epididymal WAT (EAT), BAT, Soleus muscle (SM) and Gastrocnemius muscle (GM). We found Six-week-old male C57BL/6 mice were purchased from Bcl2l13 mRNA was most highly expressed in BAT (Fig. 1A), and Shanghai SLAC Laboratory Animal Company and maintained on a protein level of Bcl2l13 were mainly expressed in both BAT and SM standard chow diet with a 12 h light/dark cycle. The mice were (Fig. 1B). Next, we detected the Bcl2l13 expression during the dif- exposed to cold (4 C) or implanted subcutaneously with mini- ferentiation of beige and white adipocyte. Our results showed that osmotic pumps (Alzet 2001) perfusing 1.0 mL/h of CL-316,243 Bcl2l13 expression was continuously increased and in parallel with (1 mg/kg, Sigma, C5976) for one week. All animal procedures the induction of Ucp1 and PPARg during both beige and white were approved by the Animal Care Committee of Shanghai Jiaotong adipocyte differentiation, respectively (Fig. 1CeF). Next we inves- University School of Medicine. tigate the regulation of Bcl2l13 expression by rosiglitazone, an antidiabetic drug inducing adipocyte remodeling, in 3T3-L1 white 2.2. Stromal Vascular Fraction (SVF) Isolation and Beige Adipocyte adipocytes, and found rosiglitazone could upregulate the Bcl2l13 differentiation expression in dose and time dependent manner (Fig. 1GeI).

SVFs were isolated from the inguinal WAT (iWAT) of C57BL/6 3.2. Bcl2l13 expression is induced in Browning of white adipose male mice as previously described [13]. For the induction of beige tissues in mice adipocytes, SVF cells were grown to confluence, then induced by incubation in 10% FBS-DMEM medium supplemented with 0.5 mM We further detected Bcl2l13 expression during the induction of isobutylmethylxanthine (Sigma, I7018), 1 mM dexamethasone the white adipose tissue browning in vivo. We induced browning of (Sigma, D4902), 5 mg/ml insulin (Lily, HI0240), 50 nM T3 (Sigma, white adipose tissue by cold challenge (4 C) and b3-adrenergic T2877) and 5 mM rosiglitazone (Sigma, R2408) for two days, and agonist CL-316,243 in mice. Our results showed mRNA and pro- subsequently in medium with insulin, T3, and rosiglitazone for tein level of Bcl2l13 increased significantly, in parallel with the another four days. induction of Ucp1 in iWAT of cold challenged mice (Fig. 2AeC). Bcl2l13 expression was upregulated in iWAT of mice stimulated by 2.3. Lentivirus transduction CL-316,243 (Fig. 2D and E). We also searched for the data available online from GSE86338 for Bcl2l13 expression profile of BAT under A lentivirus containing the Bcl2l13 expression vector were cold stimulation, and found Bcl2l13 transcriptionally upregulated purchased from Shanghai GeneChem Corporation. The cells were under room temperature and cold compared with thermoneu- infected with the lentivirus to knockdown Bcl2l13 4e6 h after trality (Fig. 2F). These results suggested that the Bcl2l13 may have seeding. potential roles in WAT browning and BAT activation.

2.4. RNA isolation and real-time PCR analysis 3.3. Knockdown of Bcl2l13 in beige adipocytes represses adipogenesis and thermogenic programing Total RNA was extracted using the Trizol reagent (Invitrogen, 15596018). Reverse transcription was performed using a Prime- To pinpoint the potential roles of Bcl2l13 in WAT browning, we ® Script RT reagent Kit (Takara, RR047B). qRT-PCR analysis was examined the effects of Bcl2l13 in beige adipocytes differentiated carried out using SYBR Premix Ex Taq (Takara, RR820A) in a from SVFs of iWAT in vitro. We used lentivirus expressing shRNAs LightCycler480 PCR system (Roche, Germany). Sequences of the against Bcl2l13 to efﬁciently knockdown its expression in SVFs primers are listed in Supplementary Table 1. during the beige adipocyte differentiation (Fig. 3A and B). Bcl2l13 reduction led to less lipid accumulation in differentiated beige 2.5. Western blot analysis and antibodies adipocyte as showed by cell morphology (Fig. 3C). However, adipocyte differentiation related transcription factors PPARg and C/ Western blotting (WB) was performed as previously described EBPa were downregulated to a certain extend by Bcl2l13 knock- [13]. Proteins were assessed with the following antibodies: Actin down at the late stage of differentiation (Fig. 3D). As a result, the (Cell Signaling Technology, 4970), Hsp90 (Cell Signaling Technol- expression of adipocyte marker Fabp4 was reduced to 0.64 of ogy, 4877), Bcl2l13 (Proteintech, 16612-1-AP), Ucp1 (Abcam, controls’ by Bcl2l13 knockdown. Remarkably, the browning related ab10983), PPARg (Santa Cruz, sc-6284), PGC-1a (Santa Cruz, sc- transcription factors Prdm16 and PGC-1a were signiﬁcantly 517380), Perilipin (Cell Signaling Technology, 9349), p62/SQSTM1 reduced by Bcl2l13 repression at the late stage of beige adipocytes (Cell Signaling Technology, 5114), LC3I/II (Cell Signaling Technology, differentiation (Fig. 3E, F). Accordingly, brown adipocyte markers 12741) and total OXPHOS rodent WB cocktail (Abcam, ab110413). Ucp1, Dio2, Adrb3 mRNA were also reduced (Fig. 3G-I).

Fig. 1. The expression and modulation of Bcl2l13 in Adipocytes.(AeB) inguinal WAT (iWAT), epididymal WAT (EAT), BAT, Soleus muscle (SM) and Gastrocnemius muscle (GM) were extracted from 8-week-old male mice. Relative mRNA levels (A) and protein levels (B) of Bcl2l13 in these tissues. (CeD) SVFs from iWAT were induced differentiate into beige adipocytes. Relative mRNA levels (C) and protein levels (D) of Bcl2l13 at different time points during beige adipocyte differentiation. (EeF) Relative mRNA levels (E) and protein levels (F) of Bcl2l13 at different time points during 3T3-L1 adipocytes differentiation. Relative mRNA levels of Bcl2l13 was detected by qRT-PCR in 3T3-L1 adipocytes, which were treated with 2 mM rosiglitazone (Rosi) for the indicated time (G) and Rosi at the indicated concentrations for 24 h (H). Bcl2l13 content was immunobloted after rosiglitazone (Rosi) treatment (2 mM) for 96 h in 3T3-L1 adipocytes (I). Data were presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

3.4. Bcl2l13 induces thermogenic programing by regulating degradation activity in adipocytes. As shown in Fig. 4A, perilipin 1 mitochondrial dynamic and biogenesis protein expression was increased by cold stress by time dependently. These results showed that the autophagy was repressed Next, we explored the possible mechanism underlying the during cold, which was inconsistent with the increased Bcl2l13 browning effect of Bcl2l13. As Bcl2l13 was identified as a expression. mammalian homolog of the yeast mitophagy receptor Atg32, first We further detected the autophagic flux during the browning we investigate the autophagic activity when the Bcl2l13 expression program in vitro, as shown in Fig. 4D, LC3-II expression was was upregulated under cold stress. Cold exposure decreased LC3-II significantly decreased as beige adipocyte differentiating in the expression in iWAT compared with control group (Fig. 4A), sug- absence or presence of chloroquine (CQ, a lysosomal inhibitor), gesting repression of autophagy. SQSTM1/p62 as one of autophagy reflecting repression of autophagic flux in the browning program. substrates, is widely used as an indicator of autophagy flux in However, after Bcl2l13 knockdown the autopagic activity was not various tissues and cells [14]. However, we found the transcription altered during beige adipocytes differentiation (Fig. 4E). Our results level of p62 was significantly upregulated in iWAT of mice under showed that the browning program was accompanied by decreased 24 h cold stimulation (Fig. 4C), suggesting p62 protein content autophagy activity and increasing levels of Bcl2l13, which sug- could not reflect autophagic flux under cold stimulation. Perilipin 1, gested that Bcl2l13 may not exert its role by regulating autophagic which was reported to be degraded through lysosomal pathway activity. [15] and not transcriptionally upregulated by cold stimulation To clarify how Bcl2l13 contributes to the browning program, we (Fig. 4B), was used to evaluate the autophagy lysosomal next examined the effect of Bcl2l13 on mitochondrial dynamic

Fig. 2. Bcl2l13 expression is induced in Browning of white adipose tissues in Mice (AeC) Six-week-old male C57BL/6 mice were exposed to cold (4 C) in an individual cage, and mice housed at room temperature served as controls. Relative mRNA levels and protein levels of Ucp1 and Bcl2l13 in iWAT. (DeE) Six-week-old male C57BL/6 mice were implanted subcutaneously with mini-osmotic pumps perfusing CL-316,243, and mice sham operated served as controls. Relative mRNA levels (D) and protein levels (E) of Ucp1 and Bcl2l13 in iWAT. (F) the mRNA expression of Bcl2l13 under different temperature from online data (GSE86338). Data were presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. related genes in beige adipocytes. Compared to control group, PGC-1a levels. In accordance with PGC-1a expression, a transcrip- mitochondrial fusion genes, Mfn2, Opa1 and mitochondrial fission tional co-regulator of mitochondrial biogenesis [18], our results genes Drp1, as well as mitochondrial biogenesis genes Tfam, were further revealed that Bcl2l13 repression reduced mitochondrial significantly inhibited by Bcl2l13 knockdown at the 4d of differ- respiratory complexes expression. Our findings also demonstrated entiation (Fig. 4F). Furthermore, a significant decrease of mito- that Bcl2l13 transcription was upregulated by PPARg agonist rosi- chondrial complexes I, II, III and V was observed in the browning glitazone, suggesting Bcl2l13 might be a key effector of PPARg program of Bcl2l13 knockdown (Fig. 4G). These results suggest that mediated adipocyte remodeling. Bcl2l13 is implicated in the regulation of mitochondrial dynamic We further explored the mechanism of these observation. It was and biogenesis. reported that Bcl2l13 is a mammalian homolog of the yeast mitophagy receptor. Bcl2l13 overexpression was sufficient to in- crease autophagic flux, as indicated by the increased LC3-II level, 4. Discussion which mainly induce autophagic mitochondria degradation (mitophagy) [6]. However, in this study LC3-II expression in iWAT In this study, we showed that Bcl2l13 expression was induced in was reduced under cold challenge, indicating repression of auto- the browning process of WATs in mice. The present studies on phagy. Besides, LC3-II levels also reduced as beige adipocytes Bcl2l13 function are limited, and the role of Bcl2l13 on apoptosis is differentiating in the absence or presence of CQ, suggesting the still controversial [16]. The only study associating Bcl2l13 with reduced autophagic flux in browning process. Cairo et al. also found fi metabolism indicated that Bcl2l13 has a speci c role in chronic that autophagy was repressed by cold stimulation, supported by exercise that requires constant maintenance of mitochondrial decreased LC3-II and increased p62 expression [19]. We found p62 quality in skeleton muscle [17]. However, it has never been re- was transcriptionally upregulated in iWAT in response to cold fi ported the function of Bcl2l13 in adipose tissue. We rst demon- challenge, suggesting p62 may not be suitable for autophagy sub- strated that Bcl2l13 is highly expressed in brown adipose tissue and strates under this condition. Perilipin 1, degraded by lysosomal soleus muscle. Consistently, Bcl2l13 expression was induced during pathway [15], was increased in protein level without being tran- the browning process of iWAT in cold and b3-adrenergic agonist- scriptionally upregulated by time dependently. As the Bcl2l13 stimulated mice, and Bcl2l13 expression was also increased dur- expression was contradictory to autophgic activity in browning ing the beige/brown adipocyte differentiation, suggesting regula- program in this study, it was difficult to evaluate the Bcl2l13 tion of Bcl2l13 expression is a key thermoregulatory mechanism. mediated mitophagy during this process. This observation prompted us to study the function of Bcl2l13 on It was reported that another Bcl-2 family protein BNIP3, serving the browning process [11]. Bcl2l13 knockdown led to less lipid as mitophagy receptor, contributes to mitochondrial bioenergetics accumulation after beige adipocyte differentiation, yet the and remodeling of adipocytes without autophagy induction dramatically inhibited browning program after Bcl2l13 knockdown [20,21]. Mitophagy is a relatively minute part of global autophagy, was more significant in vitro, reflected by downregulated UCP1 and

Fig. 3. Knockdown of Bcl2l13 in beige adipocytes represses adipogenesis and thermogenic programing (AeB) SVFs of iWAT were infected with Bcl2l13 knockdown lentiviruses and differentiated into beige adipocytes. Relative protein (A) and mRNA levels (B) of Bcl2l13 in the beige adipocyte program. (C) Cell morphology was observed by light microscopy after beige adipocyte differentiation. Original magnification, Â 400. (D) Relative mRNA expression of PPARg, C/EBPa, Fabp4 at the late stage of beige adipocyte differentiation from iWAT SVFs. (EeI) Relative mRNA expression of brown adipocyte marker genes in beige adipocytes induced from mouse iWAT SVFs for six days with the infection of mouse Bcl2l13 lentiviral shRNA. (J) The protein levels of PGC1a in beige adipocytes infected with mouse Bcl2l13 lentiviral shRNA. Data were presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. and mitophagic activity may not be reflected by markers of global interrupting the fission/fusion and biogenesis of mitochondria, autophagy [22,23], so it is important to develop an ideal approach which displays functional similarity to the BNIP3 in adipocytes. to evaluate the mitophagy distinguished from global autophagy. In summary, our study indicates the key role of Bcl2l13 in Mitochondrial fission/fusion and mitophagy governs mitochondrial mitochondrial function and thermogenesis in browning of white turnover, which is important for mitochondrial bioenergetics on adipose tissue, suggesting Bcl2l13 may be a potential therapeutic the differentiation process of brown/beige adipocytes [24,25]. We target in obesity. found Bcl2l13 knockdown repressed browning program through

Fig. 4. Bcl2l13 induces thermogenic programing by regulating mitochondrial dynamic and biogenesis. (A) Six-week-old male C57BL/6 mice were exposed to cold (4 C) in an individual cage, and mice housed at room temperature served as the controls. Protein levels of LC3-II and Perilipin 1 in iWAT. (BeC) The mRNA expression of Perilipin 1 (Plin1) and p62 under cold challenge. (D) SVFs from iWAT were differentiated into beige adipocytes. LC3-II protein levels at different time points during beige adipocyte differentiation. (EeG) SVFs of iWAT were infected with Bcl2l13 knockdown lentiviruses and differentiated into beige adipocytes. (E) Protein levels of LC3 during beige adipocyte differentiation. (F) Relative mRNA levels of Mfn1, Mfn2, Opa1, Drp1, Fis1, Tfam, Nrf1 and Nrf2. (G) Protein levels of mitochondrial OXPHOS complexes (CI, CII, CIII, CV) during beige adipocyte differentiation. Data were presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

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