Elsevier

Metabolism

Volume 107, June 2020, 154228
Metabolism

PINK1–PRKN mitophagy suppression by mangiferin promotes a brown-fat-phenotype via PKA-p38 MAPK signalling in murine C3H10T1/2 mesenchymal stem cells

https://doi.org/10.1016/j.metabol.2020.154228Get rights and content

Highlights

  • MF promotes brown and beige signatures in both murine C3H10T1/2 MSCs and human ADMSCs.

  • MF retains thermogenic capacity by down-regulating PINK1-PRKN mediated mitophagy.

  • MF improves mitochondrial mass by balancing of mitofission and fission.

  • MF improves lipolysis, mitochondrial respiration, β-oxidation, and biogenesis which increases Ucp1 expression.

  • MF activates β3-AR dependent PKA-p38 MAPK-CREB signaling during non-shivering thermogenesis and mitophagy suppression.

Abstract

Objective

Mangiferin (MF), a xanthonoid derived from Mangifera indica, has shown therapeutic effects on various human diseases including cancer, diabetes, and obesity. Nonetheless, the influence of MF on non-shivering thermogenesis and its underlying mechanism in browning remains unclear. Here, our aim was to investigate the effects of MF on browning and its molecular mechanisms in murine C3H10T1/2 mesenchymal stem cells (MSCs).

Materials/methods

To determine the function of MF on browning, murine C3H10T1/2 MSCs were treated with MF in an adipogenic differentiation cocktail and the thermogenic and correlated metabolic responses were assessed using MF-mediated signalling. Human adipose-derived MSCs were differentiated and treated with MF to confirm its role in thermogenic induction.

Results

MF treatment induced the expression of a brown-fat signature, UCP1, and reduced triglyceride (TG) in C3H10T1/2 MSCs. MF also induced the expression of major thermogenesis regulators: PGC1α, PRDM16, and PPARγ and up-regulated the expression of beiging markers CD137, HSPB7, TBX1, and COX2 in both murine C3H10T1/2 MSCs and human adipose-derived mesenchymal stem cells (hADMSC). We also observed that MF treatment increased the mitochondrial DNA and improved mitochondrial homeostasis by regulating mitofission–fusion plasticity via suppressing PINK1–PRKN-mediated mitophagy. Furthermore, MF treatment improved mitochondrial respiratory function by increasing mitochondrial oxygen consumption and expression of oxidative-phosphorylation (OXPHOS)-related proteins. Chemical-inhibition and gene knockdown experiments revealed that β3-AR-dependent PKA-p38 MAPK-CREB signalling is crucial for MF-mediated brown-fat formation via suppression of mitophagy in C3H10T1/2 MSCs.

Conclusions

MF promotes the brown adipocyte phenotype by suppressing mitophagy, which is regulated by PKA-p38MAPK-CREB signalling in C3H10T1/2 MSCs. Thus, we propose that MF may be a good browning inducer that can ameliorate obesity.

Introduction

There are two types of fat tissues in adult humans: one is an energy reservoir, white adipose tissue (WAT), and the other is an energy dissipator, brown adipose tissue (BAT). An excess of lipid accumulation in WAT causes obesity that can lead to other health problems such as type II diabetes, cardiovascular diseases, and cancer [1]. BAT burns lipids for heat in a process called non-shivering thermogenesis, which is currently believed to ameliorate obesity. Beige adipose tissue was recently defined as a new adipose tissue type located in subcutaneous WAT that is derived from a distinct cellular lineage in rodents [2]. Brown and beige adipocytes contain multi-locular lipid droplets and strongly express mitochondrial uncoupling protein 1 (UCP1). External cues such as cold exposure and treatment of β3-adrenergic receptor (β3-AR) engagement and PPARγ agonists in WAT can convert the tissue from white to brown adipocyte (browning) and promote white-to-beige transition (beiging), which causes UCP1-dependent heat generation, thereby reducing obesity [3,4].

Multifunctional mitochondrial homeostasis was recently highlighted because its functions and maintenance are directly related to human diseases [5]. Therefore, fine and tight control of mitochondrial biogenesis and degradation are important. Obesity is caused by adipocyte dysfunction following dysregulation of mitochondrial homeostasis including biogenesis, fission-fusion, and mitophagy. In adipocytes, mitochondrial clearance by mitophagy controls the differentiation and promotion of thermogenic signatures [6]. Mitophagy is an autophagic mechanism that occurs specifically in mitochondria. Mitophagy consists of three major steps: 1) fission of the mitochondrial network, 2) priming by autophagy machinery, and 3) engulfment by the autophagosome [7]. Briefly, a changed mitochondrial membrane potential leads to the recruitment of PTEN-induced putative kinase 1 (PINK1) and Parkin, E3 ubiquitin ligase that subsequently polyubiquitinates mitochondrial outer proteins [8,9]. After that, autophagy adaptors, nuclear dot protein 52 kDa (NDP52), optineurin (OPTN), and p62, bind the ubiquitinated proteins to LC3 (microtubule-associated protein 1 light chain 3), leading to the formation of double membraned mitophagosomes [10,11]. The ubiquitin-independent mechanisms that involve BCL2L13 (BCL2 like 13), BNIP3 (BCL2/E1B 19 kDa-interacting protein 3), and FUNDC1 (FUN14 domain-containing protein 1) also can activate mitophagy [12]. It remains under debate whether mitophagy induction promotes differentiation into white adipocytes and beige-to-white trans-differentiation [13]. For the maintenance of beige adipocytes, mitophagy inhibition by a genetic manipulation or pharmacological intervention is required [14]. PINK1–PARKIN (PRKN)-mediated mitophagy in adipose tissue is studied widely [15,16]. It has been reported that β3-AR–dependent PKA and p38 MAPK activations inhibit PINK1–PRKN-mediated mitophagy, thus suppressing beige-to-white reversion [15,17]. In addition, mitophagy suppression preserves a brown-fat signature, which is characterised by high UCP1 expression and mitochondrial biogenesis [18].

Despite efforts made to develop anti-obesity drugs over the past several decades, those that have been developed are limited and new approaches are highly needed. Therefore, browning WAT and activating BAT have become prominent pharmacological strategies for eradicating obesity. Many studies have shown that phytochemicals may be good candidates for anti-obesity drugs because phytochemicals promote the browning of WAT and activation of BAT [[19], [20], [21], [22]]. Researchers have also demonstrated that liensinine and raspberry ketone can inhibit beige-adipocyte reversion to the white phenotype and maintains the beige signature by suppressing mitophagy [23,24]. Nevertheless, there is only limited knowledge about the mechanisms by which phytochemicals activate the brown-fat-like signature and suppress mitophagy. Thus, discovery of potent phytochemicals that can induce the expression of the brown-fat signature and suppress mitophagy has become a popular topic and may lead to a therapeutic method for counteracting obesity.

Mangiferin (MF) from Mangifera indica is one of the widely studied phytochemicals owing to its anti-cancer [25], anti-diabetic [26], and anti-obesity [[27], [28], [29]] properties. On the other hand, the impact of MF on the brown-fat-like phenotype remains unclear. In this study, we first demonstrated that the effect of MF on browning is mediated by the suppression of mitophagy in C3H10T1/2 MSCs. Additionally, we investigated the effects of MF on mitochondrial biogenesis and homeostasis, in particular, whether these effects involve modulation of mitofission-fusion plasticity. On the basis of our findings, we can speculate that MF may be a good candidate compound for reducing obesity.

Section snippets

Materials

Reagents were obtained from the following sources: MF (cat. # M3547), H89, triiodothyronine (T3), insulin, dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), rosiglitazone (Rosi), Oil Red O dye (ORO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 4% formaldehyde, dimethyl sulfoxide (DMSO), and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP) was acquired from Cayman

MF reduces TG accumulation but induces lipolysis in C3H10T1/2 MSCs

To determine the treatment dose of MF, we measured cell viability at three doses of MF (25, 40, and 50 μM) applied to C3H10T1/2 MSCs and hADMSCs. As shown in Fig. 1C, none of the MF doses had any toxic effect on C3H10T1/2 MSCs at 24, 48, and 72 h. Nevertheless, among hADMSCs, the number of viable cells slightly diminished (Supplementary Fig. 4B); a similar observation about hADMSCs has been reported previously [29]. Besides, we determined the influence of MF on intracellular TG accumulation. As

Discussion

Inappropriate mitofission–fusion leads to mitochondrial damage that requires removal by mitophagic processing unless the mitochondrial membrane potential remains at the optimal level. Mitofission–fusion activation reportedly induces a thermogenic signature because elongated mitochondria are spared from autophagic degradation to maintain ATP levels [34]. Mitochondrial fission is a general process that is crucial for mitophagy. GTPase dynamin-related protein 1 (Drp1) plays important roles in

Conclusion

In summary, MF is a widely studied orally bioavailable phytochemical known to reduce obesity-related syndromes in both in vivo and clinical trials. However, its role non-shivering thermogenesis remains elusive. Thus, in our in vitro study, we reported the role of MF in browning and the related metabolic process. MF promotes the expression of brown and beige markers and inhibits the beige-to-white transition by suppressing PINK1–PRKN-mediated mitophagy by activating β3-AR–dependent PKA-p38

CRediT authorship contribution statement

Md. Shamim Rahman: Methodology, Formal analysis, Investigation, Writing - original draft. Yong-Sik Kim: Methodology, Formal analysis, Writing - original draft.

Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1A6A103032522; 2017R1D1A1B03032455) and partially by a research fund of Soonchunhyang University.

Declaration of competing interest

The authors declare that they have no conflicts of interest.

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