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Mark4 Inhibited the Browning of White Adipose Tissue by Promoting Adipocytes Autophagy in Mice

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Mark4 Inhibited the Browning of White Adipose Tissue by Promoting Adipocytes Autophagy in Mice International Journal of Molecular Sciences Article Mark4 Inhibited the Browning of White Adipose Tissue by Promoting Adipocytes Autophagy in Mice Kun Yang, Jiarui Cai, Miao Pan, Qian Sun and Chao Sun * Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shaanxi, China; [email protected] (K.Y.); [email protected] (J.C.); [email protected] (M.P.); [email protected] (Q.S.) * Correspondence: [email protected]; Tel./Fax: +86-29-87092164 Received: 8 March 2020; Accepted: 11 April 2020; Published: 15 April 2020 Abstract: Autophagy can remove excess or dysfunctional proteins and organelles to maintain cellular homeostasis. Browning of white adipose tissue increases the energy expenditure. Microtubules affinity regulated kinase 4 (Mark4) can regulate a variety of physiological processes. According to previous studies, we speculated that Mark4-autophagy-browning of white adipose tissue had certain linkages. Here, we established two autophagy models through serum starvation and rapamycin treatment and detected that the overexpression of Mark4 increased the expression of autophagy-related factors Beclin1, ATG7, and significantly decreased the autophagy substrate P62. Further tests showed that the overexpression of Mark4 promoted the conversion of autophagy marker protein LC3A to LC3B-II by activating the AMP-activated protein kinase (AMPK) pathway and inhibition of the AKT/mTOR signaling. Moreover, Mark4 decreased the expression of thermogenesis genes via promoting autophagy. These results indicated that Mark4 inhibited the browning of white adipose tissue via promoting autophagy. Keywords: Mark4; autophagy; browning of white adipose tissue 1. Introduction MAP/MARK4 belongs to the cell microtubule associated protein (MAPs) serine/threonine kinase (MARKs) family and involves in a variety of physiological processes [1]. The Mark4 gene encodes two alternate splicing isoforms, including L-type and S-type which differ in the C-terminal region [2]. Mark4L is highly expressed in the brain and may be involved in the development of the nervous system and the differentiation of neurons, while Mark4S is highly expressed in tumor cells and glioma cells, indicating that it may be involved in the regulation of cell cycle [3–6]. Studies indicate that Mark4 is the negative regulator of mTORC1 which plays a central role in cell growth [7,8]. Recently, the researchers found that Mark4 knockout mice increased their appetite, activity, and metabolic rate to resist obesity caused by a high-fat diet. At the same time, the protein kinase B (PKB/AKT) and AMP-activated protein kinase (AMPK) signaling pathways were activated to reduce insulin resistance and maintain glucose homeostasis [9]. Further research determined that Mark4 promoted adipogenesis by activating Jun N-terminal kinase (JNK) and p38-mitogen-activated protein kinase (MAPK) signaling pathways, it also triggered adipocytes apoptosis by activating the JNK signaling pathway [10]. Additionally, studies have found that Mark4 promoted oxidative stress and inflammation via binding to the peroxisome proliferator-activated receptor gamma (PPARγ) and activating nuclear factor-kappa B (NF-κB) pathway in mice adipocytes. These data establish a novel regulation role of Mark4 on body metabolic balance [11]. Autophagy is an evolutionarily conserved lysosome-dependent system in eukaryotes that transports cytosolic components to the lytic compartment of the cell for degradation [12,13]. Previous studies have found that nutrient deprivation-induced loss of lipid droplets was related Int. J. Mol. Sci. 2020, 21, 2752; doi:10.3390/ijms21082752 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 2752 2 of 19 to autophagy, whereas the inhibition of autophagy increased the storage of triglycerides in lipid droplets [14]. Knockdown of autophagy-related gene 7 (Atg7) in 3T3-L1 preadipocytes inhibits lipid accumulation and decreases protein levels of adipocyte differentiation factors. The adipocyte-specific knockout of Atg7 generates enhancement of insulin sensitivity and the features of brown adipocytes in mouse white adipose tissue, resulting in the lowering of white adipose mass [15]. Autophagy is the most active and essential for the initial stage of adipocyte differentiation, but it is dispensable during its later stage [16]. Studies have found that mineralocorticoid receptor (MR) antagonism induced browning of white adipose tissue through impairing autophagy and preventing adipocyte dysfunction in high-fat diet fed (HFD) mice [17]. A research found that natural plant alkaloid-berberine inhibited basal autophagy in adipocytes and adipose tissue of mice fed a high-fat diet via downregulating expression of Beclin1 [18]. Mice with skeletal muscle-specific deletion of Atg7 have a reduced fat content and been protected from diet-induced obesity and insulin resistance. This phenotype is accompanied by increased fatty acid oxidation and browning of white adipose tissue (WAT) owing to the induction of fibroblast growth factor 21 (Fgf21) [19]. Our previous research found that leptin inhibited ER stress-induced inflammation through reducing Activating transcription factor 4 (Atf4)-autophagy-related gene5 (Atg5)-mediated autophagy in adipocytes [20]. At present, the relationship among Mark4, autophagy, and browning of white adipose tissue has not been reported in adipose tissue [21,22]. Here, we demonstrated that Mark4 promoted adipocytes autophagy which was induced by serum starvation and rapamycin treatment. Furthermore, we found that the browning of white adipose tissue was inhibited via promoting autophagy. These finding elucidated a novel function of Mark4 in the regulation of cell autophagy and browning of white adipose tissue, implying a potential strategy for metabolism syndrome. 2. Results 2.1. Serum Starvation and Rapamycin Treatment Can Induce Adipocyte Autophagy To study the effects of serum starvation and rapamycin (Rapa) treatment on adipocyte autophagy, we first induced 3T3-L1 preadipocytes to differentiate into mature adipocytes (Figure1A–C). Then, we cultured mature adipocytes with serum-free medium and serum medium supplemented with a certain concentration of Rapa and selected the most appropriate culture time (8 h) and concentration (100 nM) (Figures1D and2A). After the end of the culture, the RNA and protein were extracted for related detection. The results showed that serum starvation for 8 h and 100 nM Rapa treatment for 12 h did not change cell viability (Figures1D and2A) ( p > 0.05), but the expression of autophagy-related genes Beclin1 and ATG7 were significantly higher than the control group (p < 0.05) (Figures1E and2B). A reliable marker of autophagy was the conversion of the ATG protein LC3 from a soluble form (LC3A) to a lipidized form (LC3B-II), which was stably associated with the membranes of autophagosomes. This conversion can be detected by measuring the accumulation of the LC3B-II formation. Western blot analysis showed an increase of LC3B-II upon serum starvation for 8 h and Rapa for 12 h treatment (p < 0.05) (Figures1F and2C). It is well known that Monodansylcadaverine (MDC) accumulates specifically in autophagosomes or autophagic vacuoles (AV). Next, we examined the number of AV. These results showed that both serum starvation and Rapa treatment could increase the number of AVs (p < 0.05) (Figures1G and2D). Therefore, we conclude that serum starvation and Rapa treatment can induce adipocyte autophagy. Int. J. Mol. Sci. 2020, 21, 2752 3 of 19 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 19 FigureFigure 1. 1.Adipocytes Adipocytes autophagy autophagy was was induced induced by serumby serum starvation starvation treatment. treatment. (A) Relative (A) Relative mRNA mRNA level oflevel adipogenic of adipogenic differentiation-related differentiation-related genes (n genes= 6). ( nB )= Protein6). (B) Protein level of level adipogenic of adipogenic differentiation-related differentiation- genesrelated (n genes= 6). ((Cn )=Representative 6). (C) Representative images images of differentiated of differentiated cells were cells labeled were labeled with Oil with Red Oil O Red(n = O6) (.n (D= )6). Cell (D) viability Cell viability was detectedwas detected by bycholecystokinin-8 by bycholecystokinin-8 (CCK8) (CCK8)(n = (n6) =.( 6).E )(E Relative) Relative mRNA mRNA level level ofof autophagy-related autophagy-related genesgenes withwith or without seru serumm starvation starvation for for different different lengths lengths of of time time (n (n= 6).= 6).(F) F ( Protein) Protein level level of of autophagy-related autophagy-related genes genes with with or or without without serum starvation forfor didifferentfferent lengthslengths of of time (n = 6). (G) Monodansylcadaverine (MDC) and DAPI staining by serum starvation treatment for time (n = 6). (G) Monodansylcadaverine (MDC) and DAPI staining by serum starvation treatment for 8 h. Values are means SD. vs. control group, * p < 0.05, ** p < 0.01 8 h. Values are means± ± SD. vs. control group, * p < 0.05, ** p < 0.01 Int. J. Mol. Sci. 2020, 21, 2752 4 of 19 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 19 FigureFigure 2. Adipocytes 2. Adipocytes autophagy autophagy was was induced induced by by Rapa Rapa treatment. treatment. (A) Cell (A) viability Cell viability were detected were detected by by CCK8 (CCK8n = 6). (n (=B 6).) Relative(B) Relative mRNA mRNA level level ofof autophagy-related genes genes with with or without or
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