Melatonin (N-acetyl-methoxytryptamine) is mainly synthesized by the pineal gland and maintains circadian rhythm in mammals. Melatonin presents multiple physiological functions including antioxidant, anti-inflammatory, immunomodulatory, and oncostatic effects. Many actions of melatonin are mediated through interaction with the melatonin receptors 1 and 2 (MT1 and MT2), which are protein-coupled membrane receptors found in several cell Studies demonstrate melatonin ameliorates low-grade inflammation and oxidative stress by repressing the inflammatory response in brain and peripheral tissues. Furthermore, melatonin could counteract the immune alterations and trigger an effective balance of innate and humoral immune response in immune cells.Exogenous melatonin enhances antigen presentation by splenic macrophages to T cells in mice. Recent studies suggest that melatonin suppresses lipopolysaccharide (LPS)-induced pro-inflammatory factors release in macrophages.Our previous study has proposed that melatonin promotes proliferation and alleviates inflammasome-induced pyroptosis in adipocytes. However, the effects of melatonin on the communication of adipocytes and macrophages during peripheral adipose inflammation remain elusive. Exosomes, the small vesicles, are increasingly recognized as important mediators of intercellular communication, being involved in the transmission of biological signals between cells.Therefore, this study also investigated the role of exosomes in melatonin-inhibited metabolic inflammation of adipose tissue to provide potential therapeutic applications.Adipocytes and macrophages coordinate to preserve tissue integrity while adapting to the metabolic stress in the adipose tissue of obese animals. Numerous studies indicate obesity is associated with an overall increase in the number of macrophages which formed crown-like structures (CLS) in adipose tissue of both rodents and human.nation of an increase in total macrophages and an increased ratio of M1 to M2 macrophages is a hallmark of the adipose tissue inflammation. Recent studies suggest that melatonin attenuates inflammatory response by modulating nitric oxide and arginine metabolism in macrophages. Moreover, endogenous metabolites function as sensors to transport intercellular signals and induces secretion of potent pro-inflammatory cytokines that contribute to obesity-metabolic inflammation conditions. Studies suggest that the small molecule metabolite is sufficient to regulate multiple chromatin modifications by ten-eleven translocation (Tet)-dependent DNA demethylation, which contributes to numerous inflammatory gene expression. Although several studies reveal melatonin inhibits inflammation in adipose tissue, the effects of melatonin on endogenous metabolites in adipose inflammation are still unknown.In this study, we investigated the effects of melatonin on metabolites during adipose inflammation. We further determined that melatonin could alleviate adipose inflammation though elevating -ketoglutarate ( KG) in adipocytes and delivering exosomal KG to macrophages in mice adipose MATERIAL AND METHODSAnimal studiesold C57BL/6J background male mice were purchased from the Laboratory animal center of the fourth military medical University (Xi’an, China). The use of the ing the guidelines and regulations approved by the Animal ethics committee of Northwest A&F university (Yangling, bitum with water, and a standard laboratory chow diet. The animal room was maintained constant temperature at 1°C and humidity at 555%, and 12-hour light/dark h LD cycle). Zeitgeber time (ZT) 0 corresponds lagged mice model was In brief, mice were AMPMPMAMAM to PMPMAMferred once from room 1 to 2 and then returned to room 1. The AMAMhour phase advance from room 1 to 2 (light lagged mice on the day of hour phase delay from room 2 to 1 (light lagged mice on the day of days. For the lipopolysaccharide (LPS) challenged experiment in vivo, mice were challenged via intraperitoneal (ip) injection with the indicated quantities of LPS (75Aldrich, St. Louis, MO, USA) in induced obese mice model, mice fat diet (HFD, fat provided 60% of the tech co., Ltd, Nantong, weeks, and the control mice were fed a standard diet (Chow, fat provided 10% of total energy, Trophic animal feed high-tech co., Ltd). Melatonin injection was performed The melatonin treatment and -ketoglutarate ( KG) treatment were further applied to HFD fed mice or LPS-treated mice. Melatonin (Sigma-Aldrich) was first dissolved in
| 3 of 16
amount of absolute ethanol at a final concentration of 0.5% (v/v) ethanol with melatonin and prepared as the stock melatonin solution. Separate experiments showed that there were no additional effects of the melatonin vehicle (0.05% ethanol) on any of the parameters investigated (data not shown). 24) were randomly divided into four groups (neach). One group of mice received a daily intraperitoneally L solution consisting melatonin (MT, mg/kg) in phosphate-buffered saline (PBS) before the dark onset for 14days; one group of mice was given a vehicle consisting of a solution of 0.5% ethanol in PBS; one group of mice was administered of KG (Sigma-Aldrich) at a dose g/kg body weight by means of an intragastric tube for days; one group of mice received of both melatonin and KG treatments for 14days. Jet-lagged mice were pretreated with chronic jet lag for 2weeks before the melatonin injection experiment. Mice were sacrificed by overdosed ethyl ether within 2hours after the last injection of melatonin or the vehicle. Immediately, the epididymal white adipose tissue (eWAT) was dissected and kept for the studies as following.The eWAT and liver tissues were collected immediately and fixed in 4% paraformaldehyde in phosphate buffer. After 2weeks of incubation, the samples were embedded in paraffin and sectioned. The tissue sections were used for hematoxylin and eosin (H&E) staining. The images were photographed by Cytation 3 cell imaging multimode reader (BioTek, Winooski, VT, USA). Serum aspartate transaminase (AST) and alanine aminotransferase (ALT) levels were measured by the AST and ALT activity assay kits from Sigma Aldrich, MAK055, MAK052). Serum and cell tumor necrosis factor (TNF ) and interleukin 6 (IL-6) levels were measured by commercial ELISA kits from Sigma (Sigma-Aldrich, RAB0477, RAB0308). Interleukin-1 (IL-1) level was measured by Mouse IL-1 beta ELISA Kit from Abcam (Cambridge, UK, ab100704). The citrate, malate, fumarate, and KG levels of eWAT were analyzed using the commercial assay kits from Sigma (Sigma-Aldrich, MAK057, MAK067, MAK060, MAK054) following the manufacturer’s instructions.Primary cell culturePrimary adipocytes culture was performed as previously In brief, eWAT was dissected from mice, washed with PBS, and minced. Pre-adipocytes were seeded mm culture dishes at 30% (v/v) confluence, and incubated at 37°C under a humidified atmosphere of 5% and 95% air for subsequent experiments. After reachadipocytes were induced to differentiate using Dulbecco’s modified eagle media/nutrient mixture F12 (DMEM/F12; Gibco, Grandland, NY, USA, 12500062) with 10% fetal bovine serum (FBS) and days until exhibiting a massive cn, Shanghai, China) was added into culture medium at a Aldrich, 75890) was added into culture medium at a final tion study, adipocytes were infected with overexpression adenovirus or interference lentiviral recombinant vectors Mitochondrial ) for IFU/mL, and then treated GFP. All the vectors were constructed by Primary peritoneal macrophages were obtained from days after injection of 2mL of 4% sterile thioglycolate solution as previously described by pelvic washing with PBS containing 3% FBS. Primary cultures of macrophages were maintained in RPMI 1640 medium. All culture media were supplemented by addition of 10% FBS and penicillin/streptomycin.IDH2 activity measurementActivity of IDH2 was measured by the Someya method.mediately read at 340seconds for 1Plasmid transfection and luciferase reporter assayLuc and Luc plasmid as a template; a mutagenesis kit (Invitrogen, Carlsbad, reagent (Roche, Basel, Switzerland), and the same amounts
4 of 16|
transfection 48hours, cells were harvested for luciferase Exosome isolation and transmission electron microscopy (TEM) examinationisolation procedures were all performed at 4°C. The cultured at 110PBS, and centrifuge 2hours at 110, resuspended in Isolated exosomes were analyzed per duplicate by transmission electron microscopy (TEM). Vesicles were fixed with 4% paraformaldehyde at room temperature for 10utes and then layered on carbon/formvar film-coated grids for minutes. After removing sample excess, negative staining was performed by incubation with phosphotungstic acid for seconds. After washing, grids were dried overnight at room temperature. Samples were observed with a transmission electron microscopy (TEM, HT7700, 80kV, Hitachi, Tokyo, Japan) working at 80kV. The image calculation was analyzed using Image J software (NIH).immunoprecipitation IP) analysisHEK293 cells were transfected with plasmids using transfection reagent (Roche, 06366236001) as previously described. In brief, cells were then snap frohours transfection. Whole cell lysate was harvested in lysis buffer with a protease inhibitor. seconds, and the whole cell g primary antibody overnight at 4°C. Immune complexes were pulled down with protein A agarose for hours at 4°C with shaking. Beads were washed once with lysis buffer and three times with wash buffer, and then eluted Immunocytochemical staininghydroxymethylcytosine (5hmC) in an enzymatic We well plates were incubated minutes, and permeabilized with PBS minutes. After blocking body (1:50; Cell Signaling Technology, Boston, MA, USA, #51660) at 4°C overnight. After washing for 3 times, fluoreshour. Fluorescence was examined using a Cytation 3 cell imaging multimode reader (BioTek, VT). To quantitate 5hmC levels in representative images, the images were analyzed using ferences in differences in overall DNA content, 5hmC signal Paraffin-embedded eWAT was sectioned, dewaxed, and rehydrated prior to antigen retrieval by boiling in 10sodium citrate buffer (pH 6.5). Tissue samples were blocked with 5% normal rabbit serum for 40minutes, followed by incubation for 2hours with 10 g/mL F4/80 antibody (Abcam, Cambridge, UK, ab6640). And then incubated with fluorescein isothiocyanate-conjugated goat against rabbit IgG antibody (Boster, China; diluted 1:100) for 1hour at room temperature. After washing for 3 times, the nuclei were stained with DAPI for 5minutes. Average numbers of F4/80-positive cells in the slides were calculated by scoring five random 200× microscopic fields of stained adipose tissue sections of each mouse using a Cytation 3 cell imaging multimode reader (BioTek, VT).RNA extraction and Real-Total RNA of eWAT or adipocytes was extracted with ng of MLV reverse transcriptase kit (Takara). Primers were synthesized by time PCR was performed L reaction system containing specific primers and AceQ qPCR SYBR Green Master Mix (Vazyme Biotech,
| 5 of 16
Nanjing, China). Amplification was performed in the ABI PCR System (Carlsbad, CA, USA). The
Immunoblotting analysesProtein was isolated from tissues or cultured cells. ratus, and transferred to PVDF nitrocellulose membranes 8592, CD9 CD81 (#10037, Cell Signaling Technology), STAT3 (ab68153), anti-STAT3 (ab76315), anti- B (ab207297), anti- B (ab222494), anti-Cyt C Cd11c Cd206 36494, Novus Tet1 (ab191698), DNMT1 DNMT2 (ab71015), and the appropriate conjugated secondary antibody (Baoshan, China) cent peroxidase substrate (Millipore), and then the blots Rad, Statistical analysisference (LSD). Data were presented as meanRESULTSMelatonin increases KG level in obesity-induced adipose inflammationfat diet (HFD) and then treated mice with melatonin. H&E staining showed melatonin significantly reduced adipocyte size both in 1A). ), were all increased markedly with HFD; while melatonin injection alleviated this chronic inflammation caused by
Melatonin increases KG level in obesity-induced adipose inflammation. (A) Representative hematoxylin and eosin (H&E) staining of adipose sections from high-fat diet (HFD) or chow diet mice treated with melatonin or not. The small images on the right side were the expansion (n6). (B) mRNA levels of inflammation marker genes with or without melatonin treatment of HFD and chow diet mice (n(C) Serum levels of tumor necrosis factor (TNF ), interleukin 6 (IL-6), and interleukin-1 (IL-1) of HFD and chow diet mice treated with melatonin or not (n6). (D) Concentration of key metabolites of the tricarboxylic acid (TCA) cycle of HFD and chow diet mice treated with melatonin or not (n6). (E) mRNA levels of isocitrate dehydrogenase 1 (isocitrate dehydrogenase 2) of HFD and chow diet mice treated with melatonin or not (n6). (F) Regression analysis of interleukin-66). Protein level was detected by ELISA method. Values are means.05 compared with the control group
6 of 16|
1B). Further protein level detection by 1 1C). Metabolic analyses by ELISA method identified metabolite contents such as citrate and KG were increased due to melatonin injection in HFD group; but the contents of malate and lyzed the mRNA level of rate-limiting enzymes which catalyzing KG generation. During melatonin treatment suggesting melatonin regulate adipose metabolism via 1E). As expect, correlation analysis showed were negatively latonin involved in adipose inflammation and increased Melatonin and KG coordinately reduces adipose inflammation in challenged micelike rately treated mice compared with that of control mice which 2A). treatment group contained were reduced due to the treatment of melatonin and KG in LPS-pretreated mice 2B). ELISA measurement further showed serum TNF , 1 levels were reduced with KG and melatonin 2C). KG had no effect on IDH2 activity, while melatonin injection increased IDH2 activity drastically, treatment group using ELISA
Melatonin and KG coordinately reduce adipose inflammation in LPS-challenged mice. All the experiments were performed in challenged mice. (A) Representative H&E staining of adipose sections treated with melatonin or KG. Black arrows: crown-like structures (CLS). Scale bar: 2006). (B) mRNA levels of inflammation indicators of mice adipose treated with melatonin or KG (n6). (C) Serum levels of TNF , IL-6, and IL-1 of mice treated with melatonin or KG (n6). (D) IDH2 activity of mice adipose treated with melatonin or KG (n6). (E) Representative H&E staining of liver sections treated with melatonin or KG. Scale bar: m (n6). (F) Serum levels of alanine aminotransferase (ALT) and aspartate transaminase (AST) of mice treated with melatonin or KG (n6). Protein level was detected by ELISA method. Values are means.05 compared with the control group
| 7 of 16
2D). Moreover, H&E staining demonstrated liver morphology and liver metabolic were unaltered in KG 2E,F). In short, melatonin Melatonin enhances DNA demethylation of mice adipocyteslatonin treatment drastically increased the expression of expression measurement established that fumarate hydra-melatonin significantly increased the levels of melatonin re
Melatonin enhances DNA demethylation of mice adipocytes. (A) Heatmap of genes upregulated or downregulated with melatonin treatment of mice adipocytes, along with the top affected genes with melatonin treatment (n3). (B) Gene ontology (GO) analysis of the target genes in Figure1A, showing the biology process for melatonin treatment (n3). (C) mRNA levels of melatonin receptor 1fumarate hydra-tase 1Fum1 of adipocytes treated with melatonin (n3). (D) Concentration of KG of adipocytes treated with melatonin (n3). (E) IDH2 activity of adipocytes treated with melatonin (n3). (F) mRNA levels of ten-eleven translocation 1Tet1ten-eleven translocation 2Tet2) of adipocytes treated with melatonin. Right panel: protein level of Tet3 of adipocytes treated with melatonin (n(G) mRNA levels of DNAmethyltransferase 1DNAmethyltransferase 2) of adipocytes treated with melatonin (n(H) Immunohistochemical staining of 5-hydroxymethylcytosine (5hmC) of adipocytes treated with melatonin. Scale bar: 2003). Values are .05 compared with the control group
8 of 16|
3F). The protein level of Tet3 was also elevated with methyltransferase 1 () and DNA methyltransferase 2Sirt1 and IDH2 formed a complex to increase KG levelWe then questioned how melatonin upregulated the level of KG. In fact, studies have demonstrated melatonin directly ysis and previous data sheet, our study showed Sirt1 interacted protein measurement, data indicated Sirt1 protein interacted strongly with transfected IDH2 in HEK293 cells (Figure4B). bation of melatonin, we found the level of KG was drastically enhanced; while knock down of obtained the opposite 4D). 1 by ELISA in adipocytes treated with MT and pAd- (Figure4E). Thus, these data suggest that Sirt1 and IDH2 directly bind, and then regulated KG level in adipocytes; melatonin functioned via Exosomal KG promotes M2 macrophage activation and enhances DNA demethylationTo determine whether the metabolite from adipose-) by differential ultracentrifugation (Figure5C). Electron microscopy images confirmed that exosomes were clearly separated and the size was consistlatonin treatment, KG level from adipose-derived exosome To verify that exosomes derived from melatonin-treated adipose tissue affected the macrophage population, we co-cultured macrophages with adipose tissue-derived exosomes for 24hours. Interestingly, Exos treatment significantly increased KG level of macrophages which is similarly to KG-treated group (Figure6A). Exos treatment also elevated the expression of OXGR1, the receptor of KG (Figure6B). In addition, the percentage of M2 macrophage was higher in Exos group than that in the control group from microscopy images (Figure6C), and consistent results were observed in the KG-treated group. Increased levels of specific M2 macrophage markers have been found with Exos incubation, with decreased of M1 macrophage indicators both on mRNA level and on protein level (Figure6D,E). To understand more clearly about the correlation of Exos and KG in macrophages, we then measured the DNA demethylation of macrophages. As shown in Figure6F–I, KG significantly enhanced the mRNA levels and protein levels of Tet1, Tet2, and Tet3, and reduced DNMT1 and DNMT2. And Exos treatment showed enhanced DNA demethylation either (Figure6F–I). The enrichment of 5hmC in both KG group and Exos group further confirmed the elevation of DNA demethylation (Figure6J). These data clearly indicate adipose tissue-derived exosomes, similarly as KG, increase M2 macrophage and enhance DNA demethylation in macrophages, verify the correlation between adipocytes and macrophages.Adipose-derived exosomes inactivate STAT3/NF- B signal in adipocytesWe next explore the function of exosomes derived from treated adipose tissue in adipocytes. Adipocytes . The mRNA level and protein level of TNF , IL-6, and IL-1 were greatly reduced with KG incubation, and similarly results were 7A,B). Consistently markedly elevated KG content of adipocytes (Figure7C). Further measurement showed increasing oxoglutarate receptor 1 () conment with early study that KG functioned via the inactivation B signals in mediating inflammation, our data B, pointing to the correlation between 7E). Based on these findings, the play an Melatonin drives circadian amplitude of in adipose inflammationTo further dissect the regulation of melatonin on KG, we examine the 24-hour mRNA pattern of . Interestingly, the expression profile of showed a circadian rhythm by the cosine method (Figure8A). And melatonin treatment increased the circadian amplitude of (Figure8A). Based on our previous study, we firstly considered the gene Clock
| 9 of 16
Sirt1 and IDH2 formed a complex to increase KG level. (A) IDH2 interacted with sirtuin 1 (Sirt1). (B) Co-IP analysis was performed in His-Sirt1 and Flag-IDH2 transfected HEK293 cells (n3). (C) KG concentration of adipocytes pre-infected with pAd-Sirt1Sirt1and then treated with or without melatonin (n3). (D) mRNA level of of adipocytes pre-infected with pAd-Sirt1Sirt1, and then treated with or without melatonin (n3). (E) Protein levels of TNF , IL-6, and IL-1 of adipocytes pre-infected with pAd-Sirt1Sirt1, and then treated with or without melatonin (n3). pAd-Sirt1, overexpression adenovirus vector of Sirt1Sirt1, interference lentivirus vector of Sirt1. Protein level was detected by ELISA method. Values are means.05 compared with the control group, .05 compared with the MT group
10 of 16|
as a key regulator. Dual-luciferase reporter assay demonstrated promoter contained three consensus E-box elements that were the potential targets of Clock (Figure8B). Further measurements revealed that three E-box elements, bp upstream of the initiation codon of tioned (Figure4B). Mutation of the two E-box elements in the promoter impaired the effects of Clock transcription activity (Figure8C), indicating that both of the two box elements regulated the translation of . We then overexpressed in adipocytes with or without melatonin treatment of and melatonin further elevated the content of KG, along with the reduction of IL-6mRNA level (Figure8D,E). In addition, ELISA measurement showed co-treatment of and melatonin reduced the protein levels of TNF , IL-6, and IL-1 (Figure8F). Thus, these findings suggest Clock positively regulates on transcriptional level, and melatonin functioned via the regulation of Melatonin attenuates jet-lagged-induced inflammation in mice adipose tissuestaining showed melatonin decreased the F4/80-
Isolation of exosomes from mice adipose tissue. (A) A schematic depiction of adipose exosome isolation. (B) Size distribution derived exosome as showed the peak at 30-3). (C) The exosome fractions and cell lysates of adipose-derived exosomes were analyzed by immunoblotting with antibodies against exosomal proteins CD9 and CD81 and cellular proteins Cyt c and -3). (D) Electron microscopy (EM) images of exosomes. Right panel: the expansion image of the original image. EM scale bar: 1003). (E) KG concentration from adipose-derived exosomes treated with or without melatonin (n3). Values are means.05 compared with the control group
| 11 of 16
In addition, we found melatonin also elevated the cellular indicated that KG might be involved in melatonin reducing
Exosomal KG promotes M2 macrophage activation and enhances DNA demethylation. Macrophages were pretreated with KG, derived exosome (Control), or adipose-derived exosome which had been incubated with melatonin (Exos). (A) Intracellular KG level of macrophages (n3). (B) mRNA level of oxoglutarate receptor 1OXGR) of macrophages (n3). (C) Representative pictures of M1 macrophages and M2 macrophages. Scale bar: 200 m. Black arrow: M1 macrophage, white arrow: M2 macrophages (n3). (D) mRNA levels of M1 and M2 macrophage indicators of macrophages (n3). (E) Protein levels of Cd11c, TNF , Cd206, and Cd163 of macrophages (n3). (F) mRNA levels Tet1Tet2,Tet3 of macrophages (n3). (G) Protein levels of Tet1, Tet2, and Tet3 of macrophages (n3). (H) mRNA levels of and of macrophages (n3). (I) Protein levels of DNMT1 and DNMT2 of macrophages (n3). (J) Immunohistochemical staining of 5hmC of macrophages. The right panel was the quantification of fluorescence intensity. Scale bar: 2003). Values are means.05 compared with the control group
12 of 16|
Adipose-derived exosomes inactivate STAT3/NF- B signal in adipocytes. The adipocytes were pretreated with KG, adipose-derived exosome (Control), or adipose-derived exosome which had been incubated with melatonin (Exos). (A) mRNA levels of inflammatory markers of adipocytes (n3). (B) Protein levels of TNF , IL-6, and IL-3). (C) Intracellular KG level of adipocytes (n(D) mRNA level of OXGR3). (E) Protein levels of p-STAT3, STAT3, p-NF- B, and NF-3). Protein level was detected by ELISA method. Values are means.05 compared with the control group
Melatonin drives circadian amplitude of in adipose inflammation, (A) mRNA level of at indicated times was analyzed by RT-3). (B) Fragments of promoter fused to a luciferase reporter gene were co-transfected into HEK293 cells together with PGL3-basic (control) or pc-Clock. The binding sites of Clock overlap with consensus E-boxes in promoter region. Luciferase activity was corrected for Renilla luciferase activity and normalized to the control activity (n3). (C) The strategy for generating mutant promoter-driven luciferase reporters with mutated bases in red (n3). (D) Relative KG abundance of adipocytes pre-infected with pAd- and treated with melatonin or not (n3). (E) mRNA levels of of adipocytes pre-infected with pAd- and treated with melatonin or not (n3). (F) Protein levels of TNF , IL-6, and IL-3). pAd-: overexpression adenovirus recombinant vectors of : interference lentiviral recombinant vectors of . Protein level was detected by ELISA method. Values are meanscompared with control
| 13 of 16
There exists a substantial amount of evidence supporting that melatonin can accelerate key enzymes of the Kreb’s cycle and the respiratory chain. Among the known metabolites, KG has been reported to be essential for the elicitation of desirable immune responses. Recent studies also confirm that melatonin elevates cellular KG level and prevents -ketoglutarate dehydrogenase activation in liver and kidney. To estimate the effects of melatonin on cellular KG level in adipocyte inflammation, we investigated the core metabolic and inflammatory genes expression profile in adipose tissue. We found that melatonin elevated the expression of TCA cycle genes . Moreover, the NF- B signal pathway which was correlated with inflammatory responses was significantly enriched by the gene ontology (GO) analysis. Studies have identified that KG links metabolism to epigenetic modifications in adipocytes differentiation and thermogenesis by TET-mediated DNA demethylation.In this study, our data preliminarily showed that melatonin increased cellular KG level along with the increasing of DNA demethylation during inflammatory response in adipocytes. Furthermore, melatonin could elevate IDH2 activity by Sirt1 directly interaction with IDH2 and suppressed pro-inflammatory status in adipocytes. Although the molecular mechanism of complex formation remains elusive, we suspect that it is similar to the interaction of protein Sirt3/IDH2 by directly deacetylation, and this needs further study. From these findings, we surmise that melatonin participates in the regulation of inflammatory responses by KG-mediated epigenetic reprogramming in adipose tissue.Exosomes have been considered as important mediators of cell communication by exchange of material and delivering information. Studies have determined that adipocytes can release exosomes to recipient cells and tissues thereby modifying the physiological state of the recipient cells. Here, we demonstrated that melatonin increased exosomes secretion from adipocytes and elevated the KG concentration in
Melatonin attenuates jet-lagged-induced inflammation in mice adipose tissue. The jet-lagged mice were injected with or without melatonin for 14days, and n6 in each group. (A) KG level of adipose tissue of jet-lagged mice. (B) IDH2 activity of adipose tissue of jet-lagged mice. (C) mRNA levels of and adiponectin of adipose tissue of jet-lagged mice. (D) Adipose tissue sections were stained with the macrophage-specific antibody F4/80. The right panel was the quantification of fluorescence. Values are means.05 compared with control
Melatonin coordinates inflammatory response through elevating KG and diverting to macrophages via exosomes in mice adipose tissue. Melatonin alleviated inflammatory response through increasing cellular and exosomal KG level in adipose tissue. Exosomal KG transports to recipient macrophages and performs the inflammatory function by epigenetic reprogramming
14 of 16|
derived exosomes. Notably, we are the first demonstrated that exosomes (secretion from melatonin-treated adipocytes) subsequently transported to macrophages and acted as a critical switch governing M1 and M2 macrophage polarization. In addition, further analysis suggested that KG inhibited inflammation response in macrophage through epigenetic reprogramming. These findings are consistent with other studies that KG regulated the immune responses of macrophages through metabolic and epigenetic reprogramming. Our previous study had determined that melatonin inhibited inflammasome formation and pyroptosis in adipocytes. We then assumed that exosomal KG was essential for the inhibitory role of melatonin on inflammation of neighboring adipocytes. In our study, we showed that exosomal KG decreased adipocyte inflammation along with the reduction of phosphorylation of STAT3/NF- B signal. Notably, we are the first to point out a significant correlation between circadian clock and KG concentration. The regulatory mechanism of Clock on gene transcription may depend on E-box, which is a DNA response We hypothesized that melatonin elevated cellular and exosomal KG level associated with the binding of Clockto E-box in the promoter region of during adipocyte inflammation. Although we and other studies have determined melatonin and KG coordinately alleviated inflammation, the precise mechanisms that melatonin regulates exosomal KG secretion need to be further studied.There are also several limitations in this study. First, the dose of melatonin we used was at a pharmacological level. However, we and others suggested that this dose did not affect the viability of adipocytes and could affect many biological processes in adipocytes. Second, we did not confirm whether melatonin affects other metabolites in exosomes that may play important functional roles during adipose inflammation. In addition, it is possible that multiple metabolites within the exosomes could work in a coordinated way to reduce adipose inflammation that we have observed. Future metabolomics study will be necessary to demonstrate the full set of melatonin regulating the adipose-derived exosomes.In summary, our present study demonstrates that melatonin alleviates inflammatory response through increasing cellular and exosomal KG level in adipose tissue. Moreover, adipose-derived exosomes transport to recipient macrophages and perform the anti-inflammatory function by KG-regulated epigenetic reprogramming (Figure10). Thus, our results indicate that melatonin can be potentially used in the therapy of obesity-associated metabolic disease by delivering desirable information to an accurate target ACKNOWLEDGEMENTSThis work was supported by the grants from the Major National Scientific Research Projects (2015CB943102) and the National Nature Science Foundation of China (31572365) and Key Sci-tech innovation team of Shaanxi province (2017KCT-24) and the Fundamental Research preciated Prof. Shimin Liu from School of Animal Biology, The University of Western Australia to help us improve the AUTHOR CONTRIBUTIONSCONFLICT OF INTERESTThe authors declare that there is no duality of interest associORCID
REFERENCESSzewczyk-golec K, Wozniak A, Reiter RJ. Inter-relationships of the chronobiotic, melatonin, with leptin and adiponectin: implications for obesity. J Pineal ResReilly SM, Saltiel AR. Adapting to obesity with adipose tissue inflammation. Nat Rev EndocrinolLumeng CN, Saltiel AR. Inflammatory links between obesity and metabolic disease. J Clin InvestShan B, Wang X, Wu Y, et al. The metabolic ER stress sensor IRE1alpha suppresses alternative activation of macrophages and impairs energy expenditure in obesity. Nat ImmunolBrestoff JR, Artis D. Immune regulation of metabolic homeostasis in health and disease. Mcnelis Joanne C, Olefsky-jerrold M. Macrophages, immunity, and metabolic disease. Lampropoulou V, Sergushichev A, Bambouskova M, et al. Itaconate links inhibition of succinate dehydrogenase with macrophage metabolic remodeling and regulation of inflammation. Cell MetabMills E, O’neill LA. Succinate: a metabolic signal in inflammaTrends Cell BiolHardeland R, Madrid JA, Tan DX, et al. Melatonin, the circadian multioscillator system and health: the need for detailed analyses of peripheral melatonin signaling. J Pineal ResAgil A, El-hammadi M, Jimenez-aranda A, et al. Melatonin reduces hepatic mitochondrial dysfunction in diabetic obese rats. Pineal ResAlonso-vale MI, Andreotti S, Mukai PY, et al. Melatonin and the circadian entrainment of metabolic and hormonal activities in primary isolated adipocytes. J Pineal Res
| 15 of 16
Manchester LC, Coto-montes A, Boga JA, et al. Melatonin: an ancient molecule that makes oxygen metabolically tolerable. Pineal ResMauriz JL, Collado PS, Veneroso C, et al. A review of the molecular aspects of melatonin’s anti-inflammatory actions: recent insights and new perspectives. J Pineal ResReiter RJ. Pineal melatonin: cell biology of its synthesis and of its physiological interactions. Endocr RevAlvarez-sanchez N, Cruz-chamorro I, Diaz-sanchez M, et al. Melatonin reduces inflammatory response in peripheral T helper lymphocytes from relapsing-remitting multiple sclerosis patients. J Pineal ResSlominski RM, Reiter RJ, Schlabritz-loutsevitch N, et al. Melatonin membrane receptors in peripheral tissues: distribution and functions. Mol Cell EndocrinolZhao L, An R, Yang Y, et al. Melatonin alleviates brain injury in mice subjected to cecal ligation and puncture via attenuating inflammation, apoptosis, and oxidative stress: the role of SIRT1 J Pineal ResCano Barquilla P, Pagano ES, Jimenez-ortega V, et al. Melatonin normalizes clinical and biochemical parameters of mild inflammation in diet-induced metabolic syndrome in rats. J Pineal ResChen J, Qian C, Duan H, et al. Melatonin attenuates neurogenic pulmonary edema via the regulation of inflammation and apoptosis after subarachnoid hemorrhage in rats. J Pineal ResBrazao V, Santello FH, Colato RP, et al. Melatonin: antioxidant and modulatory properties in age-related changes during trypanosoma cruzi infection. J Pineal ResMuxel SM, Laranjeira-silva MF, Carvalho-sousa CE, et al. The RelA/cRel nuclear factor-kappaB (NF-kappaB) dimer, crucial for inflammation resolution, mediates the transcription of the key enzyme in melatonin synthesis in RAW 264.7 macrophages. Pineal ResMEdrano-campillo P, Sarmiento-soto H, Alvarez-sanchez N, et al. Evaluation of the immunomodulatory effect of melatonin on the T-cell response in peripheral blood from systemic lupus erythematosus patients. J Pineal ResRadogna F, Paternoster L, Albertini MC, et al. Melatonin antagonizes apoptosis via receptor interaction in U937 monocytic cells. J Pineal ResCalvo JR, Gonzalez-yanes C, Maldonado MD. The role of melatonin in the cells of the innate immunity: a review. J Pineal ResXia MZ, Liang YL, Wang H, et al. Melatonin modulates TLR4-mediated inflammatory genes through MyD88- and TRIF-dependent signaling pathways in lipopolysaccharide-stimulated RAW264.7 cells. J Pineal ResLiu Z, Gan L, Luo D, et al. Melatonin promotes circadian rhythm-induced proliferation through Clock/histone deacetylase 3/c-Myc interaction in mouse adipose tissue. J Pineal ResLiu Z, Gan L, Xu Y, et al. Melatonin alleviates inflammasome-induced pyroptosis through inhibiting NF-kappaB/GSDMD sigJ Pineal ResEL Andaloussi S, Mager I, Breakefield XO, et al. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug DiscovTkach M, Thery C. Communication by extracellular vesicles: where we are and where we need to go. Fan R, Toubal A, Goni S, et al. Loss of the co-repressor GPS2 sensitizes macrophage activation upon metabolic stress induced by obesity and type 2 diabetes. Nat MedLackey DE, Olefsky JM. Regulation of metabolism by the innate immune system. Nat Rev EndocrinolLaranjeira-silva MF, Zampieri RA, Muxel SM, et al. Melatonin (L.) amazonensis infection by modulating arginine metabolism. J Pineal ResShi H, Chen Y, Tan DX, et al. Melatonin induces nitric oxide and the potential mechanisms relate to innate immunity against bacterial pathogen infection in ArabidopsisJ Pineal ResYoum YH, Nguyen KY, Grant RW, et al. The ketone metabolite beta-hydroxybutyrate blocks NLRP3 inflammasome-inflammatory disease. Nat MedHusted AS, Trauelsen M, Rudenko O, et al. GPCR-naling of metabolites. Cell MetabCarey BW, Finley LW, Cross JR, et al. Intracellular alpha-ketoglutarate maintains the pluripotency of embryonic stem cells. NatureEming SA, Wynn TA, Martin P. Inflammation and metabolism in tissue repair and regeneration. Kettner NM, Mayo SA, Hua J, et al. Circadian dysfunction induces leptin resistance in mice. Cell MetabGan L, Liu Z, Feng F, et al. Foxc2 coordinates inflammation and browning of white adipose by leptin-STAT3-. 2017;208: in press.Yang H, Hreggvidsdottir HS, Palmblad K, et al. A critical cysteine is required for HMGB1 binding to Toll-like receptor 4 and activation of macrophage cytokine release. Proc Natl Acad Sci USALi W, Li J, Ashok M, et al. A cardiovascular drug rescues mice from lethal sepsis by selectively attenuating a late-acting proinflammatory mediator, high mobility group box 1. Someya S, Yu W, Hallows WC, et al. Sirt3 mediates reduction of oxidative damage and prevention of age-related hearing loss under caloric restriction. Deng ZB, Poliakov A, Hardy RW, et al. Adipose tissue exosome-like vesicles mediate activation of macrophage-resistance. DiabetesCampoy I, Lanau L, Altadill T, et al. Exosome-like vesicles in uterine aspirates: a comparison of ultracentrifugation-lation protocols. J Transl MedMincheva-nilsson L, Baranov V, Nagaeva O, et al. Isolation and characterization of exosomes from cultures of tissue explants and Curr Protoc ImmunolReik W. Stability and flexibility of epigenetic gene regulation in mammalian development. NatureTahiliani M, Koh KP, Shen Y, et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner Yang Q, Liang X, Sun X, et al. AMPK/alpha-ketoglutarate axis dynamically mediates DNA demethylation in the Prdm16 promoter and brown adipogenesis. Cell Metab
16 of 16|
Yang Y, Jiang S, Dong Y, et al. Melatonin prevents cell death and mitochondrial dysfunction via a SIRT1-dependent mechanism during ischemic-stroke in mice. J Pineal ResCrooke A, Huete-toral F, Colligris B, et al. The role and therapeutic potential of melatonin in age-related ocular diseases. ResHardeland R. Melatonin and the theories of aging: a critical appraisal of melatonin’s role in antiaging mechanisms. J Pineal ResAgil A, Reiter RJ, Jimenez-aranda A, et al. Melatonin ameliorates low-grade inflammation and oxidative stress in young zucker diabetic fatty rats. J Pineal ResCarlberg C, Wiesenberg I. The orphan receptor family RZR/ROR, melatonin and 5-lipoxygenase: an unexpected relationship. J Pineal ResCervenka I, Agudelo LZ, Ruas JL. Kynurenines: tryptophan’s metabolites in exercise, inflammation, and mental health. Ungaro F, Tacconi C, Massimino L, et al. MFSD2A promotes endothelial generation of inflammation-resolving lipid mediators and reduces colitis in mice. GastroenterologyProchnicki T, Latz E. Inflammasomes on the crossroads of innate immune recognition and metabolic control. Cell MetabLiu PS, Wang H, Li X, et al. alpha-ketoglutarate orchestrates macrophage activation through metabolic and epigenetic reprogramNat ImmunolZavodnik IB, Lapshina EA, Cheshchevik VT, et al. Melatonin and succinate reduce rat liver mitochondrial dysfunction in diabetes. Physiol PharmacolDas N, Mandala A, Naaz S, et al. Melatonin protects against lipid-induced mitochondrial dysfunction in hepatocytes and inhibits stellate cell activation during hepatic fibrosis in mice. ResWu H, Zhang Y. Reversing DNA methylation: mechanisms, geZou X, Zhu Y, Park SH, et al. SIRT3-mediated dimerization of IDH2 directs cancer cell metab. and Tumor Growth. Cancer Res
Thomou T, Mori MA, Dreyfuss JM, et al. Adipose-derived circulating miRNAs regulate gene expression in other tissues. NatureHolmes D. Adipose tissue: adipocyte exosomes drive melanoma progression. Nat Rev EndocrinolHwang IY, Kwak S, Lee S, et al. Psat1-dependent fluctuations in ketoglutarate affect the timing of ESC differentiation. MetabVriend J, Reiter RJ. Melatonin feedback on clock genes: a theory involving the proteasome. J Pineal ResLi Z, Nickkholgh A, Yi X, et al. Melatonin protects kidney grafts from ischemia/reperfusion injury through inhibition of NF-and apoptosis after experimental kidney transplantation. ResKruidenier L, Chung CW, Cheng Z, et al. A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. NatureKato H, Tanaka G, Masuda S, et al. Melatonin promotes adipogenesis and mitochondrial biogenesis in 3T3-L1 preadipocytes. Pineal ResAlonso-vale MI, Peres SB, Vernochet C, et al. Adipocyte differentiation is inhibited by melatonin through the regulation of C/EBPbeta transcriptional activity. J Pineal ResWu SM, Lin WY, Shen CC, et al. Melatonin set out to ER stress signaling thwarts epithelial mesenchymal transition and peritoneal dissemination via calpain-mediated C/EBPbeta and NFkappaB cleavage. J Pineal Res Liu Z, Gan L, Zhang T, Ren Q, Sun C. Melatonin alleviates adipose ketoglutarate derived exosomes to e12455.
J. Pineal Res. wileyonlinelibrary.com/journal/jpi
Received: 4 September 2017Accepted: 6 November 2017
ORIGINAL ARTICLEMelatonin alleviates adipose inflammation through elevating ketoglutarate and diverting adipose-derived exosomes to macrophages in miceLu GanTiantian ZhangQian Ren
College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, ChinaCorrespondenceChao Sun, College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China.Email: sunchao2775@163.comFunding informationMajor National Scientific Research Projects, Grant/Award Number: 2015CB943102; National Nature Science Foundation of China, Grant/Award Number: 31572365; Key Sci-tech innovation team of Shaanxi province, Grant/Award Number: 2017KCT-24; Fundamental Research Funds for the Central Universities, Grant/Award Number:
Obesity is associated with macrophage infiltration and metabolic inflammation, both ketoglutarate ( KG) level in adipose tissue of obese mice. Mitochondrial inflammation and increased ratio of M2 to M1 macrophages by transporting of exoFurthermore, exosomal KG attenuated signal transducers and activators of trans -ketoglutarate, adipose inflammation, exosome, macrophage, melatonin
INTRODUCTIONObesity is a complex chronic disease and becomes a public health epidemic worldwide. Evidence suggests that inflammation is a central and reversible process in obesity and its comorbidities including type 2 diabetes, cardiovascular diseases, and neurodegenerative diseases. Obesity-low-grade and chronic inflammation are characterized by infiltration and activation of immune cells in adipose tissue.In particular, adipose tissue macrophages communicate with