Adipocyte microRNA-802 promotes adipose tissue inflammation and insulin resistance by modulating macrophages in obesity

Adipocyte microRNA-802 promotes adipose tissue inflammation and insulin resistance by modulating macrophages in obesity | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Adipocyte microRNA-802 promotes adipose tissue inflammation and insulin resistance by modulating macrophages in obesity Yue Yang , Bin Huang , Yimeng Qin , Danwei Wang , Yinuo Jin , Linmin Su , Yi Pan , Yanfeng Zhang , Yumeng Shen , Wenjun Hu , Zhengyu Cao , View ORCID Profile Liang Jin , View ORCID Profile Fangfang Zhang doi: https://doi.org/10.1101/2024.05.13.593999 Yue Yang 1 State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, School of life Science and Technology, China Pharmaceutical University. 24 Tongjiaxiang , Nanjing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bin Huang 1 State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, School of life Science and Technology, China Pharmaceutical University. 24 Tongjiaxiang , Nanjing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yimeng Qin 1 State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, School of life Science and Technology, China Pharmaceutical University. 24 Tongjiaxiang , Nanjing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Danwei Wang 1 State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, School of life Science and Technology, China Pharmaceutical University. 24 Tongjiaxiang , Nanjing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yinuo Jin 2 NanJing HanKai Academy , Jiangpu Street, Pukou District, Nanjing, China , 210000 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Linmin Su 1 State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, School of life Science and Technology, China Pharmaceutical University. 24 Tongjiaxiang , Nanjing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yi Pan 1 State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, School of life Science and Technology, China Pharmaceutical University. 24 Tongjiaxiang , Nanjing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yanfeng Zhang 1 State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, School of life Science and Technology, China Pharmaceutical University. 24 Tongjiaxiang , Nanjing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yumeng Shen 4 State Key Laboratory of Natural Medicines, China Pharmaceutical University , Nanjing 211198, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Wenjun Hu 1 State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, School of life Science and Technology, China Pharmaceutical University. 24 Tongjiaxiang , Nanjing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Zhengyu Cao 3 Jiangsu Key Laboratory of TCM Evaluation and Translational Research, School of Traditional Chinese Pharmacy, China Pharmaceutical University , Nanjing 211198, PR China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: 1620194592{at}cpu.edu.cn ljstemcell{at}cpu.edu.cn zycao1999{at}hotmail.com Liang Jin 1 State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, School of life Science and Technology, China Pharmaceutical University. 24 Tongjiaxiang , Nanjing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Liang Jin For correspondence: 1620194592{at}cpu.edu.cn ljstemcell{at}cpu.edu.cn zycao1999{at}hotmail.com Fangfang Zhang 1 State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, School of life Science and Technology, China Pharmaceutical University. 24 Tongjiaxiang , Nanjing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Fangfang Zhang For correspondence: 1620194592{at}cpu.edu.cn ljstemcell{at}cpu.edu.cn zycao1999{at}hotmail.com Abstract Full Text Info/History Metrics Preview PDF Abstract Adipose tissue inflammation is now considered to be a key process underlying metabolic diseases in obese individuals. However, it remains unclear how adipose inflammation is initiated and maintained or the mechanism by which inflammation develops. We found that microRNA-802 ( miR-802 ) expression in adipose tissue is progressively increased with the development of dietary obesity in obese mice and humans. The increasing trend of miR-802 preceded the accumulation of macrophages. Adipose tissue-specific knockout of miR-802 lowered macrophage infiltration and ameliorated systemic insulin resistance. Conversely, the specific overexpression of miR-802 in adipose tissue aggravated adipose inflammation in mice fed a high-fat diet. Mechanistically, miR-802 activates noncanonical and canonical NF-κB pathways by targeting its negative regulator, TRAF3. Next, NF-κB orchestrated the expression of chemokine and SREBP1, which translated into strong recruitment and M1-like polarization of macrophages. Our findings indicate that miR-802 endows adipose tissue with the ability to recruit and polarize macrophages, which underscores miR-802 as an innovative and attractive candidate for miRNA-based immune therapy for adipose inflammation. Introduction Obesity is a very powerful health determinant or indicator that facilitates the development and progression of several metabolic diseases, including insulin resistance and type 2 diabetes( 1 , 2 ). Adipose tissue is a highly dynamic metabolic organ that plays a central role in the regulation of energy homeostasis and controls glucose metabolism and insulin sensitivity( 3 , 4 ). A hallmark of obesity is low-grade chronic inflammation in adipose tissue, marked by the accumulation of macrophages and other immune cells and by an increase in the levels of pro-inflammatory cytokines( 5 - 7 ). Persistent adipose tissue inflammation is now considered to have a pivotal role in obesity-associated insulin resistance( 8 , 9 ). Resetting the immunological balance in obesity could represent an innovative approach for the management of insulin resistance and diabetes( 10 , 11 ). However, the early triggers and signals that sustain adipose tissue inflammation in obesity remain elusive, limiting our ability to effectively intervene this growing public health issue. Macrophages accumulate in the adipose tissue of obese mice and humans, where they form crown-like structures surrounding dying or dead adipocytes and are key contributors to inflammation and obesity-induced insulin resistance( 12 , 13 ). The number of adipose tissue macrophages is tightly linked to the degree of insulin resistance and metabolic dysregulation( 14 , 15 ). Ablation of pro-inflammatory adipose tissue macrophages leads to a rapid improvement in insulin sensitivity and glucose tolerance, associated with marked decreases in local and systemic inflammation in obese mice( 16 , 17 ). Targeting the major inflammatory pathways is sufficient to counteract obesity-related systemic inflammation and insulin resistance( 18 , 19 ). However, the molecular links between lipid-overloaded adipocytes and inflammatory macrophages in obese adipose tissue remain elusive. MicroRNAs (miRNAs) are small non-coding RNAs that post transcriptionally regulate gene expression by binding to specific regions of target genes to prevent translation or promote mRNA degradation( 20 ). Emerging evidence suggests that miRNAs are key regulators in a variety of important metabolic organs and substantial contributors to the pathogenesis of complex diseases, including obesity-associated metabolic diseases( 21 , 22 ). In the adipose tissue, miRNAs have dramatic effects on regulating the pathways that control a range of processes including lipogenesis, inflammation, and insulin signaling( 23 , 24 ). Moreover, mice with alterations in the levels of miRNAs in adipocytes show significantly enhanced inflammation and insulin resistance after feeding with a high-fat diet (HFD), further confirming the contribution of miRNAs to obesity-induced phenotypes( 25 , 26 ). Therefore, adipose-derived miRNAs hold great promise for understanding adipose tissue dysfunction and the relationship between chronic inflammation and obesity and insulin resistance. In this study, we demonstrated that microRNA-802 ( miR-802 ) promotes inter-cellular communication between lipid-overloaded adipocytes and macrophages, ultimately leading to adipose tissue inflammation and insulin resistance. Adipocyte miR-802 levels are positively associated with obesity in mice and humans. Adipose tissue-specific overexpression of miR-802 in mice fed an HFD exhibited increased severity of systemic insulin resistance compared with wild-type (WT) mice, which was accompanied by macrophage infiltration and a marked increase in adipose tissue inflammation. Adipose tissue-specific knockout of miR-802 achieved the opposite result. Co-culture and other in vitro experiments revealed a vicious cycle of interactions between macrophages and adipocytes ectopically expressing miR-802 . We established that miR-802 expression is an inflammatory signal in adipocytes, and this effect occurs through sensitization of the NF-κB signaling pathway. Altogether, our data raise the possibility that manipulation of this microRNA action axis has therapeutic potential for treating adipose inflammation. Results miR-802 elevation precedes macrophage accumulation Consistent with previous studies from our and other laboratories( 27 , 28 ), adipose from obesity mice showed significantly higher miR-802 expression than those from normal mice. To evaluate whether miR-802 is involved in adipose inflammation and insulin resistance, we examined the expression profile of miR-802 . we observed that miR-802 progressively increased in adipose tissue from week 4 with the development of obesity in mouse models of genetic and dietary obesity ( Figure 1A, B and Figure S1A, B ). We next compared the expression of miR-802 in different adipose depots and found that it was the highest in epididymal white adipose tissue (epiWAT) ( Figure 1C ). We further isolated mature adipose tissue and stromal vascular fraction (SVF) from epiWAT to examine the expression of miR-802 . We found that miR-802 expression was substantially higher in mature adipocytes than in SVF in both mice fed a normal chow diet (NCD) and those fed an HFD ( Figure 1D ). Through in vitro experiments, we found that miR-802 was dramatically increased in the insulin resistance cell models ( Figure 1E, F and Figure S1C, D ). These findings suggest that upregulation of miR-802 in adipocytes may be functionally involved in the pathogenesis of obesity-associated disorders. Download figure Open in new tab Figure 1 Obesity induced miR-802 elevation precedes macrophage accumulation (A) mRNA abundance of miR-802 in the epiWAT of db/db or control mice at 4, 6, 8, 12, and 16 weeks ( n= 5). (B) mRNA abundance of miR-802 in the epiWAT of mice fed a normal chow diet (NCD) or HFD for 0, 2, 4, 8, 16, 24, and 32 weeks ( n= 5). (C) The expression level of miR-802 in epiWAT, scWAT and BAT isolated from mice on HFD for 16 weeks or 10 weeks db/db mice ( n =7). (D) Copy number of miR-802 in mature adipocytes and stromal vascular fraction (SVF) of epiWAT isolated from mice on NCD or HFD for 16 weeks ( n= 5). (E–F) miR-802 expression levels in insulin resistance 3T3-L1 cell models (E) and insulin resistance WAT SVF cells models (F). (G) F4/80 and CD11b positive cells in SVFs isolated from the epiWAT of mice fed an HFD for 2, 4, 6, 8, 16, and 24 weeks ( n =5). (H) Representative images of F4/80 staining (left) and quantification of crown-like structures (CLSs; right) in the epiWAT of mice fed an HFD ( n =5). (I) Expression levels of miR-802 in human subcutaneous adipose tissue ( n normal =25, n obesity & IR =70). Scatter plots of miR-802 expression versus BMI (J) and HOMA-IR (K). Pearson’s correlation coefficients (r) are shown. The fold of miR-802 was calculated using 2 -ΔΔCt . Data represent mean ± SEM. P -values obtained using a two-tailed unpaired Student’s t -test (E, F, I) or two-way ANOVA (A–D, G) are indicated. *P <0.05, **P <0.01, ***P <0.001. Relative levels of miR-802 were normalized to U6 . epiWAT: epididymal white adipose tissue, scWAT: subcutaneous white adipose tissue, BAT: brown adipose tissue. Initial studies have indicated that macrophages are responsible for most inflammatory events in adipose tissue ( 12 , 13 ). However, what initiates macrophage infiltration or the resultant inflammatory cascade is still not well defined. We hypothesized that the elevation of miR-802 in adipocytes is associated with adipose inflammation and insulin resistance. To test this idea, we examined the correlation between miR-802 elevation and macrophage infiltration during the progression of diet-induced obesity (DIO). We first carried out a set of flow cytometric analyses to determine the dynamic alterations of macrophages in collagenase-digested SVF from epiWAT. From week 8, the number of double positive CD11b/F4/80 macrophages gradually increased in obese mice ( Figure 1G , Figure S1E ). Immunohistochemical analysis of F4/80 expression also revealed that the number of macrophages continued to increase in the epididymal fat pads of obese mice as compared to that in mice on a normal diet ( Figure 1H ). The dynamic increase in miR-802 preceded the infiltration of macrophages, indicating that miR-802 may play a critical role in the occurrence of adipose inflammation. To gain additional insight into the clinical importance of miR-802 in obese fat, we analyzed the expression of miR-802 in samples of human subcutaneous adipose tissue. Levels of miR-802 expression were significantly higher in obese subjects (body mass index [BMI]=38.30±5.82 kg/m 2 , fasting plasma glucose=8.39±1.54 mM, homeostatic model assessment for insulin resistance (HOMA-IR)=3.77±1.97) than in lean ones (BMI=20.55±0.97 kg/m 2 , fasting plasma glucose=4.84±0.53 mM, HOMA-IR=0.21±0.06) ( Figure 1I and Figure S1F ). Pearson’s correlation analysis showed that the BMI and HOMA-IR were positively associated with miR-802 abundance in subcutaneous fat ( Figure 1J, K ). The same phenomenon was also observed in RNA-FISH analysis ( Figure S1G ), indicating that upregulation of miR-802 in the adipose tissue during obesity is conserved in humans. Adipose-selective overexpression of miR-802 aggravates inflammatory cascade in obese mice To further assess the role of adipocyte miR-802 , we generated adipose-selective miR-802 KI mice by crossing miR-802 ki/ki mice ( 27 ) with animals expressing Cre recombinase under the control of the promoter of adiponectin ( Figure S2A, B ). Real-time PCR analysis confirmed that the overexpression of miR-802 was restricted in the adipose tissues of the miR-802 KI mice, miR-802 expressions were up-regulated about 150 times, whereas its expression in other organs was not affected ( Figure S2C ), and the upregulation of miR-802 was limited to adipocytes and were not observed in SVFs ( Figure S2D ). There was no obvious difference in food intake, body weight, glucose content, and adiposity between miR-802 KI mice and their WT littermates in both male and female when they were fed with NCD ( Figure S2E-H ). We then fed the mice an HFD and performed metabolic and histological analyses. We detected the presence of adipose inflammation, typified by macrophage crown-like structures (CLSs) in epiWAT at 8 weeks in miR-802 KI mice, which was earlier than their WT littermates, and the number of CLSs was almost doubled at 16 weeks ( Figure 2A ). No change of CLSs was between two groups fed with NCD ( Figure S2I ). Consistently, flow cytometric analysis showed that HFD-induced elevation in the number of CD11b + F4/80 + macrophages in the SVF of epiWAT in adipose-specific miR-802 KI mice was significantly higher than that in WT littermates in both male and female ( Figure 2B and Figure S2J ). In miR-802 KI mice fed on HFD for 16 weeks, the number of classically activated proinflammatory M1 macrophages (defined as CD86 + CD206 - ) was significantly higher than that of alternatively activated anti-inflammatory M2 macrophages (defined as CD86 - CD206 + ) in epiWAT ( Figure 2C and Figure S2K ). In line with this finding, epiWAT from dietary-obese miR-802 KI mice exhibited obviously higher mRNA expression of the M1 macrophage–related genes ( Ccl2 , Il-1β, Il-6, Tnf-α, Inos, and Ifn-γ ) but significant reductions of M2 macrophage–related genes ( Il-10, Ym1, Arg1 , and Fizz1 ) ( Figure 2D ). Similarly, HFD also increased the level of several inflammatory factors (chemokine ligand 2 [CCL2], interleukin [IL]-1β, IL-6, and tumor necrosis factor [TNF]-α) in the serum of miR-802 KI mice ( Figure 2E-H ). Download figure Open in new tab Figure 2 Adipose tissue-specific overexpression of miR-802 exacerbates adipose tissue inflammation and leads to metabolic dysfunction (A) Representative images of F4/80 staining (top) and quantification of CLSs (bottom) in epiWAT of WT or miR-802 KI mice on HFD for 0, 8, and 16 weeks ( n =5). Scale bar: 40 μm. (B) Percentage of F4/80 + /CD11b + total macrophages in the epiWAT of miR-802 KI and KI-control mice fed with HFD ( n =5). (C) M1 (CD86 + CD206 - ) and M2 (CD206 + CD86 - ) within the macrophage population ( n =5). (D) qRT-PCR analysis for mRNA levels of the M1 and M2 markers in the epiWAT of mice on KI-control or miR-802 KI at 16 weeks ( n =5). (E–H) Serum levels of CCL2 (E), IL-1β (F), IL-6 (G), and TNF-α (H) of miR-802 KI and control mice fed with HFD for 0, 8, 16, and 30 weeks ( n =5). (I, J) Dynamic changes in body weight (I) and glucose (J) in WT and miR-802 KI mice during 30 weeks of HFD feeding ( n =5). (K, L) Fat mass of whole body (K) and individual tissues (L) ( n= 7). (M) Representative coronal section MRI images and visceral and subcutaneous adipose tissue volume of HFD-fed control and miR-802 KI mice ( n= 5). (N, O) Area over the curve (AOC) of the blood glucose level was calculated via intraperitoneal glucose tolerance tests (IPGTTs, 2 g/kg, N, n =5) or intraperitoneal insulin tolerance tests (IPITTs; 0.75 U/kg, O, n =5). (P) Fasting insulin (FINS) levels of HFD-fed mice were measured by ELISA ( n =7). (Q) HOMA-IR was calculated with the equation FBG (mmol l −1 ) × FINS (mIU l −1 ))/22.5. Data represent mean ± SEM. Differences between groups were determined by ANOVA (B–J, L, and N–Q) or two-tailed unpaired Student’s t -test (K). *P <0.05, ***P <0.001. Gene levels were normalized to 18S RNA abundance. We next explored whether the aggravation of adipose inflammation in adipose-selective miR-802 KI mice in both male and female were associated with exacerbation of metabolism and insulin sensitivity. We found that in miR-802 KI mice, HFD induced weight gain ( Figure 2I and Figure S2L ) and hyperglycemia ( Figure 2J ) both in male and female. HFD also induced adiposity in miR-802 KI mice, which mainly manifested in the expansion of visceral WAT ( Figure 2K, L ). MRI analysis confirmed that HFD induced an increase in visceral WAT in miR-802 KI mice ( Figure 2M ). We next monitored the dynamic changes in insulin sensitivity at different time points (0, 4, 8, 16, and 30 weeks) after feeding the two groups of mice with an HFD. As expected, miR-802 KI mice on a HFD exhibited progressive development of glucose intolerance ( Figure 2N and Figure S2M-Q ) and insulin resistance ( Figure 2O and Figure S2R-V ) at 8 weeks, as compared to their WT littermates. These differences became even more obvious after 16 and 30 weeks, coupled with an increase in fasting insulin levels ( Figure 2P ) and HOMA-IR ( Figure 2Q ). Collectively, these effects of adipose-selective overexpression of miR-802 show that miR-802 is required for the recruitment of macrophages into obese adipose tissue and for the initiation and propagation of the inflammatory cascade. miR-802 depletion ameliorates obesity-induced metabolic dysfunction Given the striking effects of adipose-selective overexpression of miR-802 on metabolism, we next investigated whether selectively ablated miR-802 in adipose tissue could improve metabolic disturbance and inflammation induced by obesity. We generated miR-802 conditional knockout mice using the Cre/Lox system ( Figure S3A ). miR-802 fl/fl were crossed with Adipoq -Cre transgenic animals to selectively ablate miR-802 in adipose tissues ( Figure S3B ). Expression analysis showed that total miR-802 levels were reduced by approximately 70% in the adipose tissue but not in SVFs of miR-802 KO mice compared with WT littermates ( Figure S3C, D ). The knockout of miR-802 in adipose tissue did not alter food intake, body weight, glucose level, and adiposity (data not shown); however, this approach could prevent HFD-induced weight gain and hyperglycemia ( Figure 3A, B and Figure S3E ). Adipose-selective ablation of miR-802 also alleviated HFD-induced adiposity, mainly by reducing the expansion of visceral WAT, including epiWAT and retro-peritoneal WAT ( Figure 3C, D ). MRI analysis confirmed this result ( Figure 3E ). Histological and FACS analysis showed that miR-802 depletion reduced macrophage infiltration, which mainly manifested as a decrease in the number of CLSs and macrophages ( Figure 3F, G and Figure S3F ), but had little effect between two groups fed with NCD ( Figure S3G ). Notably, the miR-802 KO mice exhibited obvious reductions in mRNA expression of the M1 macrophage-related genes ( Ccl2 , Il-1β, Il-6, Tnf-α, Inos, and Ifn-γ ) but significant upregulation of M2 macrophage-related genes ( Fizz1, Ym1, Arg1 , and Il-10 ) ( Figure 3H ). The miR-802 KO mice also markedly blunted HFD-induced elevation in serum levels of several inflammatory factors (TNF-α, IL-6, IL-1β, and CCL2) ( Figure 3I ). In addition, the insulin resistance and glucose intolerance induced by an HFD were ameliorated by miR-802 depletion ( Figure 3J, K and Figure S3H-M ). These phenomena were the same both in male and female miR-802 KO mice. Download figure Open in new tab Download figure Open in new tab Figure 3 Adipose tissue–specific ablation of miR-802 protects mice from obesity-induced metabolic dysfunction (A-B) Dynamic changes in body weight (A) and glucose (B) of KO control and miR-802 KO mice during 30 weeks of HFD feeding ( n =7). (C-D) Fat mass of whole body (C) and individual tissues (D) ( n =7). (E) Representative coronal section MRI images and visceral and subcutaneous adipose tissue volume of HFD-fed control and miR-802 KO mice ( n =5). (F) Representative images of F4/80 staining (left) and quantification of CLSs (right) in epiWAT of WT or miR-802 KO mice on HFD for 0, 8, and 16 weeks ( n =5). Scale bar: 40 μm. (G) Cells isolated from SVFs of epiWAT in miR-802 KO and WT mice fed with HFD for 8, 16, and 24 weeks were subjected to flow cytometry analysis for percentage of CD11b + /F4/80 + total macrophages ( n =5). (H) qRT-PCR analysis for the mRNA levels of the M1 and M2 markers in epiWAT of mice on HFD 16 weeks ( n =5). (I) Serum levels of CCL2, IL-1β, IL-6, TNF-α determined with ELISA ( n =5). (J-K) AOC of the blood glucose level was calculated via IPGTT (1.5 g/kg, J, n =5) or IPITT (0.75 U/kg, K, n =5). (L) Representative images of F4/80 staining and quantification of CLSs ( n =5) in the epiWAT of WT or miR-802 KO mice. Scale bar: 40 μm. (M-N) The percentage of CD11b + /F4/80 + total macrophages (M, n =5) and M1 (CD86 + CD206 - ), and M2 (CD206 + CD86 - ) within the macrophage population (N, n =5) in the SVFs isolated from epiWAT in the HFD-control or miR-802 eWAT KO /HFD mice. (O) Serum levels of TNF-α, IL-6, IL-1β, CCL2 determined with ELISA ( n =6). (P-Q) IPGTT (P) and IPITT (Q) were performed in HFD-control mice or miR-802 eWAT KO /HFD mice( n =5). Data represent mean ± SEM. Differences between groups were determined by ANOVA (A-B, D, E-K, N-Q) or two-tailed unpaired Student’s t test (C, L-M). ***P < 0.001. Gene levels were normalized to 18S rRNA abundance. We next examined the activity of miR-802 in obese adipose tissues in which inflammation had already been established. We performed the acute deletion of adipocyte miR-802 that did not influence whole-body weight. To address this question, we depleted miR-802 in eWAT using an approach of adeno-associated virus (AAV, miR-802 eWAT KO ) to 16-week-old DIO mice that had been fed an HFD since they were 4 weeks old ( Figure S3N ). After 7 days, we detected 70% lower expression of miR-802 compared with the control in the epididymal fat pad; miR-802 expression was unaffected in other organs ( Figure S3O ). The weight and the number of CLSs were lowered with miR-802 sponge treatment ( Figure S3P , Figure 3L ), and the reduction in macrophage infiltration was confirmed by CD11b and F4/80 flow cytometry analysis ( Figure 3M , Figure S3Q ). Phenotypic analysis indicated that miR-802 inhibitor also lowered the M1 (CD86 + CD206 - ) macrophage fraction, while it increased the M2 macrophage (CD206 + CD86 - ) fraction ( Figure 3N ). DIO led to upregulated mRNA expression of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) in the adipose tissue that was suppressed in the miR-802 eWAT KO mice ( Figure 3O ). miR-802 inhibitor treatment also ameliorated insulin resistance and glucose intolerance in DIO mice ( Figure 3P, Q ). These results clearly show that miR-802 inhibitor treatment suppresses preexisting adipose inflammation, which strongly suggests that miR-802 is required for the maintenance of inflammatory reactions in obese adipose tissue. Interplay between miR-802 ectopically expressed adipocytes and macrophages We next analyzed the cellular interplay via which inflammation develops in obese adipose tissue. Based on the findings of the in vivo experiments summarized above, we hypothesized that obese adipose tissue upregulates miR-802 , and miR-802- overexpressing adipocytes in turn recruit and activate macrophages. To test this hypothesis, we first co-cultured isolated primary macrophages with WAT SVF cells isolated from lean or obese mice to determine whether obese adipose tissue can affect macrophages ( Figure 4A ). EdU assays and flow cytometric analysis showed that obese WAT SVF induced the proliferation of macrophages, whereas lean fat did so only mildly ( Figure S4A, B ). Transwell co-culture further showed that obese WAT SVF also promoted macrophage migration and invasion ( Figure 4B ). We next explored the effects of obese WAT SVF on the characteristics of macrophages. After co-culture macrophages and WAT SVF of obese mice, isolated primary macrophages had elevated expression of classical activation (M1-like) marker CD86, whereas the alternative activation marker (M2-like) CD206 was decreased ( Figure 4C ). The results of ELISA indicated that obese WAT SVF-induced macrophages were predominantly polarized to pro-inflammatory macrophages ( Figure 4D ). When we plated primary macrophages in Boyden chambers and treated them with a medium conditioned with obese WAT SVF or lean WAT SVF, the number of macrophages that migrated through the pores between chamber wells with obese WAT SVF conditioned medium was significantly higher than the number of cells cultured in lean WAT SVF conditioned medium ( Figure 4E ). ELISA results showed that conditioned medium of obese WAT SVF can secrete more humoral factors known to induce macrophage migration, especially CCL2 ( Figure 4F ). Download figure Open in new tab Figure 4 Interplay between miR-802 ectopically expressed adipocytes and macrophages (A) Flowchart of the co-culture experiments designed for determining WAT SVF of obese adipose tissue can affect macrophages. (B) Obesity promoted macrophage migration and invasion in transwell migration and invasion assay. (C) M1 (CD86 + CD206 - ) and M2 (CD206 + CD86 - ) within the macrophage population. (D) The levels of TNF-α, IL-6, IL-1β, and CCL2 determined with ELISA. (E) Migration and invasion ability of macrophages treated with a medium conditioned with obese or lean SVF cells. (F) Chemokine levels in the medium conditioned with obese or lean SVF cells. (G) miR-802 induced 3T3-L1 cells recruitment more RAW 264.7 cells in transwell migration and invasion assay. (H) miR-802 mimics-transfected 3T3-L1 cells promoted RAW 264.7 cells M1-like polarization. Data represent mean ± SEM. Differences between groups were determined by ANOVA (D, F). **P <0.01, ***P <0.001. To further confirm the function of miR-802 in adipose tissue, the adipocyte cell line 3T3-L1 was transfected with miR-802 mimics ( miR-802 ) or miR-802 inhibitor ( anti-miR-802 ). We then explored the effect of miR-802 ectopically expressed 3T3-L1 cells on the macrophage cell line RAW 264.7 in co-culture. The knockdown and overexpression efficiencies were approximately 80% and 240-fold, respectively ( Figure S4C ). First, we found that miR-802 -overexpressing 3T3-L1 cells had no effect on the proliferation and lipid droplet production of RAW 264.7 cells ( Figure S4D, E, F ). However, miR-802 -overexpressing 3T3-L1 cells promoted the migration and invasion of RAW 264.7 cells, whereas 3T3-L1 cells knocked down by anti-miR-802 had the opposite effect ( Figure 4G ). miR-802 mimics-transfected 3T3-L1 cells also promoted RAW 264.7 cells M1-like polarization ( Figure 4H ). We also found higher level of CCL2 in the medium conditioned with miR-802 -overexpressed 3T3-L1 cells ( Figure S4G ). Collectively, the results of the co-culture experiments showed that the interaction between miR-802 ectopically expressed adipocytes and macrophages is crucial for the initiation and propagation of adipose tissue inflammatory cascades. miRNA-802 promotes adipose tissue inflammation and insulin resistance by targeting TRAF3 To better understand the role of miR-802 in regulating macrophage-mediated adipose tissue inflammation and insulin resistance, we next set out to identify the target genes of miR-802 in adipocytes. For that, we utilized RNA-sequencing of samples derived from the epiWAT of miR-802 KI mice and their WT littermates. A total of 191 differentially expressed genes were identified. The cutoff criteria for significant differentially expressed genes were log fold change > 2 and adjusted p value < 0.05. We identified 29 upregulated genes and 57 downregulated genes ( Figure 5A left, Supplementary Table 2 ). Then, we combined the multiMiR database( 29 ) with prediction programs (TargetScan Release 7.0 and miRPathDB) to predict possible targets of miR-802 . Among 18 tested potential targets, TNF receptor-associated factor 3 ( Traf3 ) was identified as a genuine target of miR-802 , which was among the genes that were significantly downregulated in miR-802 KI versus WT epiWAT ( Figure 5A right, Figure S5A ). Indeed, we observed that TRAF3 was decreased in both mRNA and protein levels in obese humans and in various obese mice ( Figure 5B, C and Figure S5B, C ). The targeting potential between miR-802 and Traf3 was also observed in miR-802 KI and miR-802 KO mice ( Figure 5D and Figure S5D ). We then demonstrated miR-802 binding to the Traf3 3’-UTR by transiently co-expressing luciferase reporter fusions of Traf3 and miR-802 mimics in 3T3-L1 cells. The results of these co-transfection experiments indicated that the relative luciferase activity in Traf3 3’-UTR-expressing cells was significantly inhibited by miR-802 , whereas other Traf3 3’-UTR fusions that contained mutations ( Traf3 -MUT) in miR-802 binding sites were unaffected ( Figure 5E ). Consistent with these findings, the ectopic expression of miR-802 in 3T3-L1 cells effectively regulated the mRNA and protein levels of endogenous Traf3 ( Figure 5F ). Moreover, we conducted anti-Ago2 RIP in 3T3-L1 cells, which transiently overexpressed miR-802 . Endogenous Traf3 pulldown by Ago2 was specifically enriched in miR-802 -transfected cells ( Figure 5G ) and vice versa ( Figure S5E ). Overall, these data suggest that Traf3 is a direct target of miR-802 . Download figure Open in new tab Figure 5 Adipose miR-802 modulates infiltration and polarization of macrophages by directly targeting Traf3 (A) Heat map illustrating the differential expression of mRNAs in the epiWAT of miR-802 KI mice compared to their WT miR-802 ki/ki littermates ( n =3). (B) mRNA and protein levels of TRAF3 in human subcutaneous adipose tissues from obese and normal individuals ( n normal =4 and n obesity&IR =9). (C, D) mRNA and protein levels of TRAF3 in the epiWAT of HFD mice (C, n =3) or miR-802 KI mice (D, n =3). (E) Relative luciferase activity in 3T3-L1 cells co-transfected with miR-802 mimics and a luciferase reporter containing either Traf3 - WT or Traf3 - MUT . Data are presented as the relative ratio of Renilla luciferase activity to firefly luciferase activity. (F) mRNA and protein levels of TRAF3 in 3T3-L1 cells transfected with miR-802 mimics or miR-802 inhibitor. (G) Anti-Ago2 RIP was performed in 3T3-L1 cells transiently overexpressing miR-802 , followed by qRT-PCR to detect Traf3 associated with Ago2 (nonspecific IgG served as a negative control). (H) mRNA and protein levels of TRAF3 in the epiWAT of control, miR-802 KI, Traf3 eWAT KI , and miR-802 KI & Traf3 eWAT KI mice ( n =3–5). (I) Representative images of F4/80 staining and quantification of CLSs ( n =5). (J) M1 (CD86 + CD206 - ) and M2 (CD206 + CD86 - ) within the macrophage population ( n= 5). (K) qRT-PCR analysis of the mRNA levels of M1 and M2 markers in the epiWAT of HFD-fed control, Traf3 eWAT KI , miR-802 KI, and miR-802 KI & Traf3 eWAT KI ( n= 5). (L, M) Dynamic changes in body weight (L), glucose level (M), fat mass (N), glucose tolerance (O), and HOMA-IR (P) of control, miR-802 KI, Traf3 eWAT KI , and miR-802 KI & Traf3 eWAT KI mice during 30 weeks of HFD feeding ( n= 7). Data represent mean ±SEM. Differences between groups were determined by ANOVA (E, F, J–P). ***P <0.001. MiR-802 abundance was normalized to U6 level, and other genes levels were normalized to 18S rRNA abundance. To address whether the increase in inflammation and insulin resistance in miR-802 KI mice was attributable to decreased Traf3 , 8-week-old male miR-802 KI mice were given AAV- Adipoq - Traf3 ( miR-802 KI & Traf3 eWAT KI ) through epididymal fat pad. At 1 week after injection of adeno-associated virus (AAV) expressing Traf3 , Traf3 expression in the epiWAT of miR-802 -KI mice was increased to a level similar to that in WT mice ( Figure 5H ). Notably, upregulation of Traf3 led to significant decreases in the counts of total macrophages ( Figure 5I and Figure S5F ) and M1 macrophages ( Figure 5J and Figure S5G ) in the epiWAT of HFD-fed miR-802 KI mice compared with those treated with AAV8-vector. Coherently, the increased expression of M1 macrophage-associated proinflammatory factors ( Tnfα, Il-6, Inos, Il-1β , and Ifn-γ ) in the epiWAT of HFD-fed miR-802 KI mice was reversed by the AAV-mediated upregulation of Traf3 ( Figure 5K ). In addition, Traf3 eWAT KI reversed the weight gain ( Figure 5L ), hyperglycemia ( Figure 5M ), and adiposity ( Figure 5N ) induced by overexpression of miR-802. MRI analysis further confirmed that Traf3 can reverse the increase in visceral fat caused by miR-802 ( Figure S5H ). Consistent with these findings, upregulation of Traf3 led to restoration of glucose intolerance ( Figure 5O ) and insulin resistance ( Figure S5I ) after 16 weeks of Traf3 eWAT treatment in HFD-fed miR-802 KI mice, coupled with a decrease in fasting insulin levels ( Figure S5J ) and ameliorative HOMA-IR ( Figure 5P ). Taken together, these findings support the notion that elevated miR-802 induces macrophage recruitment and polarization at least partly via downregulation of Traf3 , thereby leading to adipose tissue inflammation and insulin resistance. miR-802 activates noncanonical and canonical NF-κB pathways leading to macrophage recruitment To further unravel the mechanism by which inhibition of TRAF3 expression induces adipose tissue inflammation, we looked for TRAF3 downstream cascades. Several studies have suggested that TRAF3 negatively regulates the noncanonical NF-κB pathway( 30 - 32 ), which is consistent with the KEGG analysis based on our RNA-seq results ( Figure S6A ). This prompted us to measure NF-κB inducing kinase (NIK) protein levels and to explore the processing of p100 to p52. To test whether miR-802 is required for the suppression of NIK protein levels, western blot analysis of NIK was performed on miR-802 -overexpressed 3T3-L1 cells and miR-802 ectopically expressed adipose tissue. As shown in Figure 6A and B , profound accumulation of NIK was observed in all cells with overexpression of miR-802 , which correlated well with decreased TRAF3. miR-802 selectively ablated adipose tissues showed the opposite result ( Figure S6B ). Processing of the p100 precursor to p52, the hallmark of noncanonical NF-κB activation, was also assessed by immunoblotting. Although 3T3-L1 cells exhibited the normal kinetics of p100 processing with substantial p52 accumulation by 48 h after treatment with the empty vector, miR-802 -overexpressing 3T3-L1 cells and miR-802 selectively overexpressed adipose tissues showed constitutive and total processing of the p100 precursor protein ( Figure 6C, D ). On the contrary, there was less accumulation of p52 in miR-802 selectively deleted adipose tissue ( Figure S6C ). As expected, IKK-α phosphorylation levels were also enhanced in 3T3-L1 cells and in the epiWAT of miR-802 KI mice ( Figure 6E, F and Figure S6D ). To confirm that miR-802 activates the noncanonical NF-κB pathway through TRAF3, Traf3 plasmid was transfected into miR-802 -overexpressing 3T3-L1 cells, then NIK protein levels and processing of p100 to p52 were again assessed by immunoblotting. As shown in Figure 6G , Traf3 restored the levels of NIK and the processing of p100 to p52 in miR-802 -overexpressing 3T3-L1 cells. Moreover, these results were confirmed in miR-802 KI mice ( Figure 6H ), indicating that miR-802 regulated the noncanonical NF-κB pathway via TRAF3. Download figure Open in new tab Figure 6 miR-802 activates noncanonical and canonical NF-κB pathways by recruiting macrophages (A, B) NIK protein levels in 3T3-L1 cells transfected with miR-802 mimics or miR-802 inhibitor (A) in the epiWAT of miR-802 KI mice (B, n =3). (C, D) P100/52 protein levels in 3T3-L1 cells transfected with miR-802 mimics or miR-802 inhibitor (C), and in the epiWAT of miR-802 KI mice (D, n =3). (E, F) Protein levels of IKK-α and P-IKK-α in 3T3-L1 cells transfected with miR-802 mimics or miR-802 inhibitor (E) in the epiWAT of miR-802 KI mice (F, n =3). (G, H) Overexpression of Traf3 reverses the protein levels of NIK, P-IKK-α, and P100/52 in 3T3-L1 cells (G) and in the epiWAT of miR-802 KI mice (H, n =3). (I, J) Protein levels of some major canonical NF-κB signaling targets in the epiWAT of miR-802 KI mice (I, n =3) and Traf3 eWAT KI rescued mice (J, n =3). (K, L) qRT-PCR (K) and ELISA (L) were performed to detect major chemokine levels. Data represent mean ± SEM. Differences between groups were determined by ANOVA (K–L). ***P <0.001. Genes levels were normalized to 18S rRNA abundance. Previous studies have suggested that TRAF3 also suppresses activation of the canonical NF-κB pathway ( 33 , 34 ). To verify whether miR-802 can regulate the canonical NF-κB pathway through TRAF3, nuclear extract was harvested from the adipose tissue of 16-week-old WT and mice with adipose-selective forced expression of miR-802 . NF-κB activation status was then assessed by measuring p65, IκBα, and some major targets associated with the pathway. As shown in Figure 6I , p65 and IκBα were phosphorylated, and some major targets of canonical NF-κB signaling, such as MnSOD, FAS, and iNOS, were activated in miR-802 KI mice. As expected, the activation of canonical NF-κB signaling in miR-802 KI mice was partially reversed by overexpression of Traf3 ( Figure 6J ). To assess the potential impact of heightened NF-κB activity, we harvested mRNA from the adipose tissue of WT and miR-802 KI mice and analyzed the expression levels of multiple noncanonical and canonical NF-κB pathway target genes( 35 , 36 ) using qRT-PCR. Here, we observed that the expression levels of Ccl2 , Ccl3 , Ccl5 , Ccl20 , and Cxcl2 were elevated in the adipose tissue of miR-802 KI mice ( Figure 6K ), which is consistent with ELISA results using the serum of miR-802 KI mice ( Figure 6L ). Taken together, these data indicate that miR-802 activates the noncanonical and canonical NF-κB pathways via TRAF3, leading to macrophage recruitment. miR-802 promotes lipid synthesis and M1 macrophage polarization in adipose tissue through activating SREBP1 The mechanism of an miR-802 increasing M1 polarization by inducing NF-κB pathways remains unclear. To better understand the role of miR-802 in regulating macrophage polarization, we performed transcriptome sequencing using epiWAT derived from miR-802 KI mice. In the adipose tissues of miR-802 KI mice, the expression of 191 mRNAs was significantly altered compared to that of mRNAs in WT mice, of which the expression of 75 mRNAs increased ( Figure 7A , Supplementary Table 2 ). We found that seven mRNAs were upregulated more than 10-fold ( Figure 7A , right), and these mRNAs were annotated using UCSC and Ensemble. Among these upregulated genes, we focused on the lipogenic gene sterol regulatory element-binding protein 1 a (SREBP1a), which is involved in fatty acid synthesis and lipid droplet formation( 37 ). We verified that the mRNA levels of Srebp1a were increased in both the epiWAT of miR-802 KI mice and in 3T3-L1 cells transfected with miR-802 mimics in qRT-PCR analysis ( Figure S7A and B ). We also found that the mature form of the SREBP-1 protein (m-SREBP1) was significantly higher in the epiWAT of miR-802 KI and 3T3-L1 cells transfected with miR-802 mimics ( Figure 7B and C ). As expected, overexpression of Traf3 reduced the upregulation level of mature SREBP1 induced by miR-802 ( Figure 7D ). However, target gene prediction algorithms as well as luciferase reporter and Ago2-RIP assays confirmed that Srebp1a is not the direct target gene for miR-802 (data not shown). This prompted us to verify whether Srebp1a is a downstream gene in the NF-κB pathway. Download figure Open in new tab Figure 7 miR-802 promotes lipogenesis and induces M1 macrophage polarization in adipose tissue through activating SREBP1 (A) Heat map illustrating the top 20 upregulated mRNAs in the epiWAT of miR-802 KI mice compared to their WT miR-802 fl/fl littermates ( n =3). (B–C) Protein levels of the mature form of SREBP-1 protein (m-SREBP1) and the precursor form of SREBP-1 (P-SREBP1) in mature 3T3-L1 cells transfected with miR-802 mimics or miR-802 inhibitor (B), in the epiWAT of miR-802 KI mice (C, n =3). (D) The protein levels of m-SREBP1 and P-SREBP1 were reversed by Traf3 . (E) Srebp1a mRNA levels in 3T3-L1 cells transfected with p65 -overexpressing plasmid or p65 shRNA plasmid. (F, G) ChIP-qPCR assays were conducted to verify that p65 binds to the Srebp1 promoter in 3T3-L1 cells transfected with miR-802 mimics or miR-802 inhibitor (F) and in the epiWAT of miR-802 KI mice (G, n =3). (H) DNA pull-down assay using a biotinylated DNA probe corresponding to the −360 to −400 or −1198 to −1237 region of the Srebp1 promoter in 3T3-L1 cells transfected with miR-802 mimics or miR-802 inhibitor. (I) DNA pull-down assay using a biotinylated DNA probe corresponding to the −1198 to −1237 region of the wild-type (WT) or a mutant sequence of the Srebp1 promoter in 3T3-L1 cells stimulated with p65 plasmid for 48 h. (J) Luciferase reporter assays in 3T3-L1 cells transfected with the indicated plasmids for 48 h. Dual-luciferase activity was determined. (K) Schematic illustration for the mechanism of miR-802 increased Srebp1 expression by activating canonical NF-κB pathways. (L) Representative images of the immunofluorescence of lipid droplets (HCS LipidTOX TM , Red) and DAPI (Blue). Scale bar: 20 μm. (M) Oil red O staining was performed to assess the number of lipid droplets in 3T3-L1 cells transfected with miR-802 mimics. Scale bar: 200 μm. (N) Representative images of the immunofluorescence of lipid droplets (HCS LipidTOX TM , Red) and F4/80 (Green, n =3). Scale bar: 20 μm. (O) Transmission electron microscopy (TEM) was performed to detect the contact between lipid droplets and macrophages ( n =3). Data represent mean ±SEM. Differences between groups were determined by ANOVA (E–G and J). ***P <0.001. Genes levels were normalized to 18S rRNA abundance. For this purpose, we first predicted the nuclear factor-κB (NF-κB) family ( p65 , RelB , C-rel , p50 , and p52 ) sites in the promoter of Srebp1a using JASPAR and the Promo database. Two p65 potential binding sites (B1 and B2) were found in the promoter of Srebp1a ( Figure S7C ). We next overexpressed p65 in 3T3-L1 cells; qRT-PCR results showed that p65 could increase Srebp1a expression in 3T3-L1 cells ( Figure 7E ). To determine whether Srebp1a is a direct p65 target gene, ChIP-qPCR assays were used. The results showed that occupancy of p65 binding site 2 (B2) on the Srebp1a promoter was significantly increased in miR-802 -overexpressing 3T3-L1 cells and miR-802 selectively overexpressed adipose tissues ( Figure 7F and G , Figure S7D and E ). We then conducted DNA pull-down assays to examine the binding of p65 to the Srebp1a promoter in vitro . We constructed two DNA probes containing −360 to −400 or −1198 to −1237 that contained the predicted binding site 1 (B1) and predicted binding site 2 (B2), respectively, to detect binding to p65 in nuclear extracts. Similar findings were obtained in that the B2 DNA probe, but not the B1 DNA probe, bound to p65 in the 3T3-L1 cell line overexpressing miR-802 ( Figure 7H ). However, with mutant B2 (agggaatgct, Mut2), DNA pull-down results showed that p65 could not bind to Mut2 ( Figure 7I ). Moreover, we constructed a luciferase reporter plasmid containing the Srebp1a promoter region from −1295 to +1 WT and two mutant reporter plasmids mutated in −375 to −385 (Mut1) or in −1211 to −1221 (Mut2). Overexpression of p65 significantly enhanced WT and Mut1, but not Mut2-driven luciferase in 3T3-L1 cells ( Figure 7J ). Taken together, these results indicated that miR-802 indirectly stimulates Srebp1a expression via the canonical NF-κB signaling pathway ( Figure 7K ). As previously described, Srebp1a is a well-established regulator of lipid synthesis( 37 ). Accordingly, miR-802 overexpression significantly increased the number and fluorescence intensity of lipid droplets, which correlated well with increased SREBP1 ( Figure 7L-M ). Conversely, knockdown of miR-802 strongly reduced lipid droplet formation in 3T3-L1 cells and miR-802 -KO mouse adipose tissue ( Figure S7F and G ). Lipid droplets have been shown to play a crucial role in M1 macrophage polarization ( 38 , 39 ). Consistent with this, we observed that adipose tissue macrophages (ATMs) of the miR-802 KI mice could engulf more lipid droplets ( Figure 7N, O ). The elevated expression of the classical activation marker further indicated that lipid droplets induced the ATMs in miR-802 KI mice to the pro-inflammatory phenotype ( Figure S7H ). Altogether, these data show that miR-802 indirectly regulates lipid droplet formation through SREBP1 and ultimately promotes macrophage M1 polarization. Discussion Macrophage infiltration of adipose tissue has been described in both mice and humans during obesity. However, how lipid-loaded hypertrophic adipocytes send signals to trigger infiltration and alter the polarization of macrophages in obesity remains poorly understood. In this study, we found that miR-802 endows adipose tissue with the ability to interact with macrophages and regulate the inflammatory cascade. Mechanistically, miR-802 recruits macrophages and drives the polarization program toward proinflammatory M1 phenotype by targeting the cytoplasmic adaptor protein TRAF3 ( Figure 8 ). Our findings indicate that miR-802 has essential roles in the initiation and maintenance of adipose tissue inflammation and systemic insulin resistance. Download figure Open in new tab Figure 8 Schematic illustration for the mechanism of miR-802 exacerbates adipose tissue inflammation and leads to metabolic dysfunction during obesity. We found that miR-802 endows adipose tissue with the ability to interact with macrophages and regulate the inflammatory cascade. During obesity, miR-802 promotes adipose tissue secretion more chemokines recruiting macrophages by targeting Traf3 activating canonical and noncanonical NF-κB signaling pathways; and miR-802 increases lipogenesis through promoting Srebp1 transcription, then, macrophages toward proinflammatory M1 phenotype by engulfing lipid droplet. Adipose tissue inflammation is a hallmark of obesity and a causal factor of metabolic disorders such as insulin resistance. Mice fed an HFD frequently develop chronic low-grade inflammation within adipose tissues, characterized by increased infiltration of macrophages and the production of pro-inflammatory cytokines. Here, we showed that the increasing trend of miR-8 02 in adipocytes is an early event during the development of adipose tissue obesity induced by an HFD. miR-802 expression in visceral fat was progressively increased with the development of dietary obesity, whereas adipose-selective ablation of miR-802 protected mice from exacerbation of meta-inflammation and insulin resistance caused by dietary stress. The high level of miR-802 expression in visceral fat may partly explain why this adipose depot is more prone to inflammation and is closely related to insulin resistance. miR-802 is required for adipose tissue inflammation and has major roles in macrophage recruitment and polarization. Thus, miR-802 is crucially involved in initiating inflammatory cascades in obese adipose tissue. Moreover, the finding that miR-802 inhibitor treatment ameliorated pre-established adipose inflammation in DIO mice indicates that miR-802 is also essential for maintenance of the inflammatory response. Although previously studies have found that miR-802 was up-regulated in the adipose tissue during obesity ( 27 , 28 , 40 , 41 ), while the function of miR-802 was focused on cancers( 42 ), liver( 43 , 44 ), small intestine( 41 ) and pancreas( 40 ). Whether miR-802 can regulate adipose function is still confused. To our knowledge, the present study is the first to directly address the functional role of miR-802 in adipose tissue inflammation. The findings that systemic insulin resistance is ameliorated by miR-802 depletion and is aggravated by adoptive transfer of miR-802 mimics strongly suggest that miR-802 -dependent adipose inflammation has an impact on systemic metabolism. Like most other miRNAs, miR-802 regulates the expression of multiple genes in different tissues. In the liver, miR-802 is induced by obesity and impaired glucose tolerance, and it attenuates insulin sensitivity by downregulation of Hnf1b ( 28 ). Genetic ablation of miR-802 in the small intestine of mice leads to decreased glucose uptake, impaired enterocyte differentiation, increased Paneth cell function, and intestinal epithelial proliferation through derepression of Tmed9 ( 41 ). We recently discovered that in pancreatic islet cells, elevated miR-802 causes impaired insulin transcription and secretion by targeting NeuroD1 and Fzd5 ( 27 ). In this study, we found that miR-802 promotes adipose tissue inflammation and insulin resistance by targeting TRAF3 in adipocytes. As a member of the TNF receptor (TNFR) superfamily, TRAF3 plays vital roles in inflammatory responses via activation of both the canonical and noncanonical NF-κB signaling pathways( 32 , 33 ) following engagement of a variety of TNFR superfamily members such as Baff receptor, lymphotoxin β receptor, and CD40( 45 ). Here, we found that miR-802 can regulate the NF-κB pathway by directly targeting TRAF3 rather than by activating the classic receptor, which enriches the understanding of the NF-κB pathway. Macrophage accumulation was significantly higher in adipose tissue from HFD-fed miR-802 KI mice than in WT mice, suggesting that overexpression of miR-802 enhances the infiltration ability of macrophages. Correspondingly, we observed that miR-802- overexpressing adipocytes released more chemokines by activating NF-κB pathway, such as CCL2, CCL5, CCL20, and CXCL2. Adipose tissue inflammation is well documented as an important contributor to systemic insulin resistance( 46 ). This was further validated by our enhanced adipose tissue inflammatory responses in miR-802 KI mice. Moreover, HFD-fed miR-802 KI mice exhibited adipose tissue macrophage infiltration, proinflammatory cytokine expression, and NF-κB pathway activation. Genes that are crucial for meta-inflammation and insulin resistance were directly affected by the enhancement of miR-802 in adipose tissue. Thus, increased adipose tissue inflammation resulting from miR-802 overexpression contributed, in large part, to systemic insulin resistance in miR-802 KI mice. The chronic inflammation microenvironment is one of the main features of obesity. A recent study found that adipocytes can release lipid-filled vesicles that become a source of lipids for local macrophages( 47 ). Phagocytosis or excessive accumulation of lipid droplets can induce macrophage M1 polarization( 47 , 48 ). In our study, we observed the same phenomenon, that is, in miR-802 KI mice, macrophages accumulated more lipid droplets and exhibited an inflammatory phenotype. SREBP1 has been found to promote the acute inflammatory response and lipogenesis ( 49 , 50 ). Here, we found that miR-802 increased SREBP1 expression inducing lipogenesis by activating canonical NF-κB signaling pathways, then macrophage engulf lipid droplet promoting macrophage M1 polarization. This has enriched our understanding of the functionality of SREBP1 to some extent. However, miR-802 only indirectly regulates SREBP1, but it still has a considerable impact on macrophages, indicating the importance of miRNA positive or indirect regulation. Taken together, our results support the idea that obese adipose tissue activates miR-802 , which, in turn, initiates and propagates inflammatory cascades, including the recruitment of macrophages into obese adipose tissues and their subsequent induction of the inflammatory phenotype. Thus, miR-802 appears to have a primary role in obese adipose tissue inflammation. However, future studies are needed to clarify which environmental cues within obese adipose tissue initiate miR-802 elevation. The present observations indicate that miR-802 inhibitors might offer a novel approach to prevent diseases associated with insulin resistance. Materials and methods Animal studies All mice used were of mixed strain backgrounds with approximately equal contributions from C57BL/6J, with the exceptions of db/db mice (C57BLKS/J). MiR-802 fl/fl and miR-802 ki/ki in mice were initially described in( 27 ). To generate adipose-specific miR-802 knockout and miR-802 knockin animals, we used Adipoq -Cre mice on a C57BL/6J background purchased from Jackson Laboratories. Mice were crossed with homozygous for miR-802 fl/fl or miR-802 ki/ki and heterozygous for Adipoq -Cre to generate Adipoq-miR-802 KO mice ( miR-802 KO), Adipoq - miR-802 KI mice ( miR-802 KI), control miR-802 fl/fl littermates or control m iR-802 ki/ki littermates. Studies were performed on 8-week-old male and female mice initially housed under standard conditions with full access to standard mouse chow and water. After this time, mice were switched to a 60% high-fat diet (HFD) or normal chow diet (NCD) consisting of a 10% fat diet for 30 weeks. All mice had free access to food and water ad libitum. Animals were housed in a temperature-controlled environment with a12 h dark–light cycle. At the end of the 30-week period, mice were euthanized via overdose of isoflurane anesthesia, and tissues were immediately weighed, dissected, and frozen in liquid nitrogen. Tissue samples were stored at -80 °C until use. Care of all animals was within institutional animal-care committee guidelines, and all procedures were approved by the animal ethics committee of China Pharmaceutical University (Permit Number: 2162326) and were in accordance with the international laws and policies (EEC Council Directive 86/609,1987). For administration of AAV8-Adipoq -miR-802 sponge vector, AAV8-Adipoq- Traf3 vector to epididymal adipose tissue, mice were anesthetized with pentobarbital sodium (60 mg/kg) intraperitoneally and the laparotomy was performed. Each epididymal fat pad was given 8 injections of 5 uL (1 × 10 13 viral genome copies) of AAV solution. Human adipose samples of lean and overweight individuals Adipose and clinicopathological data were collected from Sir Run Run Hospital, Nanjing Medical University (Nanjing, China). All patients enrolled in this study were obese (BMI > 25). The negative controls were normal-weight individuals (20 ≤ BMI ≤ 25). All human subjects provided informed consent. All human studies were conducted according to the principles of the Declaration of Helsinki and were approved by the Ethics Committees of the Department Sir Run Run Hospital (Nanjing, China, 2023-SR-046). The clinical features of patients are listed in Supplementary Table 1 . Adipose sample preparation SVF and mature adipocytes were obtained as follows: adipose tissue samples were digested with collagenase type 1 in Krebs-RingerHenseleit (KRH) buffer for 30 min at 37°C. Cell suspensions containing mature adipocytes and SVF were then filtered with nylon mesh and washed three times with KRH buffer. Mature adipose was floated to the surface and the remaining solution containing the SVF was centrifuged at 1500 rpm for 5 min. The pellet was washed with pre-adipocyte growth medium (DMEM-F12 supplemented with 10% calf serum and 1% penicillin-streptomycin), followed by a second centrifugation. SVF cells were then cryopreserved using a freezing medium (DMEM-F12 supplemented with 60% FBS and 10% DMSO). The medium was added to the pellet and frozen with a temperature gradient (−1°C/minute) and stored in liquid nitrogen until analysis. Following collection, whole adipose tissue samples were quickly frozen in liquid nitrogen and stored until analysis. 3T3-L1 cell culture and differentiation 3T3-L1 cells were cultured in DMEM (Gibco) containing 10% calf serum with high glucose at 37 °C, 5% CO 2 and full saturation humidity until they reached 80%-90% confluence, at which point the media was changed to the first differentiation medium containing high glucose DMEM, 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 μM dexamethasone and 10 μg/ml insulin for 48 h, then the media was changed to the terminal differentiation cocktail containing high glucose DMEM, 10% FBS, and 10 μg/ml insulin for 48 h. The insulin-resistant cell models were established in mature 3T3-L1 and mature WAT SVF cells by 0.5 mM palmitate, 10 μg/ml insulin and 25 mM glucose for 24 h. Resident peritoneal macrophage isolation and culture For 2 d, 1.0 ml of sterile 4% thioglycolate medium (Sangon Biotech, China) was injected into the peritoneal cavity of C57BL/6J mice. Then, resident peritoneal macrophages were obtained via peritoneal lavage with 5 ml lavage solution (PBS (Sangon Biotech) supplemented with 5 mM EDTA and 4% FBS). Lavages of the same genotype were pooled and resuspended in complete medium (RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin, 10 μg/ml streptomycin, and 400 μM L-glutamine [Invitrogen]). Typically, the cells were plated and left to adhere for 3 h at 37°C, 5% CO 2 before being washed two times with warm complete medium. The cells were plated on transwell permeable supports or 24-well plates and co-cultured with SVF cells. Migration and invasion assays The 3T3-L1 cells or SVF cells were evenly plated in 24-well plates. To differentiate mature cells, migration and invasion assays were performed using a transwell chamber (Millipore, Billerica, MA, USA). For the migration assay, RAW 264.7 macrophage cells were seeded in the upper chamber with serum-free medium (1.0×10 5 cells); the bottom chamber contained mature 3T3-L1 cells. For the invasion assay, the chamber was coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA); the subsequent steps were similar to the migration assay. After the cells migrated or invaded for 24 h, they were fixed and stained with crystal violet. Migrated and invaded RAW 264.7 cells were counted under an inverted light microscope. The number of migrated or invaded cells was quantified by counting the number of cells from 10 random fields at ×100 magnification. RNA-sequencing analysis Total RNA from epididymis white adipose tissue of wide type control mice ( n =3) and miR-802 KI mice ( n =3) was isolated using the RNeasy mini kit (Qiagen) following the protocol. The quality of the samples, the experiment, and the analysis data was completely finished by the HaploX (Shangrao, China). Cuffdiff (v2.2.1) 51 was used to calculate the fragments per kilobase million (FPKM) for mRNAs in each group. A difference in gene expression with a p value ≤ 0.05 was considered significant. The raw data is presented in Supplementary Table 2 . The RNA-seq raw data that support the findings of this study has been deposited in the NCBI’s Sequence Read Archive (SRA) database (PRJNA1021754). Fluorescence in situ hybridization (FISH) Cy3 labeled miR-802 probe was designed and synthesized by GenePharma (Shanghai, China). The frozen sections of adipose tissue from obese patients, normal persons or obese mice were fixed with 4% formaldehyde at room temperature for 10 min. The probe was hybridized at 37℃ for 16 h. DAPI was added at 1:5000 for 15 min after washing with probe detergent. Images were obtained with confocal laser scanning microscope (CLSM, LSM800, Zeiss, Germany) and processed using the ZEN imaging software. Plasmid and shRNA construction The coding sequences for Traf3 (NM_001286122.1), p65 (NM_ 009045.5), RelB (001290457.2), Srebp1 (001313979.1) were amplified by PCR from full-length cDNA of mice, and then cloned in pcDNA 3.1 (+) vector (Addgene, Watertown, MA, USA). All plasmids were confirmed to be correct by sequencing. The primer sequences for PCR are listed in Supplementary Table 3 . The shRNA of Traf3 , Srebp1 , p65 , RelB were constructed in plvx-shRNA2 lentivirus vector (Takara). The plvx-shRNA2 lentivirus vector was digested with EcoR I and BamH I . The shRNA primer sequences are listed in Supplementary Table 4 . Luciferase assay miR-802 mimics/ miR-802 inhibitor ( anti-miR-802 )/miRNA NC (NC)/miRNA inhibitor NC was purchased from GenePharma (Shanghai, China). The construction of Traf3 (both wild type and mutants) was achieved by digestion of pmir-PGLO vector (Addgene, Watertown, MA, USA) with double restriction enzymes ( Xhol I and Xbal I ), followed by ligation of sequences encoding the corresponding 3’UTR of the target genes. Sequences of the synthetic oligonucleotides encoding the 3’UTR of the target genes and their mutants are listed in Supplementary Table 3 . 3T3-L1 cells were transfected with one of the above-mentioned plasmids using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. At 48 h after transfection, the cells were lysed and the luciferase activity was assayed with a dual-luciferase reporter assay kit (Vazyme, Nanjing, China). Data are presented as the ratio of Renilla luciferase activity to firefly luciferase activity. Flow cytometric analysis of macrophage polarization SVF was resuspended in 1 ml of Live/Dead Fixable Dead Cell stain (Molecular Probes) and incubated on ice for 30 min. Afterward, the cells were washed once with FACS buffer (1% BSA in 1×PBS), followed by staining with different antibodies. For flow cytometry analysis of macrophages, 1×10 6 freshly isolated cells were triple stained with CD11b-Apc (#101211, Biolegend; 1:100), Zombie NIR TM Fixable Viability Kit (#423105, Biolegend; 1:200) and F4/80-PE (#157304, Biolegend; 1:100), or stained with F4/80-FITC (#123108, Biolegend; 1:100), Cd206-APC (#141708, Biolegend;1:100), Cd86-PE (#105007, Biolegend; 1:100), or their isotype controls (Biolegend) on ice for 30 min in the dark. After staining, the cells were fixed with 2% (w/v) paraformaldehyde and stored at 4°C before analysis with FACS Celesta Cell Analyzer (BD Biosciences). Data were analyzed using FlowJo software version X.0.7 (Tree Star, Inc.). Mouse metabolic studies After 12 h fasting treatment, mice fasting blood glucose (FBG) levels were examined via using a glucometer (OMRON, Japan) and fasting serum insulin (FINS) levels were tested by insulin ELISA kit (Crystal Chem, USA). And the homeostatic model assessment indices of insulin resistance (HOMA-IR) was calculated with the equation (FBG (mmol/l) ×FINS (mIU/l))/22.5. To perform the glucose tolerance tests, 2 g/kg glucose (Sigma-Aldrich, StLouis, MO, USA) was intraperitoneal (i.p.) injected into mice, whereas 0.75 U/kg insulin (Novolin R, Novo Nordisk, Bagsvaerd, Denmark) was i.p. injected into mice for insulin tolerance tests. Blood glucose levels were examined at 0, 15, 30, 60, 90 and 120 min after glucose or insulin injection and serum sample was collected from eye canthus blood at 0, 5, 15, and 30 min after glucose injection. Insulin level was evaluated using mice insulin ELISA kit (Crystal Chem, USA), according to the manufacturer’s instructions. Subtracting the baseline area, by subtracting the starting glucose value from the value at each time point, generates the area of the curve (AOC)( 51 ). Body Composition The changes in body composition were assessed as we have previously describe( 52 ). In brief, mice were anesthetized with 2% isoflurane by volume in a box and fixed on an MRI platform (Bruker BioSpec 7T/20 USR). Anesthesia was also maintained with isoflurane of 1% by volume. After turning on the instrument, the mice were scanned layer by layer according to the cross-section of their internal adipose tissue content, using ImageJ software analysis and statistics of the lipid distribution in mice. RNA isolation and qRT-PCR analysis Total RNA from adipose tissues or its fractions or 3T3-L1 cells was extracted with TRIzol reagent (Invitrogen). For mRNA expression analysis, 500 ng of total RNA was used for synthesis of cDNA using PrimeScriptTM RT reagent Kit (Takara, Tokyo, Japan). For miRNA expression analysis, 150 ng total RNA was reverse-transcription into cDNA using miRNA-specific primers supplied with TaqMan MicroRNA Reverse Transcription kit. The quantitative real-time PCR was performed using the LightCycle 480 (Roche). The relative level of gene expressions were calculated by the 2 -ΔΔCT method, after normalization with the abundance of 18S rRNA or U6 . For miR-802-5p and U6 , TaqMan probes (Ambion) were used to confirm our results. The sequences of genes were listed in Supplementary Table 5 . Western blot analysis Proteins were extracted from tissues or cells in radioimmunoprecipitation assay (RIPA) buffer (Beyotime) containing a complete protease inhibitor cocktail (Roche), resolved by SDSPAGE, transferred onto polyvinlidene fluoride (PVDF) membranes (Bio-Rad), and then probed with primary antibodies against TRAF3 (#ab36988), NIK (#ab314146) were from Abcam, NF-κB2 p100/p52 (#4882), NF-κB p65 (#8242) were from CST, and RelB (#A23389), P52 (#ab125611), IKK-α (#A2062), phospho-IKK-α (#AP0546), IKBα (#A19714), phosho-IKBα (#AP0614), iNOS (#A14031), phosho-p65 (#AP0123), Tubulin (#AC008), β-Actin (#AC038) and Histone H3 (#A2348) were from ABclonal. The protein bands were visualized with enhanced chemiluminescence reagents (GE Healthcare) and quantified by using the ImageJ software. RNA immunoprecipitation (RIP) RNA immunoprecipitation was performed using an EZMagna RIP Kit (Millipore, Billerica, MA, USA) following the manufacturer’s protocol. 3T3-L1 cells transfected with miR-802 or oe-Traf3 were lysed in complete RIP lysis buffer, and then, 100 μl of whole cell extract was incubated with RIP buffer containing magnetic beads conjugated with anti-Ago2 (#ab186733, Abcam) antibody or negative control normal mouse IgG (#ab172730, Abcam). Furthermore, purified RNA was subjected to qRT-PCR analysis to demonstrate the presence of the binding targets using the respective primers. The primer sequences are listed in supplementary table 5 . Chromatin immunoprecipitation assay (ChIP) ChIP experiments were strictly performed according to the manual for the ChIP Assay Kit (#17-10086, Millipore) and the manufacturer’s protocol. 3T3-L1 cells transfected with miR-802 mimics, miR-802 inhibitor, oe-Traf3 , or miR-802 & oe-Traf3 were fixed with 37% formaldehyde for 10 min, followed by 30 rounds of sonication, each for 3 s, to fragment the chromatin. The chromatin was incubated with NF-κB p65 antibody (#8242, CST) at 4°C overnight and then immunoprecipitated with Proteinase K (Millipore). Purified DNA was amplified by PCR using primer pairs that spanned the predicted p65 binding sites on the Srebp1 promoter. The primer sequences are listed in Supplementary Table 5 . Agarose-oligonucleotide pull-down assay The oligonucleotides for the mouse Srebp1 a promoter and their complementary strands were synthesized by GenePharma (Shanghai, China) and biotinylated using a Pierce™ Biotin 3’ End DNA Labeling Kit (Cat. #89818; Thermo Scientific). These oligonucleotides were annealed to form double-stranded oligonucleotides, which were then incubated with streptavidin-conjugated agarose beads at 4°C for 60 min and washed twice with IP lysis buffer. Next, the nuclear extract (50 μg each) in 200 μl IP lysis buffer was pre-cleared with agarose beads at 4°C for 90 min to reduce any nonspecific binding and then incubated with oligo/streptavidin-conjugated beads at 4°C overnight. The mixtures were washed three times with IP lysis buffer via centrifugation the following day, and the affinity-purified proteins were eluted by boiling in SDS sample buffer for 10 min. Samples were then subjected to analysis by western blot. Primer sequences are listed in Supplementary Table 5 . Histological and immunochemical analysis The white adipose tissue was fixed in 4 % formalin solution at 4°C for 24 hours, embedded in paraffin, and sectioned at 5 μm. Deparaffinized and rehydrated sections were stained with haemotoxylin and eosin (Sigma), or with reagents for Sirius red staining, or immunohistological staining of HCS LipidTOX™ red neutral stain (#H34467, Invitrogen) and F4/80 (#GB113373, Servicebio, china). The slides were analyzed using a confocal laser scanning microscope (CLSM, Carl Zeiss LSM800) at ×20 magnification. Transmission electron microscopy For transmission electron microscopy, mouse epiWAT was dissected, sliced into small fragments of 1–2 mm each, and then fixed in 5% glutaraldehyde for 2 days. Specimens were post-fixed in 1% osmium tetroxide. After staining with 2% aqueous uranyl acetate for 2 h, the samples were dehydrated in a series of ethanol up to 100% and embedded in epoxy resin. Ultrathin sections were cut with an EM UC7 ultramicrotome (Leica) and poststained with lead nitrate. Ultrathin sections were mounted in formvar-coated nickel grids and observed under an FEI Tecnai G2 Electron Microscope (FEI Tecnai G2). EdU labeling Cell proliferation was detected with BeyoClick™ EdU Alexa Fluor 488 Imaging Kit (Beyotine, China). Briefly, 1×10 5 macrophage cells or 1×10 4 RAW264.7 cells were plated on twenty-four well plates. After co-cultured with 3T3-L1 cells or SVF cells, cells were gently washed twice with PBS, and further incubated with 10 μM EdU for 4 h. Treated cells were fixed in 4% paraformaldehyde solution at room temperature for 15 min and EdU detection was carried out according to manufacturer’s instructions. Sample size and replication Sample size varied between experiments, depending on the number of mice allocated for each experiment. The minimum sample size was three. Data inclusion/exclusion criteria All patients enrolled in this study were obese (BMI > 25). The negative controls were normal-weight individuals (20 ≤ BMI ≤ 25). Data or samples were not excluded from analysis for other reasons. Randomization Mice used for the experiments were randomly selected and randomly assigned to experimental groups. There was no requirement for randomization of cell selection. Blinding During experimentation and data acquisition, blinding was not applied to ensure tractability. Statistical analysis All in vivo experiments represent individual mice as biological replicates. The exact values of n are reported in figure legends. Data are presented as mean ± SEM. Comparisons were performed using the Student’s t test between two groups or ANOVA in multiple groups. Dunn’s multiple comparisons for one-way ANOVA and Fisher’s least significant difference (LSD) for two-way ANOVA were used. The level of significance was set at *p < 0.05, **p < 0.01, ***p < 0.001. Graphpad prism 8 (GraphPad, San Diego, CA, USA) was used for all calculation. Author contributions Y. Y, B. H and YM. Q performed the experiments; DW. W performed partial experiments on animals; YN. J and LM. S collected all the human samples; Y. P, YF. Z, YM. S and WJ. Hu analyzed data; FF. Z and L.J designed the project, FF. Z, L.J and ZY. C interpreted the data and wrote the manuscript. Declaration of interests The authors declare no competing interests exist. Figure 5-source data 1: The original files of the full raw unedited blots of TRAF3 and β-Actin in human subcutaneous adipose tissues from obese and normal individuals ( n normal =4 and n obesity&IR =9). Figure 5-source data 2: The original files of the full raw unedited blots of TRAF3 and β-Actin in the epiWAT of HFD mice ( n =3). Figure 5-source data 3: The original files of the full raw unedited blots of TRAF3 and β-Actin in the epiWAT of miR-802 KI mice ( n =3). Figure 5-source data 4: The original files of the full raw unedited blots of TRAF3 and β-Actin in 3T3-L1 cells transfected with miR-802 mimics or miR-802 inhibitor. Figure 5-source data 5 : The original files of the full raw unedited blots of TRAF3 and β-Actin in the epiWAT of control, miR-802 KI, Traf3 eWAT KI , and miR-802 KI & Traf3 eWAT KI mice ( n =3). Figure 6-source data 1: The original files of the full raw unedited blots of TRAF3, NIK and β-Actin in 3T3-L1 cells transfected with miR-802 mimics or miR-802 inhibitor. Figure 6-source data 2: The original files of the full raw unedited blots of TRAF3, NIK and β-Actin in the epiWAT of miR-802 KI mice ( n =3). Figure 6-source data 3: The original files of the full raw unedited blots of p100/p52 and β-Actin in 3T3-L1 cells transfected with miR-802 mimics or miR-802 inhibitor. Figure 6-source data 4: The original files of the full raw unedited blots of p100/p52 and β-Actin in the epiWAT of miR-802 KI mice ( n =3). Figure 6-source data 5 : The original files of the full raw unedited blots of P-IKK-α, IKK-α and β-Actin in 3T3-L1 cells transfected with miR-802 mimics or miR-802 inhibitor. Figure 6-source data 6: The original files of the full raw unedited blots of P-IKK-α, IKK-α and β-Actin in the epiWAT of miR-802 KI mice ( n =3). Figure 6-source data 7: The original files of the full raw unedited blots of p100/p52, P-IKK-α, IKK-α, NIK and β-Actin in the 3T3-L1 cells. Figure 6-source data 8: The original files of the full raw unedited blots of p100/p52, P-IKK-α, IKK-α, NIK and β-Actin in the epiWAT of miR-802 KI and Traf3 eWAT KI mice ( n =3). Figure 6-source data 9: The original files of the full raw unedited blots of some major canonical NF-κB signaling targets in the epiWAT of miR-802 KI mice( n =3). Figure 6-source data 10 : The original files of the full raw unedited blots of some major canonical NF-κB signaling targets in the epiWAT of miR-802 KI mice and Traf3 eWAT KI rescued mice ( n =3). Figure 7-source data 1: The original files of the full raw unedited blots of m-SREBP1, P-SREBP1 and β-Actin in mature 3T3-L1 cells transfected with miR-802 mimics or miR-802 inhibitor. Figure 7-source data 2: The original files of the full raw unedited blots of m-SREBP1, P-SREBP1 and β-Actin in in the epiWAT of miR-802 KI mice ( n =3). Figure 7-source data 3: The original files of the full raw unedited blots of m-SREBP1, P-SREBP1 and β-Actin in the 3T3-L1 cells. Figure 7-source data 4: The original files of the full raw unedited blots of p65 and β- Actin in 3T3-L1 cells transfected with miR-802 mimics or miR-802 inhibitor. Figure 7-source data 5 : The original files of the full raw unedited blots of p65 and β- Actin in 3T3-L1 cells stimulated with p65 plasmid for 48 h. Figure 2-figure supplement 2-source data 1 : The original files of the full raw unedited gels of miR-802 KI mice. Figure 3-figure supplement 3-source data 1 : The original files of the full raw unedited gels of miR-802 KO mice. Figure 5-figure supplement 5-source data 1: The original files of the full raw unedited blots of TRAF3 and β-Actin in the epiWAT of ob/ob mice ( n =3). Figure 5-figure supplement 5-source data 2: The original files of the full raw unedited blots of TRAF3 and β-Actin in the epiWAT of db/db mice ( n =3). Figure 5-figure supplement 5-source data 3: The original files of the full raw unedited blots of TRAF3 and β-Actin in the epiWAT of miR-802 KO mice ( n =3). Figure 6-figure supplement 6-source data 1: The original files of the full raw unedited blots of TRAF3, NIK and β-Actin in in the epiWAT of miR-802 KO mice ( n =3). Figure 6-figure supplement 6-source data 2: The original files of the full raw unedited blots of p100/p52 and β-Actin in the epiWAT of miR-802 KO mice ( n =3). Figure 6-figure supplement 6-source data 3: The original files of the full raw unedited blots of P-IKK-α, IKK-α and β-Actin in the epiWAT of miR-802 KO mice ( n =3). Figure 7-figure supplement 7-source data 1: The original files of the full raw unedited gels by ChIP-PCR experiments in the 3T3-L1 cells. Figure 7-figure supplement 7-source data 2: The original files of the full raw unedited gels by ChIP-PCR experiments in the epiWAT of miR-802 KI mice ( n =3). Supplemental information Download figure Open in new tab Supplementary Figure 1 (A) The mRNA abundance of pri-miR-802 in epiWAT of 4, 6, 8, 12, 16 weeks db/db mice or control mice ( n =5). (B) The mRNA abundance of pri-miR-802 in epiWAT of mice fed with normal chow diet (NCD) or HFD for 0, 2, 4, 8, 16, 24 and 32 weeks ( n =5). (C-D) The insulin-resistant cell models were established in 3T3-L1 (C) and WAT SVF cells (D) by 0.5 mM palmitate, 10 μg/ml insulin and 25 mM glucose for 24 h, and qRT-PCR was performed to measure the expression levels of pri-miR-802 . (E) Representative images of flow cytometric analysis of CD11b + /F4/80 + in the SVFs isolated from eipWAT in mice fed with HFD ( n =5). (F) The expression levels of pri-miR-802 in the human subcutaneous adipose tissues ( n normal =25, n obesity & IR =70). (G) FISH analysis of miR-802 in the human subcutaneous adipose tissues of obese patient or normal patient ( n =7). The nuclei were stained with DAPI. Magnification: ×20, scale bar, 20 μm. Data represent mean ± SEM. The p-values by two-tailed unpaired Student’s t test (C-D, F), or two-way ANOVA (A-B) are indicated. **P < 0.01, ***P < 0.001. Relative levels of pri-miR-802 were normalized to U6 . Download figure Open in new tab Download figure Open in new tab Supplementary Figure 2 (A) Schematic diagram showing the strategy for generation of adipose-specific miR-802 KI mice. (B) Genotypic PCR analysis showing that the adipose tissue miR-802 WT mouse carrying homozygous miR-802 KI allele, while KI mouse carrying both KI and Cre allele. (C) qRT-PCR analysis showing a markedly decreased expression of miR-802 in several adipose tissues (epiWAT, scWAT, BAT), but not in liver or heart tissues ( n =3). (D) miR-802 mRNA levels in isolated adipocytes and SVF from epiWAT of miR-802 KI mice ( n =3). (E) The cumulative food intake of miR-802 KI and control mice treated with NCD feeding ( n =5). (F-G) Dynamic changes in body weight (F) and glucose (G) of control and miR-802 KI mice during 30 weeks of NCD feeding ( n =7). (H) Fat mass of whole body of control and miR-802 KI mice of NCD feeding ( n =7). (I) Representative images of F4/80 in epiWAT of WT or miR-802 KI mice on NCD for 0, 8, and 16 weeks ( n =5). Scale bar: 40 μm. (J) Representative images of flow cytometric analysis of CD11b + /F4/80 + cells in the SVFs isolated from eipWAT in control or miR-802 KI mice fed with HFD ( n =5). (K) Representative images of flow cytometric analysis of CD86 or CD206 in the SVFs isolate from eipWAT in control or miR-802 KI mice fed with HFD ( n =5). (L) Representative photos of adipose-specific miR-802 KI mice and their WT miR-802 ki/ki littermates fed with either HFD for 16 weeks ( n =3). (M-Q) IPGTT (1.5 g/kg, K-O) and IPITT (0.75 U/kg, R-V) were performed in miR-802 KI mice and control mice at the 0th, 4th, 8th, 16th or 30th week of high-fat diet administration, respectively ( n =5). Data represent mean ± SEM. Differences between groups were determined by ANOVA (C-G, M-V) or two-tailed unpaired Student’s t test (H). ***P < 0.001. miR-802 abundance was normalized to U6 level. Download figure Open in new tab Supplementary Figure 3 (A) Schematic diagram showing the strategy for generation of adipose-specific miR-802 KO mice. (B) Genotypic PCR analysis showing that the adipose tissue miR-802 WT mouse carrying homozygous miR-802 KO allele, while miR-802 KO mouse carrying both miR-802 KO and Cre allele. (C) qRT-PCR analysis showing a markedly decreased expression of miR-802 in several adipose tissues (epiWAT, scWAT, BAT), but not in liver or heart tissues ( n =3). (D) miR-802 mRNA levels in isolated adipocytes and SVF from epiWAT of miR-802 KO mice ( n =3). (E) Representative photos of adipose-specific miR-802 KO mice and their WT miR-802 fl/fl littermates fed with either HFD for 16 weeks ( n =3). (F) Cells isolated from SVFs of epiWAT in m iR-802 KO and KO control mice fed with HFD for 8, 16, and 24 weeks were subjected to flow cytometry analysis for percentage of CD11b + /F4/80 + total macrophages ( n =5). (G) Representative images of F4/80 in epiWAT of WT or miR-802 KO mice on NCD for 0, 8, and 16 weeks ( n =5). Scale bar: 40 μm. IPGTT (H-J) and IPITT (K-M) were performed in miR-802 KO mice and control mice at the 8th, 16th or 30th week of high-fat diet administration, respectively ( n =5). (N) Flowchart of the in vivo experiments designed for detecting adipose tissue inflammation and metabolic function via inguinal fat pad infusion of AAV- Adipoq-anti-miR-802 ( n =10). (O) The expression levels of miR-802 in the different tissue of HFD-control mice or miR-802 eWAT KO /HFD mice ( n =3). (P) Dynamic changes in body weight of miR-802 eWAT KO /HFD mice and control during 8 weeks of HFD feeding. (Q) Representative images of flow cytometric analysis of CD11b + /F4/80 + in the SVFs isolated from eipWAT in HFD-control or miR-802 eWAT KO /HFD mice ( n =3). Data represent mean ± SEM. Differences between groups were determined by ANOVA (C-D, H-M, O). **P < 0.01, ***P < 0.001. miR-802 abundance was normalized to U6 level. Download figure Open in new tab Supplementary Figure 4 (A-B) obesity induced macrophages proliferation tested by EdU staining (A) and flow cytometry analysis (FACS, B). (C) qRT-PCR was performed to test the miR-802 expression levels in the 3T3-L1 cells transfected with miR-802 mimics or miR-802 inhibitor. (D-E) EdU staining (D) and FACS analysis (E) were used to detect the proliferation of RAW264.7 cells. (F) FACS analysis of LipidTOX TM in RAW 264.7 cells. (G) The CCL2 levels were determined with ELISA. Data represent mean ± SEM. Differences between groups were determined by ANOVA (C-D, G). **P < 0.01, ***P < 0.001. miR-802 abundance was normalized to U6 level. Download figure Open in new tab Supplementary Figure 5 (A) miRPathDB, Targetscanand and multiMiR were used to predict the target genes of miR-802 . (B-D) The mRNA and protein levels of TRAF3 in the epiWAT of ob/ob mice (B, n =3), db/db mice (C, n =3) or miR-802 KO mice (D, n =3). (E) Anti-Ago2 RIP was performed in 3T3-L1 cells transiently overexpressing Traf3 , followed by qRT-PCR to detect miR-802 associated with Ago2 (nonspecific IgG served as a negative control). (F-G) Cells isolated from SVFs of epiWAT in control, Traf3 eWAT KI , miR-802 KI and miR-802 KI & Traf3 eWAT KI , mice fed with HFD 16 weeks were subjected to flow cytometry analysis for percentage of CD11b + /F4/80 + total macrophages (F, n =3) and M1 (CD86 + CD206 - ) and M2 (CD206 + CD86 - ) within the macrophage population (G, n =3). (H) Representative coronal section MRI images and visceral and subcutaneous adipose tissue volume of HFD-fed control, Traf3 eWAT KI , miR-802 KI and miR-802 KI & Traf3 eWAT KI mice. (I) Insulin tolerance test after mice were fed with HFD 16 weeks. (J) Serum insulin levels of control, miR-802 KI, Traf3 eWAT KI and miR-802 KI & Traf3 eWAT KI mice during 30 weeks of NCD or HFD feeding ( n =7). Data represent mean ± SEM. Differences between groups were determined by ANOVA (E-F, I-J). ***P < 0.001. miR-802 abundance was normalized to U6 level, and other genes levels were normalized to 18S rRNA abundance. Download figure Open in new tab Supplementary Figure 6 (A) GO analysis of RNA sequencing in epiWAT of miR-802 KI mice compared to their WT miR-802 fl/fl littermates. (B) NIK protein levels in the epiWAT of miR-802 KO mice ( n =3). (C) P100/52 protein levels in the epiWAT of miR-802 KO mice ( n =3). (D) The protein levels of IKK-α and P-IKK-α in the epiWAT of miR-802 KO mice ( n =3). Data represent mean ± SEM. Download figure Open in new tab Supplementary Figure 7 (A-B) qRT-PCR was performed to detect Srebp1a mRNA levels in the 3T3-L1 cells miR-802 mimics or miR-802 inhibitor (A) and in the epiWAT of miR-802 KI mice (B, n =3). (C) The predicted binding site of p65 on the Srebp1 promoter. (D-E) ChIP-PCR experiments were conducted to verify that p65 binds to the promoter of Srebp1 in the 3T3-L1 cells (D) and in the epiWAT of miR-802 KI mice (E, n = 3). (F) Representative images of immunofluorescence of lipid drop (HCS LipidTOXTM, Red) and DAPI (Blue). Scale bar: 20 μm. (G) Oil red O staining was performed to test the lipid droplet number in the 3T3-L1 cells transfected with miR-802 inhibitor. (H) Immunohistochemical analysis was performed to test F4/80, CD86 and CD206 levels in the epiWAT of miR-802 KI mice ( n =3), Scale bar: 20 μm. Data represent mean ± SEM. Differences between groups were determined by ANOVA (A-B). ***P < 0.001. Genes levels were normalized to 18S rRNA abundance. View this table: View inline View popup Download powerpoint Supplementary Table 1 Clinical characteristics of the patients with obese patients and normal individuals View this table: View inline View popup Supplementary Table 2 RNA islolated from epiWAT of wide type mice and miR-802 KI mice, this table shows significantly changed mRNA (Log2 (FPKM ( miR-802 KI/WT)) ≥1). View this table: View inline View popup Download powerpoint Supplementary Table 3 Primer sequences used for RT-PCR View this table: View inline View popup Download powerpoint Supplementary Table 4 Oligo sequences used for shRNA. View this table: View inline View popup Supplementary Table 5 The primers used in Real-time PCR (5’-3’). Acknowledgements This work was supported by the National Natural Science Foundation of China: Grant No. 82373925, 82070801 (To L.J.), 82100858, 82370804 (To FF.Z.), 82073227 (To Y.P). Supported by Natural Science Foundation of Jiangsu Province, BK20221520(To L.J.), BK20200569 (To FF.Z.). Supported by grants from the ‘111’ project, B16046 (To L.J.). Supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions, PAPD (To L.J.), 2632023TD03 (to FF Z). Supported by China Postdoctoral Science Foundation, 2022T150726, Supported by the Fundamental Research Funds for the Central Universities 2020M671661 (To FF.Z.) and 2632023GR07 (To Y. Y). Supported by Jiangsu Province Research Founding for Postdoctoral, 1412000016 (To FF.Z.). We would like to thank Xiaonan Ma for providing technical assistance of Carl Zeiss LSM 800 on the Public Experimental Platform of China Pharmaceutical University. We thank Yumeng Shen (Public Platform of State Key Laboratory of Natural Medicines, China Pharmaceutical University) for her assistance with flow analysis. We thank LetPub ( www.letpub.com ) for its linguistic assistance during the preparation of this manuscript. References 1. ↵ Ling C , Rönn T . ( 2019 ). Epigenetics in Human Obesity and Type 2 Diabetes . 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