Adipocyte ZAG improves obesity-triggered insulin resistance by reshaping macrophages populations in adipose tissue

doi:10.21203/rs.3.rs-1963317/v1

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Adipocyte ZAG improves obesity-triggered insulin resistance by reshaping macrophages populations in adipose tissue

https://doi.org/10.21203/rs.3.rs-1963317/v1

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Adipose tissues macrophages (ATMs) serve as a critical effector in the mediating occurrence of metabolic inflammation to impact whole-body insulin sensitivity in obesity. Discovering the key adipokines mediating crosstalk of adipocytes-macrophages and understanding the molecular mechanism of ATMs polarization and function have become hot topic issues in the immunometabolism fields. Zinc-α2-glycoprotein (ZAG) as a anti-inflammatory adipokines plays important roles in obesity-related metabolic diseases. We attempt to explore the precise role of adipose ZAG in metabolic inflammation and obesity-associated insulin resistance. Here we showed that Omental ZAG was positively associated with insulin sensitivity and M2 macrophages markers. ZAG-specific ablation in adipocyte aggravated insulin resistance and adipose tissues inflammation as evidenced by enhanced M1 macrophages proportion and inhibited AKT signaling pathway in mice fed with a high-fat diet. Exogenous ZAG inhibits PA-induced M1 macrophage polarization via β3-AR/PKA/STAT3 signaling in RAW264.7 macrophages.These findings suggest that adipocyte ZAG maintain insulin sensitivity via the cross talk with adipose-resident macrophages.

ZAG

inflammation

insulin resistance

obesity

adipose tissue

macrophages

Obesity is a primary risk factor for the development of type 2 diabetes [1]. The existence of a low-grade, chronic inflammation within hypertrophic adipose tissue is the most important cause of insulin resistance [2, 3]. ATMs (adipose tissue macrophages) are one of the most abundant immune cell types present in visceral adipose tissues and can be categorized as M1 macrophages (pro-inflammatory) and M2 macrophages (anti-inflammatory) [4]. Obesity induces the accumulation of M1 macrophages, aggravates tissues inflammation, and then contributes to local, as well as systemic insulin resistance through disrupting insulin signaling pathways [5]. Strategies to elevate the ratio of the M2/M1 macrophage are beneficial for adipose tissues and whole-body metabolism. Obesity-induced adipokines Imbalance can drive the specific metabolic reprogramming of ATMs in a paracrine manner [6]. Hence, the identification of key protective adipokines reversing macrophage invasion and reshaping macrophage phenotype may provide treatment targets against insulin resistance along with type 2 diabetes.

Zinc-alpha2 glycoprotein (ZAG) is a newly discovered adipokine with pro-lipolysis and anti-insulin resistance effect [7–9]. Yang. et al. proposed the ZAG index promised to be a simple index to evaluate insulin sensitivity [10]. We also found that ZAG can alleviate hepatic inflammatory response and inhibit the translocation of NF-κB to enhance hepatic insulin sensitivity [11–13]. The resolution of inflammation in critically ill patients with sepsis is related to a significant increase in ZAG, highlighting that ZAG has anti-inflammatory properties [14]. M2 polarization of ATMs favors browning remodeling of white adipocytes[15, 16] Interestingly, We have confirmed that ZAG browns white fat by activating PKA signaling (17). Although there is strong evidence ZAG serves as a molecular link between insulin resistance and metabolic inflammation, however, whether ZAG has a protective role in adipose tissues inflammation remains poorly understood.

Hence, we created adipocyte-specific ZAG knockout mice to explore the role of ZAG signaling in adipose tissues inflammation. We established that during the onset of obesity, ZAG deficiency in adipocytes augmented peripheral insulin resistance through regulation of macrophage invasion, as well as polarization via reducing inflammatory phenotype in ATMs. Furthermore, we have discovered the activation of the β3-AR/PKA/STAT3 signaling cascade by ZAG as a prospective mechanism through which adipocyte ZAG modulates macrophage polarization. This is the first report showing that ZAG deficiency in adipose tissues is a pivotal event of obesity-associated insulin resistance, strengthening ZAG serves as a therapeutic target to ameliorate adipose tissues inflammation in obesity and to improve the associated metabolic complications.

2.1 Animal Experiments

ZAG^FKO mice were generated via crossbreeding ZAG^flox/flox mice with AdipoQ-Cre mice. ZAG^flox/flox: AdipoQ-Cre⁻ mice were utilized as WT controls. ZAG^FKO mice or control mice at the age of 8 weeks were fed a standard diet (SFD, 14.7 kJ/g, 13% of energy as fat) or a high-fat diet (HFD, 21.8 kJ/g, 60% of energy as fat) for 30 weeks. Male C57BL/6J, db/db, and ob/ob mice were obtained from the experimental animal center of Nanjing University. Mice were keeped on a 12-h light/dark cycle and given adlibitum access to food and water. All procedures were conducted as per the standards in the guidelines of the Animal Care and Use Committee of the First Affiliated Hospital of the University of South China.

2.2 Isolation of mature adipocytes along with SVF in adipose tissue

SVF and primary adipocytes from epididymal white adipose tissues(eWAT)from Loxp mice and ZAG^FKO and mice were isolated as previous studies [18] with minor modifications. Firstly, the eWAT pad was digested with type I collagenase (Invitrogen) for one hour at 37°C. The digestion mixture was filtered by a 100-lm cell strainer (BD Biosciences) and span at 800 g for ten minutes. The mature adipocytes from upper floating layer, and the pelleted SVF from lower layer were collected and rinsed twice before further analysis.

2.2 Histology and Immunohistochemistry(IHC)

The eWAT sections were embeded by Paraffin, and then stained in H&E or inoculated with antibodies against F4/80, CD206, CD11c (Servicebio, Wuhan, China). OD density and CLSs in the WAT sections were assessed with the Image-Pro Plus software (Media Cybernetics, Rockville, MD).

2.3 Flow cytometry

Isolation of SVFs from eWAT was done via type I collagenase digestion [19, 20], inoculated with an antibody cocktail consisting of F4/80–PE-Cyanine7, CD206-FITC, CD11b-APC (allophycocyanin), as well as CD11c-PE (eBioscience, Thermo Fisher Scientific). Identification of M1 along with M2 macrophages was done via FACS assessment through the gating approach as documented previously [21].

2.4 Metabolic Studies

Insulin tolerance test (ITT) along with the glucose tolerance test (GTT) were carried out as documented previously [22, 23]. After ZAG^FKO mice and Loxp mice were accept overnight fasting (from 20:00 to 10:00 AM), the mice were intra-peritoneal inoculation with insulin (0.75 units/kg body weight) or D-glucose (0.9 g/kg body weight). Contents of blood glucose in the tail vein blood samples were monitored at the specified times (0,15, 30, 60, or 120 min). For insulin-induced phosphorylation detection, fasted mice for four hours were given intraperitoneal inoculation with insulin (2 U kg − 1 body weight). After 15 min, The mice were sacrificed for liver ,eWAT, and muscle collection and protein extraction

2.5 Western blot and RT-PCR analysis

RT-qPCR was conducted as documented previously [24]. The primer sequences are given in the Supplementary Table 2. The information of anti-body were used for Western blot, Flow cytometry and IHC were summarized in Supplementary Table.1. visualization of protein bands was done with the ECL Plus Western Blotting Detection System (Beyotime Institute of Biotechnology, China).

2.6 Cell culture and treatment

RAW264.7 cell line were acquired from ATCC (Manassas, VA, USA), and inoculated in DMEM culture medium augmented with 10% FBS, 2 mM glutamine, 100 units/ml penicillin along with 100 µg/ml streptomycin. For induction of M1 macrophages polarization, RAW264.7 cells were inoculated with 150 µm for palmitate acid (PA) for 24 hours [25]. To further investigate the mechanisms involved in ZAG-induced macrophage polarization, PA-treated macrophages were transfected with LV-STAT3 RNAi or LV-β3-RNAi as described by the manufacturer. Additionally, were pre-teated for 72h with PA and then incubated with pka inhibitors (H89 20 µM) for 2h.

2.7 Human Samples

Omental fat were acquired from age and gender-matched human subjects undergoing non-acute surgical interventions, for instance hernia or cholecystectomy in a scheduled routine surgery at the Third Xiangya Hospital Affiliated to Central South University (Hunan china) from March 1 in 2018 to August 31(Registration number: ChiCTR1800016839). The study was granted approval by the Ethics Committees of the hospital, with written informed consent from all patients. All subjects granted informed consent and the study was reviewed and approved by the Ethics and Research Committee of the Third Xiangya Hospital Affiliated to Central South University and the first affiliated hospital of the university of south china. Subjects were categorized as lean or obese as per BMI on the basis of the World Health.

2.8 Statistical analyses

Independent experiments were carried out at least three times. All data are given as mean ± SEM. Statistical analysis were implemented in SPSS software. Normality was tested with Kolmogorov-Smirnov test, before The student’s t-test was employed to compare two groups. Multiple groups comparison was determined via one-way ANOVA with Tukey’s posthoctest. The assumption of normality was cheked by the Shapiro − Wilks test, with P < .05 signifying statistical significance.

3.1 ZAG expression is reduced in visceral adipose tissues in humans or mice with obesity

We first attempted to explore the ZAG expression alteration in the different fat pads of obese models and found that ZAG content was remarkably down-regulated in eWAT of HFD mice, (Fig. 1A). After that, we explored the cellular source of ZAG in WAT and established that HFD mice had elevated level of ZAG mRNA in mature adipocytes (Fig. 1B). These data demonstrate that ZAG content is remarkably reduced in the eWAT of obese mice, especially in mature adipocytes. Previous investigations have confirmed that plasma ZAG levels are lower in obese in contrast with lean humans [26, 27]. To define whether obesity modulate ZAG expressio in human visceral adipose tissues, we detected ZAG content in omental fat samples from lean subjects, overweight subjects and obese subjects classified on the basis of body mass index (BMI). The basic information of the subjects has been summarized in Table.1. Omental fat of ZAG mRNA content in obese subjects exhibited the lowest content in contrast with lean or overweight controls and correlated inversely with BMI (Fig. 1C-D). Notably, we observed a positive correlation between the content of ZAG in visceral fat and clinical parameters linked to M2 macrophage markers (Arg-1, IL-10) (Fig. 1F-G). However, the decrease in ZAG mRNA in omental fat was negatively correlated with increased M1 macrophages markers ( TNF-α, IL-6)(Fig. 1H-I) and (Fig. 1E). These clinical data suggest that obesity may perturb the anti-inflammatory and anti-insulin resistance function of omental adipocyte ZAG.

3.2 Adipose-specific ZAG deficiency exacerbates adiposity and insulin resistance

To clarify the physiological regulation of adipocyte ZAG on insulin sensitivity and metabolic inflammation, adipose-specific (FKO) ZAG knockout mice were generated as described in methods sections. The gene and protein for ZAG content confirmed by RT-PCR and Western blotting, suggested that adipocyte ZAG was deleted in ZAG^FKO mice (sFig.1A-B). When FKO and control mice were fed SFD, no detectable difference was seen in body weight, fat mass, and glucose tolerance among Loxp mice and ZAG^FKO mice (sFig.1D-E). But when the mice were challenged with HFD, obese ZAG^FKO mice had a slight increase in body weight and body fat (Fig. 2A-2D) without alteration of food intake (sFig.1A). Despite the obesity, ZAG^FKO mice demonstrated a significant deterioration in the metabolic phenotype compared with controls. Intraperitoneal GTTs and ITTs were performed in ZAG^FKO and control mice on both the HFD, insulin resistance was remarkably worsened in the HFD ZAG^FKOmice compared with controls(Fig. 2E-2F). Since plasma nonesterified fatty acids (NEFA) mediate insulin resistance, we sought to determine whether differences in serum TG or NEFA levels could account for ZAG depletion-induced insulin resistance. As shown in the Fig. 2G-H, the serum NEFA and TG levels was distinctly elevated in obese ZAG^FKO mice. To further explain the above unexpected findings, We investigate the periphral insulin sensitivity in metabolic tissues of ZAG^FKO and control mice. Consistently, insulin-triggered phosphorylation of Akt displayed substantial blunted in muscle, liver and eWAT of ZAG^FKO mice (Fig. 2I).

3.3 Ablation of adipocyte ZAG deteriorated obesity-induced inflammatory macrophage invasion and polarization in adipose tissues

Chronic inflammation at adipose tissue, typified by increased invasion of M1 macrophages, is a primary contributor to obesity-triggered insulin resistance[28]. ATMs infiltration will aggregate a crown-like structure (CLS) around adipocytes ^[29]. ZAG^FKO mice displayed a more pronounced ATMs recruitment with increased numbers of macrophage crown-like structures (Fig. 3A). Next, we determined whether ZAG deletion in adipocyte result in a deterioration in inflammation of the adipose tissue. Immuno-histochemical staining with anti-F4/80 (a cell surface biomarker of macrophages), anti-CD206 (a cell surface maker of M2 macrophages), and anti-CD11c ((a cell surface maker of M2 macrophage) demonstrated that increased the number of invading pro-inflammatory in the eWAT of ZAG^FKO mice was higher than Loxp mice after 30 weeks of HFD (Fig. 3B). Likewise, The content of markers for pro-inflammatory M1 macrophages (TNF-α, MCP-1, and CD11c) were more higher in obese ZAG^FKO mice .(Fig. 3D, 3F). On the other side, The eWAT of obese ZAG^FKO mice displayed the dampened levels of expression for M2 macrophage markers (CD206, Retnla, IL10) (Fig. 3C, 3E). Consistently, quantified results of the flow cytometry also revealed the elevated composition of M1 macrophages(CD11c^-CD206^་) and reduced composition of M2 macrophages (CD11c^།CD206^་)in contrast with the control group (Fig. 3G), suggesting that adipocyte ZAG ablation triggers the polarization of ATMs toward the pro-inflammatory subpopulations.

3.4 ZAG regulates macrophages phenotypic switching in vitro

Given ZAG deficiency drives metabolic inflammation and macrophage polarization toward M1 phenotype in adipose tissues, we are curious whether exogenous ZAG could reverse PA-induced inflammation and alter phenotype of RAW264.7 macrophages. We found that ZAG not only inhibited the mRNA content of M1 genes (IL-1β, TNF-α, IL-6) but also augmented M2 gene content(CD206, TGF-β, Arg-1) in PA-treated RAW264.7 macrophages(Fig. 4D-E). Flow cytometry analysis had demonstrated that ZAG remarkably enhances the proportion of M2/M1 macrophages in PA-treated RAW246.7 macrophages (Fig. 4F). Additionally, ZAG elicits an anti-inflammatory effect by induction of M2-like polarization in unstimulated M0 RAW246.7 macrophages (Fig. 4A-B). Similarly, recombinant ZAG protein enhanced the ratio of M2-like macrophage subset in unstimulated RAW246.7 macrophages(Fig. 4C). These results highlight that ZAG could directly act as a switcher of macrophages phenotype.

3.5 ZAG drives macrophages M2 polarization via activation STAT3 signaling pathway

It is known that both STAT3 and STAT6 play a particularly crucial role in the suppression of inflammatory responses and M1 macrophage accumulation [31, 32]. Hence, we first determined whether ZAG had a positive regulatory role on the STAT family in macrophages. Unexpectedly, exogenous ZAG had no clear effect on STAT6 signaling (Fig. 5B), but induced activation of STAT3 signaling in RAW264.7 macrophages (Fig. 5A). Moreover, lentivirus-mediated silence of STAT3 could partially block a switching effect of ZAG on the macrophage phenotype (Fig. 5C-5E). Altogether, these data confirm that ZAG drives macrophages M2 polarization via activation STAT3 signaling pathway.

3.6 β3-AR/PKA signaling is required for ZAG activating pSTAT3 signaling pathway in RAW264.7 macrophages

It was recently reported that activation β3-AR or β2-AR by macrophages caused a distinct adipose anti-inflammatory signature and prevented the progress of T2DM in obesity [33, 34]. The previous study has confirmed that ZAG induces lipolysis and browning process of WAT by binding to β3-AR of adipocytes. To further investigate the regulatory mechanisms of ZAG in controlling STAT3 signaling, we investigated the influence of ZAG overexpression on β3-AR and β2-AR signaling. As Fig. 6A-6B shown, ZAG induced the protein along with mRNA expression of β3-AR rather than β2-AR. Furthermore, ZAG-mediated STAT3 signaling activation was abrogated when β3-AR signaling was silenced (Fig. 6C, 6F). PKA is a pivotal adapter protein of β3-AR, that mediates a series of physiological effects of β3-AR. We previously demonstrated that ZAG induces the browning process in a PKA-dependent manner. However, whether PKA is necessary for ZAG-induced phosphorylation of STAT3 remains unknown. As shown in Fig. 6D, ZAG upregulated p-PKA protein content in RAW246.7 macrophages, and then blockade of PKA signaling also dampened p-STAT3 expression in RAW246.7 macrophages (Fig. 6E). Altogether, β3-AR /PKA signaling is required for ZAG activating the pSTAT3 signaling pathway in RAW264.7 macrophages

ATMs serve as a critical effector in the mediating occurrence of metabolic inflammation to impact whole-body insulin sensitivity in obesity [35]. Discovering the key adipokines mediating crosstalk of adipocytes-macrophages and understanding the molecular mechanism of ATMs polarization and function have become hot topic issues in the immunometabolism fields [36]. It is urgent to identify pivotal regulators of ATMs polarization for contributing to developing therapeutic targets of obesity and obesity-associated complication. We have focused on the function of adipokines in metabolic disease, ZAG in particular, as a driver of lipid mobilization in adipocytes and hepatocytes, however, The precise role of adipose ZAG in metabolic inflammation and obesity-associated insulin resistance remains poorly understood.

Hence, We first investigate the clinical correlation between visceral ZAG expression and macrophage polarization. As expected, decreased omental ZAG expression was associated with M2 macrophage markers (Arg-1, IL-10), M1 macrophages markers (IL-6 along with TNF-α), and insulin sensitivity in patients with obesity [37, 38], indicating that ZAG may act as a bioactive messenger to guide the ATMs metabolic reprogramming. Subsequently, we extend the study to investigate whether adipokines ZAG have a therapeutic effect in adipose tissues inflammation and insulin resistance by use of adipose-specific deletion mice. Consistently, we have confirmed the metabolic benefits for the adipocytes ZAG in resisting insulin resistance. Obesity leads to a decrease in the expression of endogenous adipocytes ZAG while repairing the expression of ZAG in adipocytes could inhibit ATMs accumulation, enhance the insulin signaling pathway. But ZAG^FKO mice exhibited adverse metabolic phenotype consisting of impaired glucose tolerance, more pronounced adipose inflammation, and inactivation of insulin signaling in metabolic tissues. Therefore, reduced ZAG activity in adipocytes is a remarkable trigger for adipose tissues inflammation and insulin resistance in obese status

Apart from the above-mentioned metabolic improvement, ZAG induces a biased polarization for the M1 phenotype to the M2 phenotype in mice with HFD. Our in vitro study has shown that ZAG not only enhances the polarization of unstimulated macrophages toward the M2 subsets but is also critically involved in switching of polarization phenotypes between M1 and M2 macrophages. Although ZAG-modulated macrophage polarization is important for adipose tissue function, the key molecular switches responsible for ZAG controlling polarization of ATMs remain poorly obscure. Several pieces of literature have clarified the transcription activators (STAT) family, especially STAT3 and STAT6, are the most dominant signaling cascades to activation of M2 macrophages [39, 40]. In this regard, our present study demonstrated that ZAG enhanced expression of p-STAT3 was validated in our loss-of-function analyses in eWAT. Of note, interference with STAT3 can abolish enhanced M2 polarization in ZAG-treated macrophages. Altogether, we provide strong evidence supporting that STAT3 rather than Stat6 as a downstream target of ZAG in adipocytes.

In previous work, we found that ZAG promotes white fat browning through activation of the cAMP/PKA or p38 MAPK pathway [17]. It is recently reported that cAMP/PKA can mediate exenatide-induced M2-like macrophages polarization [41], but the downstream of a transcription factor for M2 polarization activation by cAMP/PKA is not clear. In our study, blockade of PKA signaling prevents ZAG-induced conversion of macrophages phenotype, indicating that ZAG activates the STAT3 signaling pathway to induce ATMs M2 polarization in a PKA-dependent manner.

β-adrenergic receptor (β-AR), as a primary member of the G protein-coupled receptor family, contains three sub-Type, in which β3-AR is closely correlated with adipose tissue browning. ZAG as natural ligands of β3-AR could enhance energy consumption and lipid catabolism in adipocytes of obese mice [8, 17]. Recent studies have shown that both β3-AR and β2-AR could be detected in the surface of the macrophages membrane, exerting a facilitating role on the polarization of ATMs toward an M2 phenotype in white adipose tissue [42,43]. Our in vitro study has demonstrated that ZAG enhances the M2 macrophages population through binding to β3-AR but not the β2-AR pathway, this result may be conflicted with some previous studies which believe that β2-AR is the main receptor modulating phenotype of ATMs[34]. But the surface of ATMs are overexpressed of sympathetic neurotransmitter catecholamine receptors [42]. Once sympathetic nerves on ATMs are stimulated, ATMs will polarize towards an anti-inflammatory phenotype [44, 45]. ZAG may act as a natural ligand of β3-AR to metabolically reprogram ATMs by activating sympathetic nerves and releasing catecholamine neurotransmitter. PKA, a downstream protein of β3-AR has been reported involved in exenatide-induced M2 polarization [46, 47], Our study has demonstrated that PKA has an activated role on STAT3 signaling, while blockade of PKA could reverse ZAG-mediated phosphorylation of STAT3. These observations further uncover ZAG/β3-AR/PKA/STAT3 as the mechanisms of a novel for the regulatory ATMs polarization.

Despite the obvious findings acquired in this present research, there were several shortcomings including the following point: whether ZAG modulates macrophage polarization in a paracrine manner; is there another regulatory mechanism involved in ZAG alleviating adipose tissues inflammation independent of β3-AR?; The treatment effect of ZAG on obesity-related insulin resistance should further be validated in clinical research;Apart from macrophages, adipose tissues contain a multitude of other immune cell also play important role in metabolic inflammation and insulin resistance[48] .Whether ZAG can act on other immune cells remains to be further investigated. We intend to address this issue in more detail in follow-up studies. In summary, the results unmask the beneficial role of ZAG on macrophages polarization and recruitment and highlight that supplement of ZAG in adipose tissues may serve as a new therapy in the intervention of adipose tissues inflammation and insulin resistance in obesity.

Authorship

All of the authors have read and approved the final version of the manuscript. Zhe-zhen Liao and Ya-Di Wang performed literature. Can Hu, Yun-Ni Mai, Ran-Li drew the figures. Xiao-Yan Qi ,Yuan-Yuan Wang, Qi-Yu Wang, Jing Yang, Jiang-Hua Liu contributed to discussion. Xin-hua Xiao provide the guidance on experimental design. Li-Yong Zhu, Ting Peng and Liang-Liang Wu collected and analysed clinical specimens

Findings

This systematically received funding from the National Natural Science Foundation of China

(82070873, 82000813), the Project of Hunan Provincial Clinical Research Center for Diabetes （2019SK4005), Natural Science Foundation of Hunan province(2020JJ8097, 2021JJ40496, 2020SK51814) and Major special projects of Hunan provincial health and family planning commission (A2017011).

Conflict of Interest

The authors have nothing to disclose

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Table.1 Baseline data of the patients with obesity

Variables	Control	Overweight	Obesity	P-value
Sample (n)	8	8	8	-
Age, years	51.13 ± 9.14	47.38 ± 13.91	29.88 ± 12.51	< 0.001
Gender, male/female	4/4	4/4	4/4	1.0
BMI (kg/m²)	21.86 ± 1.83	25.60 ± 1.31	38.61 ± 8.42	< 0.001
WC (cm)	83 ± 5	91 ± 6	112 ± 34	0.024
SBP (mmHg)	142 ± 22	125 ± 17	133 ± 21	0.259
DBP (mmHg)	80 ± 10	74 ± 9	84 ± 12	0.206
FPG (mmol/l)	5.34 ± 0.72	5.38 ± 0.67	4.96 ± 0.60	0.391
ALT (U/L)	13.3 (10.2,24.0)	25.7 (17.4,59.1)	40.0 (17.6,64.8)	0.238
AST (U/L)	19.0 (11.2,33.2)	18.6 (17.0,28.9)	26.0 (16.1,33.7)	0.969
TC (mmol/l)	3.80 ± 0.77	4.51 ± 0.72	4.63 ± 0.44	0.042
TG (mmol/l)	1.74 (1.15,2.27)	1.67 (1.11,2.76)	1.40 (1.25,2.29)	0.866
HDL-C (mmol/l)	1.38 ± 0.26	1.36 ± 0.40	1.11 ± 0.13	0.135
LDL-C (mmol/l)	2.10 ± 0.90	2.53 ± 0.59	2.56 ± 0.31	0.302

Values are expressed as the mean ± SD or the median.BMI body mass index; WC waist circumference; SBP systolic blood pressure; DBP diastolic blood pressure; FPG fasting blood glucose; ALT alanine aminotransferase; AST aspartate aminotransferase; TC total cholesterol; TG triglycerides; LDL-C low-density lipoprotein cholesterol; HDL-C high-density lipoprotein cholesterol

Supplementary Tables 1-2 are not available with this version

There is NO Competing Interest.

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Graphical abstract
S1.png
sFig.1 the metabolic phenotype of ZAG^FKOmice with SFD or HFD(A) The mRNA expression in different organs of ZAG^FKO mice and Loxp mice (n=6).(B) The protein expression in eWAT of ZAG^FKO mice and Loxp mice (n=3)(C) The food intake curve of Loxp and ZAG^FKOmice after feeding with HFD (n = 6).(D) Weight gain curve of male Loxp and ZAG^FKO mice after feeding with SFD (n = 6).(E) Glucose tolerance test (GTT) of male Loxp and ZAG^FKO mice after SFD (n = 6).(F-G) Weight gain curve and GTT of male Loxp and ZAG^FKO mice after feeding with SFD (n = 6).Data are represented as mean ± SEM.*P < 0.05, **P < 0.01 for the comparison between Loxp mice.
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Adipocyte ZAG improves obesity-triggered insulin resistance by reshaping macrophages populations in adipose tissue

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Abstract

Figures

1. Introduction

2. Research Design And Method

2.1 Animal Experiments

2.2 Isolation of mature adipocytes along with SVF in adipose tissue

2.2 Histology and Immunohistochemistry(IHC)

2.3 Flow cytometry

2.4 Metabolic Studies

2.5 Western blot and RT-PCR analysis

2.6 Cell culture and treatment

2.7 Human Samples

2.8 Statistical analyses

3. Results

3.1 ZAG expression is reduced in visceral adipose tissues in humans or mice with obesity

3.2 Adipose-specific ZAG deficiency exacerbates adiposity and insulin resistance

3.3 Ablation of adipocyte ZAG deteriorated obesity-induced inflammatory macrophage invasion and polarization in adipose tissues

3.4 ZAG regulates macrophages phenotypic switching in vitro

3.5 ZAG drives macrophages M2 polarization via activation STAT3 signaling pathway

3.6 β3-AR/PKA signaling is required for ZAG activating pSTAT3 signaling pathway in RAW264.7 macrophages

4. Discussion

5. Conclusion

Declarations

References

Table

Supplementary Tables

Additional Declarations

Supplementary Files

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