Regulatory T cells differentiation in visceral adipose tissues contributes to insulin resistance by regulating JAZF‐1/PPAR‐γ pathway

Abstract Regulatory T cell (Treg) activity and differentiation in visceral adipose tissue (VAT) play an important role in inhibiting chronic inflammation and insulin resistance. Whether JAZF‐1 and PPAR‐γ mediate VAT Treg differentiation to promote the inhibition of chronic inflammation and insulin resistance remains unclear. Here, we investigated the roles of JAZF‐1 and PPAR‐γ in VAT Treg differentiation, inflammation and insulin resistance using a transgenic mouse model. First, we determined that the levels of glucose and insulin biochemical markers in the JAZF‐1 transgenic general feeding or high‐fat groups were lower than those in the wild‐type general feeding or high‐fat groups. Second, the levels of CD4+, CD25+, and FOXP3+ differentiation markers in the JAZF‐1 transgenic general feeding or high‐fat groups were significantly higher than those in the wild‐type groups. PPAR‐γ inhibition was associated with low levels of CD4+, CD25+ and FOXP3+ differentiation markers. Third, the levels of TNF‐α, IL‐1β and IL‐6 in the JAZF‐1 transgenic groups were lower than those in the wild‐type groups, whereas IL‐10 and TGF‐β levels were higher in the JAZF‐1 transgenic groups than in the wild‐type groups. After using the PPAR‐γ inhibitor, we observed that TNF‐α, IL‐1β and IL‐6 increased, while IL‐10 and TGF‐β decreased. We found that JAZF‐1 and PPAR‐γ could promote Tregs differentiation and regulate insulin resistance by synergistically decreasing the expression levels of TNF‐α, IL‐1β and IL‐6 and increasing those of IL‐10 and TGF‐β.

promote inflammation and lead to insulin resistance. Understanding the mechanisms underlying the progression of insulin resistance can lead to the development of new strategies that relieve insulin resistance and subsequent metabolic diseases.
Local inflammation can occur in visceral adipose tissue (VAT) with the secretion of various fat-specific inflammatory factors and is involved in glucose and lipid metabolism. 8 Regulatory T cells (Tregs) express CD4, CD25 and forkhead box P3 (FOXP3), inhibiting immune and inflammatory responses. 9 Numerous Tregs in VAT can suppress the inflammatory response by secreting cytokines. 10 Moreover, a reduction in the number of Tregs can give rise to metabolic disorders. 11,12 Therefore, regulating the differentiation and activity of VAT Tregs plays an essential role in inhibiting chronic inflammation and insulin resistance.
Peroxisome proliferator-activated receptor gamma (PPARγ) is a proliferator-activated receptor in adipose tissues with antiinflammatory properties and is an important transcription factor that regulates fat remodelling and macrophage polarization. 13 PPARγ activates the transcription of multiple genes and regulates adipocyte, T cell, and macrophage functions. A previous study demonstrated that PPARγ selective knockout in macrophages is significantly related to insulin resistance progression. 14 PPARγ is considered a key factor for regulating Tregs in adipose tissue, promoting the differentiation and activity of VAT Tregs. 15,16 PPARγ can affect the inflammatory response by regulating T cells from the early stages of inflammation into the antioxidant stage. 17 In addition, juxtaposed with another zinc finger gene 1 (JAZF-1) is highly expressed in adipose tissues and is involved in regulating gluconeogenesis, insulin sensitivity, lipid metabolism and inflammatory responses. 18 Studies have demonstrated that JAZF-1 polymorphism is significantly associated with the progression of type 2 diabetes, 19 and JAZF-1 overexpression enhances glucose tolerance and insulin sensitivity. 20 However, the role of JAZF-1 and its downstream pathway molecules in Treg differentiation remains unclear. Therefore, this study aimed to assess the mechanism of action of JAZF-1 and PPARγ on the progression of insulin resistance in VAT Tregs. We found that JAZF-1 and PPARγ could activate Treg differentiation in VAT. These proteins also may work together to decrease the expression levels of TNFα, IL-1β and IL-6, and increase those of IL-10 and TGFβ. Altogether, we believe that JAZF-1 and PPARγ function synergistically to reduce insulin resistance in VATs.

| Chemicals, reagents and antibodies
The adeno-associated virus (AAV) helper-free system was purchased from Agilent Technologies (catalog # 240071), and the pAAV-JAZF-1 plasmid construction followed the AAV helper-free system instructions.
After amplifying and extracting the endotoxin-free plasmid, pAAV-JAZF-1 was transfected into AAV-293 cells. After the cells became large, round, and floated like grape clusters, the virus was collected and purified by caesium chloride (CsCl) density gradient centrifugation. The viral concentration was determined by quantitative PCR (qPCR), and the number of copies was calculated. After packaging, the virus was suspended in 4% sucrose buffer and stored in a refrigerator at −80°C.

| Animal experiments
Male C57BL/6 wild-type mice (4 weeks old, weighing 15 ± 3 g, SPF grade) were purchased from Chongqing Enswell Biotechnology Co., LTD (Chongqing, China). All protocols in this study were approved by the Laboratory Animal Care Committee of Three Gorges Hospital, affiliated with Chongqing University. The mice were fed adaptively for 2-12 h, alternating day and night, temperature of 20-22°C, humidity of 40%-60%, drinking water freely and eating ordinary feed.
After 1 week, 32 mice were randomly divided into wild-type general feeding, wild-type high-fat diet, JAZF-1 transgenic general feeding and JAZF-1 transgenic high-fat diet groups (n = 8). The weight and energy compositions between the general diet and high-fat diet are shown in Table 1. Transgenic mice overexpressing JAZF-1 were obtained by continuously injecting AAV-JAZF-1 in their tail vein for 3 days. Another 32 mice were injected with AAV-JAZF-1 and randomly assigned to general feeding plus normal saline, high-fat diet plus normal saline, general feeding plus PPARγ inhibitor (GW9662) and high-fat diet plus PPARγ inhibitor (n = 8). The insulin resistance model was constructed using a high-fat diet, while an ordinary diet  was given to the control group, and the body weight of mice was recorded weekly.

| Biochemical markers
After 12 weeks, the mice from all eight groups fasted for 12 h and intraperitoneally injected with 2 g/kg glucose. Peripheral blood was collected from the tail vein at 0, 15, 30, 60 and 120 min. Insulin and glucose tolerance were measured using an insulin tolerance test (ITT) and glucose tolerance test (GTT) respectively. Moreover, total cholesterol (TC), triglyceride (TG) and glucose (GLU) levels in the blood were measured using an E600 automatic biochemical analyzer. transfer the cells to an incubator at 37°C with 5% CO 2 . One day before induction, a phosphate buffered saline (PBS) solution containing 5 μg/mL anti-mouse CD28 and anti-mouse CD3 antibodies was added to the Petri dish and coated overnight at 4°C. The next day, CD4 + T cells were added to the pre-coated Petri dishes.

| CD4 + T cells in peripheral blood and Tregs differentiation
Meanwhile, 100 nM recombinant IL-2 was added to induce differentiation. Three days later, differentiation rate peak was reached, and the following experiments were performed. Mice cells from each one of the eight study groups were evenly divided into three tubes for immunofluorescence staining. Anti-mouse CD4-FITC, anti-mouse CD25-APC and anti-mouse FOXP3-PE were added to all the eight group tubes, and the Treg cells differentiation status was detected by flow cytometry.

| Inflammatory markers
For all eight study groups, we collected the serum of the mice's tail vein or the homogenate supernatant of peritoneal omental adipocytes. PBS was added to process the tissue samples into a singlecell suspension. The cytokines in the serum or tissue homogenate supernatant were detected, including TNFα, IL-1β, IL-6, IL-10 and TGFβ. The flow cytometry was performed according to the operating procedures of the cytometric bead array (CBA) kit.

| JAZF-1 and PPARγ expression and their interaction
After 12 weeks of differential feeding, mice in each group were  mean ± standard deviation (SD), and the differences among groups

| Statistical analyses
were assessed using one-way analysis of variance (anova), and Tukey's post hoc test was applied to perform pair comparisons. The inspection level was two sided, and statistical significance was set at p < 0.05.

| JAZF-1 promotes insulin sensitivity
The biochemical markers in the wild-type general feeding, wild-type high-fat, JAZF-1 transgenic general feeding and JAZF-1 transgenic high-fat groups are shown in Figure 1 and Appendix S1. We noted that the weight graduaally increased from 1 to 12 week, and the JAZF-1 transgenic group were associated with lower weight when compared with the wild-type groups at various time points ( Figure S1 in Appendix S1). The TC, TG and GLU levels in high-fat group was significantly increased, while JAZF-1 transgenic mice were associated with lower TC, TG and GLU levels ( Figure 1A). Moreover, the ITT test indicated that the blood glucose content was reduced after insulin injection, and that it was lower in the JAZF-1 transgenic general feeding or high-fat groups than that in the wild-type general feeding or high-fat groups at various time points respectively ( Figure 1B).
Furthermore, the GTT test indicated that blood glucose and insulin contents were significantly increased within 15 min after glucose injection that were shown to be reduced at later time points. The blood glucose and insulin contents in the JAZF-1 transgenic groups were lower than those in the wild-type groups at various time points ( Figure 1C,D).

| JAZF-1 and PPARγ regulate VAT Tregs differentiation
Treg differentiation in VAT was assessed using CD4 + , CD25 + and FOXP3 + antibodies, and the results are shown in Figure 2 and Appendix S2 (Figures S1-S3). By comparing the same feeding treatments, we observed a higher percentage of VAT Tregs in the JAZF-1 transgenic groups than that in the wild-type groups.
These results suggest that JAZF-1 promotes Treg differentiation in VAT (Figure 2A). To assess the role of PPARγ on VAT Tregs, we noted the percentage of VAT Tregs in the JAZF-1 transgenic high-fat diet PPARγ agonist group was significantly higher than that in the PPARγ inhibitor and control groups, while the PPARγ inhibitor group was associated with a lower percentage of VAT Tregs than the control group ( Figure 2B). Finally, the percentage of VAT Tregs in PPARγ inhibitor groups was significantly lower than F I G U R E 1 The biochemical markers in the wild-type general feeding, wild-type high-fat, JAZF-1 transgenic general feeding and JAZF-1 transgenic high-fat groups. (A) TC, TG and GLU levels in the wild-type general feeding, wild-type high-fat, JAZF-1 transgenic general feeding and JAZF-1 transgenic high-fat groups; (B) Blood glucose content assessed by ITT test in the wild-type general feeding, wild-type high-fat, JAZF-1 transgenic general feeding and JAZF-1 transgenic high-fat groups; (C) Blood glucose content assessed by GTT test in the wild-type general feeding, wild-type high-fat, JAZF-1 transgenic general feeding, and JAZF-1 transgenic high-fat groups; (D) Blood insulin content assessed by GTT test in the wild-type general feeding, wild-type high-fat, JAZF-1 transgenic general feeding and JAZF-1 transgenic high-fat groups.

| JAZF-1 and PPARγ affect inflammatory markers expression
The TNFα, IL-1β, IL-6, IL-10 and TGFβ levels in each group are shown in Figure 3 and the Appendix S1-S4. We observed that TNFα, IL-1β and IL-6 levels in the JAZF-1 transgenic general feeding group were lower than those in the corresponding wild-type group. Similarly, the JAZF-1 transgenic high-fat group showed lower TNFα, IL-1β and IL-6 levels as compared to those in the wild-type high-fat group. Contrarily, IL-10 and TGFβ levels in the JAZF-1 transgenic general feeding and high-fat groups were significantly higher than those in the wild-type general feeding and high-fat groups respectively ( Figure 3A). Furthermore, we noted that TNFα, IL-1β and IL-6 levels in the JAZF-1 transgenic PPARγ agonist group were significantly lower than those in the PPARγ inhibitor and control groups. However, the levels of IL-10 and TGFβ in the PPARγ agonist group were higher than those in the PPARγ inhibitor and control groups ( Figure 3B). Finally, TNFα, IL-1β and IL-6 levels in the JAZF-1 transgenic general or high-fat diets plus PPARγ inhibitor groups were significantly lower than those in the general or high-fat diet control groups respectively.
However, the IL-10 and TGFβ levels were significantly higher in mice fed with general or high-fat diets and PPARγ inhibition than those in the respective control groups with the same feeding and without PPARγ inhibition ( Figure 3C).

| Co-expression and interaction of JAZF-1 and PPARγ
The mRNA and protein expression levels of JAZF-1 and PPARγ are shown in Figure 4. As expected, the JAZF-1 transgenic mice showed upregulation of JAZF-1 mRNA and protein when compared to the wild-type general feeding mice, independent of the feeding treatment. PPARγ was also induced in JAZF-1 transgenic mice, suggesting a potential synergistic effect between JAZF-1 and PPARγ ( Figure 4A and Figures S1-S9 in Appendix S3). Moreover, the JAZF-1 and PPARγ mRNA and protein expression levels in the JAZF-1 transgenic mice agonist to PPARγ were significantly higher than those in the control group. Notably, the PPARγ inhibitor was associated with low PPARγ expression ( Figure 4B). These results encourage the hypothesis of synergistic function between JAZF-1 and PPARγ. Furthermore, PPARγ mRNA and protein expression in mice under general diet or high-fat were significantly lower in the PPARγ inhibitor group than those in the respective normal saline groups ( Figure 4C). Finally, the interaction between JAZF-1 and PPARγ proteins was further investigated, and co-IP results did not find interactions between JAZF-1 and PPARγ ( Figure 5 and Tables S1 and S2 in Appendix S4).

| DISCUSS ION
In this study, we investigated the mechanism of action of JAZF-1 The mRNA and protein expression levels of JAZF-1 and PPARγ in general feeding plus normal saline, high-fat plus normal saline, general feeding plus PPARγ inhibitor and high-fat plus PPARγ inhibitor groups.
rs849334 of JAZF-1 is significantly associated with insulin clearance, which could predict the progression of diabetes. 27 In our results, Treg differentiation could be activated by JAZF-1 and PPARγ, and PPARγ inhibitors could inhibit Treg differentiation, consistent with previous studies. 28,29 The PPARγ signalling pathway plays an essential role in regulating various biological processes, especially in the liver. 30 The proliferation and differentiation of adipose tissues can be activated by PPARγ signalling. 31 PPARγ agonists can improve hepatic steatosis and liver lesions, 32 while PPARγ inhibitors significantly increase fat cell necrosis. 33 Furthermore, JAZF-1 can prevent lipogenesis and systematic inflammatory response, which play a role in glucose homeostasis, enhancing glucose tolerance and insulin sensitivity. 20,34 The expression of inflammatory cytokines was inhibited by JAZF-1 and PPARγ, while JAZF-1 and PPARγ activated IL-10 and TGFβ inhibitory cytokines. A previous study has demonstrated that Tregs regulate immune responses and have a protective role in hepatic damage. 35 Moreover, JAZF-1 could promote macrophage polarization from the M1 to M2 phenotype and increase the number of Tregs, which could induce the production of anti-inflammatory cytokines. 20 Furthermore, PPARγ downregulates innate and adaptive immune cells, and PPARγ expression is associated with reduced inflammation and hyperresponsiveness. [36][37][38] Our study found a potential synergistic effect between JAZF-1 and PPARγ. The possible mechanism may involve PPARs binding to PPAR response element (PPRE) to regulate the transcription and activation of target genes. 39 The testicular receptor 4 (TR4) competitively binds to PPRE and inhibits PPARs transcriptional activity, which could be reversed by JAZF-1 mediated transcriptional repression of TR4. 39 Furthermore, JAZF-1 induces carnitines palmitoyl transferase-1a expression in the liver by enhancing hepatic PPARs. 18 After activation of PPARs, visfatin transcription is indirectly promoted by JAZF-1, which plays an important role in insulin sensitivity. 39 Although above, several limitations of this study should be acknowledged. First, Treg cells resident in different non-lymphoid tissues exhibit tissue-specific properties, while our study focused on Tregs differentiation in VAT. 40 Second, it is well-established that a large fraction of VAT Treg cells express the IL-33 receptor ST2 as well as KLRG1 and CCR2, especially in male mice. An additional subset of VAT Treg cells is characterized by a naive-like phenotype, expressing CD73 and TCF-1, 41 while the current study assessed Treg differentiation in VAT using CD4 + , CD25 + and FOXP3 + antibodies. Finally, the IgG immunoprecipitation for negative control in Co-IP was not applied, and the interactions between JAZF-1 and PPARγ was restricted.

| CON CLUS ION
In summary, this study found that JAZF-1 inhibits TC, TG and GLU, and JAZF-1 and PPARγ activation promote Treg differentiation.
Moreover, JAZF-1 and PPARγ activation inhibited TNFα, IL-1β and IL-6, and promoted IL-10 and TGFβ expression. Furthermore, a potential synergistic effect between JAZF-1 and PPARγ, which plays a key role in insulin resistance, was observed. Therefore, JAZF-1 could be explored as a novel therapeutic agent to prevent the progression of insulin resistance.

ACK N O WLE D G E M ENTS
Not Applicable.

This research was supported by the Chongqing Science and
Technology Bureau, China (cstc2019jcyj-msxmX0122).

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that there are no competing interests associated with the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The original contribution presented in the study are included in the article, further inquiries can be directed to the corresponding author.