Lipid-induced monokine cyclophilin-A promotes adipose tissue dysfunction implementing insulin resistance and type 2 diabetes in zebrafish and mice models of obesity

Abstract

Several studies have implicated obesity-induced macrophage–adipocyte cross-talk in adipose tissue dysfunction and insulin resistance. However, the molecular cues involved in the cross-talk of macrophage and adipocyte causing insulin resistance are currently unknown. Here, we found that a lipid-induced monokine cyclophilin-A (CyPA) significantly attenuates adipocyte functions and insulin sensitivity. Targeted inhibition of CyPA in diet-induced obese zebrafish notably reduced adipose tissue inflammation and restored adipocyte function resulting in improvement of insulin sensitivity. Silencing of macrophage CyPA or pharmacological inhibition of CyPA by TMN355 effectively restored adipocytes’ functions and insulin sensitivity. Interestingly, CyPA incubation markedly increased adipocyte inflammation along with an impairment of adipogenesis, however, mutation of its cognate receptor CD147 at P309A and G310A significantly waived CyPA’s effect on adipocyte inflammation and its differentiation. Mechanistically, CyPA–CD147 interaction activates NF-κB signaling which promotes adipocyte inflammation by upregulating various pro-inflammatory cytokines gene expression and attenuates adipocyte differentiation by inhibiting PPARγ and C/EBPβ expression via LZTS2-mediated downregulation of β-catenin. Moreover, inhibition of CyPA or its receptor CD147 notably restored palmitate or CyPA-induced adipose tissue dysfunctions and insulin sensitivity. All these results indicate that obesity-induced macrophage–adipocyte cross-talk involving CyPA–CD147 could be a novel target for the management of insulin resistance and type 2 diabetes.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-022-04306-1.

Introduction

Adipose tissue plays a critical role in regulating both glucose and lipid homeostasis in humans [1]. Adipose tissue contains heterogeneous cell populations mainly composed of mature adipocytes and its committed preadipocytes, mesenchymal stem cells, endothelial cells with various immune cell types including macrophages [1]. Reports from the last few decades clearly indicate that obesity-induced hypertrophic expansion of adipose tissue is strongly correlated with the chronic low-grade inflammation implementing adipose tissue dysfunction and type 2 diabetes (T2D) pathogenesis [2–5]. We and several other investigators have reported that activation of toll-like receptor 4 is causally linked with adipose tissue inflammation and insulin resistance [6–8]. Moreover, the discovery of increased macrophage infiltration into the adipose tissue and proliferation of resident macrophages [9–11] provided major mechanistic insight about the propagation of obesity-induced adipose tissue inflammation. It was observed that adipose tissue macrophages (ATM) comprises about 50% of the stromal cell population in the adipose tissue of obese subjects compared with 5–10% in lean counterparts [9, 10] and also the ATM content is higher in visceral than subcutaneous adipose tissue [12] supporting the concept that visceral fat plays a major role in obesity-induced insulin resistance and T2D [13]. Subsequent studies have demonstrated that prevention of macrophage infiltration into the adipose tissue and/or the impediment of macrophages retention within adipose tissue strikingly improved insulin sensitivity [14, 15]. Studies have also demonstrated that ATMs of lean mice exhibits M2 phenotype or “alternatively activated” macrophages, while in diet-induced obesity, overexpression of various pro-inflammatory genes characteristic of M1 or “classically activated” macrophages was observed [16, 17]. Interestingly, obesity also caused macrophage phenotypic change from an anti-inflammatory M2 to a pro-inflammatory M1 polarization state in the adipose tissue contributing to the onset of insulin resistance in mice [18]. Recent evidence revealed a diverse population of ATMs in the obese patients that exhibits a mixed profile of several M1-M2 gene signatures, although, the prevalence of M1 population is higher than M2-like cells [19]. All these reports provided compelling evidence that cross-talk between inflammatory macrophages and adipocytes in the adipose tissue are paramount in the pathogenesis of obesity-induced insulin resistance and T2D. However, specific macrophage mediators or monokines involved in the cross-talk with adipocytes in obese adipose tissue triggering T2D pathogenesis are unknown and warrant further investigation.

Cyclophilins is a group of 16 distinct immunophilins initially identified as an important member involved in protein folding due to its peptidyl-prolyl isomerase activity [20]. Among the different cyclophilins, cyclophilin-A (CyPA) is most abundant in nature [21] and mainly synthesized and secreted from monocytes [22] in response to inflammatory insults such as hyperglycemia, hypoxia, infection and oxidative stress [23–25]. Secreted CyPA mediates cell–cell communication and cell trafficking upon binding with its cognate receptor CD147 on the target cells [26, 27]. Increased level of circulatory CyPA is known to be associated with various inflammatory diseases including cardiovascular diseases and T2D [28–30] and considered a novel biomarker for patients with T2D [22]. However, the role of CyPA on obesity-induced adipose tissue dysfunction and insulin resistance has never been explored.

Although adipose tissue dysfunction and insulin resistance are markedly correlated with the macrophage–adipocyte cross-talk in the obese adipose tissue microenvironment, however, the causal relationship has not yet been established. In this study, we have identified an obesity-induced monokine CyPA that attenuates adipocytes insulin sensitivity through the induction of adipose tissue inflammation and its dysfunction.

Research design and methods

Reagents and antibodies

All tissue culture materials were obtained from Gibco™, Thermo-Scientific, Grand Island, NY. Please refer to Supplementary Table 1 for key resources table for details of resources used in this study. Different gene-specific primers were procured from Integrated DNA Technologies, India. The detailed gene-specific primer sequences were presented in Supplementary Table 2. All other chemicals and reagents used were purchased from Sigma Chemical Co., St. Louis MO, USA.

Zebrafish husbandry and treatments

In this study, three groups of healthy wild-type IND strain of adult (7 months old) male zebrafish (Danio rerio), collected from the College of Fisheries Scientific Farm, Assam Agricultural University, were used. Prior to the experiment, fishes were acclimatized for 14 days under laboratory condition. The experiment was carried out in 50 L Aquarias containing 30 L of water with continuous aeration. Congenial environmental parameters for the fishes such as photoperiod 14:10 (L:D), water temperature 28 ± 2 °C and pH 7.6 ± 4 was maintained during the entire course of the experiment. The diet-induced obesity and type 2 diabetes model was developed following the procedure described previously [31]. The fishes were fed twice a day with the zebrafish feed pellets (MicroDot M3) in different quantities in different treatment groups as mentioned below: Group I: normal feeding group (20 mg/fish/day for 4 weeks); Group II: overfeeding group (120 mg/fish/day for 4 weeks); and Group III: overfeeding group with TMN355 treatment (120 mg/fish/day for 4 weeks + i.p. administration of TMN355 (10 mg/kg bw) on day 22, day 24 and day 26. On the 28th day, the zebrafish were anesthetized using ice-chilled water and blood was collected from the caudal peduncle as per Babaei et al. [32]. Blood glucose was immediately measured by Accu-Chek glucometer (Roche, Germany) and blood plasma was collected for the measurement of CyPA through ELISA. Intraperitoneal glucose tolerance test (IP-GTT), and intraperitoneal insulin tolerance test (IP-ITT) were performed on these zebrafish after 6 h fasting, following the method described previously [6, 31]. For the IP-GTT, 500 mg glucose/kg bw was administered intraperitoneally, and for the IP-ITT, we injected 1 IU insulin/kg bw. Blood glucose level was determined at 0, 30, 60, and 120 min with an Accu-Chek glucometer. Adipose tissue was harvested from zebrafish, fixed in 10% neutral buffered formalin, and used for immunohistofluorescence analysis of pNF-κB. The animal experiment protocol was carried out following the guidelines and protocol of the Institutional Animal Ethics Committee (AAU/FY/AEM/NAR-01/2021-22/1310).

Mice models and treatments

Wild-type C57BL/6 J male mice aged 5–6 weeks and weighed 20–24 g were procured from the IISER Mohali animal facility and kept in the National Toxicological Center at NIPER S.A.S. Nagar animal house for 7 days in 12-h light/dark cycle at 23 ± 2 °C with relative humidity 55 ± 5% and fed with normal rodent pellet diet and water ad libitum. For the palmitate-infused mouse model, palmitate (100 mM/day/mice) or saline was administered intraperitoneally for 7 days. For studies involving CyPA inhibitor TMN355, mice were intraperitoneally administered with TMN355 (10 mg/kg bw/day) for 3 days after 4 days of palmitate challenge. For the CyPA-infused mouse model, CyPA (500 μg/ kg bw/day) or saline was administered in mice by intraperitoneal injection for 7 days. The role of CD147 was investigated in this mouse model by delivering an anti-CD147 antibody (1 mg/kg bw) for 3 days after 4 days of CyPA challenge. The blood glucose level has been measured regularly with an Accu-Chek glucometer (Roche, Germany). We determined the oral glucose tolerance test (OGTT) by measuring blood glucose levels before and after oral gavages of 1 g glucose/kg bw at the indicated time points. Plasma insulin level was estimated using a Mouse Insulin ELISA kit (Elabscience, USA). Insulin function was evaluated by HOMA-IR and calculated as fasting insulin (mU/l) × fasting glucose (mmol/l)/22.5 [6]. On day 8, mice were sacrificed for different experiments. The visceral adipose tissue was immediately used for FACS analyses and also fixed in 10% neutral phosphate-buffered formalin for histological analysis or frozen in liquid nitrogen for gene and protein expression analysis. The serum samples were used for CyPA, TNFα, and IL-6 ELISA assay. All animal experiments were performed following the guidelines prescribed by and with the approval of the Institutional Animal Ethics Committee, NIPER S.A.S. Nagar, Punjab (Protocol No.: IAEC/21/62).

Cell culture and treatments

Mouse 3T3-L1 preadipocyte cell line (Cat. No. #CL-173) was procured from the ATCC, USA. THP-1 monocyte cell line (Cat. No. #TIB-202, ATCC, USA) was a kind gift from Dr. Rupak Mukhopahyay, Tezpur University and RAW264.7 macrophage cell line was procured from the National Center of Cell Science, Pune, India. 3T3-L1 preadipocyte and RAW264.7 macrophage cells were cultured in DMEM containing 1% Penicillin–Streptomycin and supplemented with 10% BCS or 10% FBS, respectively, in a humidified 5% CO₂ environment at 37 °C as described by us previously [6]. Two days after confluence, preadipocytes were stimulated to differentiate over 7 days in a differentiation medium supplemented with insulin (1 μg/ml), 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM), and dexamethasone (1 μM). Mature adipocytes were used in different experiments. Human monocytic THP-1 cells were cultured in RPMI1640 medium containing 1% Penicillin–Streptomycin and supplemented with 10% FBS in a humidified 5% CO₂ environment at 37 °C. Two days post-confluence, THP-1 monocytes were differentiated to macrophages by 48 h incubation with PMA (5 ng/ml). We have prepared a 50 mM palmitate-5% BSA stock solution as described by us previously [33]. Briefly, palmitate was dissolved in ethanol and diluted 1:100 in 1% FBS (v/v) and 5% BSA (w/v) containing DMEM. In case of cell incubations with inhibitors, CyPA inhibitor TMN355 (1 μM) or NF-κB inhibitor Bay11-7082 (20 μM) or Wnt signaling inhibitor IWR-1-endo (15 μM) were added 1 h before the addition of CyPA (10 ng/ml). Upon termination of incubations, cells were washed twice with ice-cold DPBS and harvested with trypsin–EDTA solution using a cell scraper. Harvested cell pellets were used for protein or total RNA extraction. For protein extraction, cell pellets were re-suspended in NP-40 lysis buffer supplemented with Halt protease and phosphatase inhibitor cocktail, vigorously vortexed every 10 min for 30 min, centrifuged for 10 min at 17,950g at 4 °C, and the supernatant was collected. The protein concentration of the supernatant was determined by following the method of Lowry et.al. [34]. For total RNA extraction, TRI reagent (Sigma, USA) or RNeasy Lipid Tissue Mini Kit (Qiagen, Germany) were used.

Transwell coculture of macrophages and adipocytes

RAW264.7 macrophages (1 × 10⁵ cells) were cultured on a transwell cell culture insert (0.4-μm pore size) transfected without or with control siRNA or Cyclophilin-A siRNA for 48 h. Cells were then treated with BSA or palmitate (0.75 mM) for 8 h and then washed several times with PBS to remove the residual palmitate, if any, and placed on a plate containing 3T3-L1 adipocytes (1 × 10⁵ cells) and incubated for 8 h. After 8 h, the 3T3-L1 adipocytes were used for glucose uptake assay.

RNA interference study

3T3-L1 adipocytes (1 × 10⁵ cells/insert) were transfected with Control siRNA or Cyclophilin-A siRNA using Lipofectamine™ RNAiMAX Transfection Reagent following the manufacturer’s protocol. The transfection mixture prepared in Opti-MEM was added to the cells and incubated for 6 h at 37 °C. After the addition of 20% FBS in culture media, cells were kept for an additional 18 h. The media was replaced with a fresh culture medium containing 10% FBS and incubated for 48 h. After 48 h of transfection, knockdown efficiency was analyzed by RT-qPCR.

Glucose uptake assay

Glucose uptake assay was performed using a glucose uptake cell-based assay kit following the manufacturer’s instructions. Briefly, 3T3-L1 adipocytes (1 × 10⁴ cells/well) were serum-starved overnight in Kreb’s Ringer Bicarbonate Buffer (Cat. No. #TL-1097; HiMedia Laboratories, Mumbai, India) supplemented with 0.2% bovine serum albumin (BSA). Cells were pretreated with TMN355 for 1 h followed by the incubation of CyPA at various concentrations for different time periods. In a separate set of experiment, 3T3-L1 adipocytes were incubated with the conditional media of macrophages for 8 h. Insulin (100 nM) was added 30 min before the termination of incubations. Addition of supraphysiological concentration of insulin (100 nM) is a well-accepted model typically used in cell cultures for the induction of insulin-stimulated glucose uptake in 3T3-L1 adipocytes by several investigators [35, 36] including us [6]. Fluorescent-labeled glucose analog 2-NBDG was added to each of the incubations for 10 min before the termination of the experiment. Cells were then lysed and fluorescent intensity was measured by Varioskan LUX Multimode Microplate Reader (Thermo Scientific, Finland).

Luciferase reporter assay

The RAW264.7 and THP-1 macrophages, (1 × 10⁴ cells/well) were transfected with CyPA promoter luciferase plasmid (0.1 μg/well) and 3T3-L1 adipocytes (1 × 10⁴ cells/well) were transfected with CyPA promoter luciferase plasmid or κB-luciferase or PPRE-luciferase plasmids (0.1 μg/well) using Lipofectamine 3000 Transfection Reagent following manufacturer’s protocol. Briefly, 0.3 µl of Lipofectamine 3000 Transfection Reagent and 0.1 μg respective plasmids were added separately into 10 μl of Opti-MEM media; both of these solutions were mixed and incubated for 10 min. The transfection mixture was added to the cells containing DMEM media without antibiotics. After incubation at 37 °C for 6 h, the culture medium was changed to DMEM containing 20% FBS. After 48 h of transfection, cells were washed with DMEM and used for different incubations. On termination of incubations, 3T3-L1 cells were lysed and luciferase activity was measured using Steady-Glo Luciferase Assay System with the help of Varioskan LUX Multimode Microplate Reader (Thermo Scientific, Finland).

Adipogenesis and Oil-Red O staining

To investigate the effect of CyPA and TMN355 on adipogenesis, 3T3-L1 preadipocytes (1 × 10⁴ cells/well) were incubated without or with CyPA in the absence or presence of TMN355 followed by the incubation of insulin, IBMX and dexamethasone. On termination of incubations, cells were fixed in 4% paraformaldehyde for 10 min and stained with Oil-Red O stain for 30 min at room temperature. Cells were rinsed in 60% isopropanol and then washed in PBS for three times. Cellular images were taken using an inverted fluorescent microscope (Leica DMi8, Germany) and lipid accumulation was measured by a spectrophotometer at 492 nm.

Immunoblotting

Immunoblot analysis was performed following our previously described method [6]. Briefly, cell or tissue lysates (50 µg of protein) were subjected to either 10% or 15% SDS–PAGE and transferred onto Immobilon E PVDF membranes (Millipore, Bedford, MA) with the help of Wet/Tank Blotting System (Bio-Rad Laboratories, Hercules, CA). 5% BSA in TBST (TBS containing 0.1% Tween 20) buffer was used to block the membranes for 1 h followed by the overnight incubation with primary antibodies (1:500 or 1:1000 dilutions) in a rotating shaker at 4 °C. The membranes were then washed three times with TBST buffer for 10 min intervals and incubated with peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (1:20,000 dilution) for 2 h at room temperature. Membranes were then washed three times with TBST for 10-min interval and TBS for 5-min interval. After washing, membranes were subjected to Clarity™ Western ECL Substrate incubation for 5 min at room temperature. Protein bands were visualized and quantified in Chemidoc XRS + System (Bio-Rad Laboratories, USA) using Image Lab Software.

Semi-quantitative and real-time quantitative PCR

Total RNA was extracted from the THP-1 macrophages or 3T3-L1 adipocytes using TRI reagent (Sigma, USA) or RNeasy Lipid Tissue Mini Kit (Qiagen, Germany), respectively, following the manufacturer’s instructions. RNA was then treated with DNase I and reverse transcribed using the iScript Reverse Transcription Supermix. We used 2× PCR Master Mix for semi-quantitative RT-PCR in BioRad C-1000 Thermal Cycler and PowerUP SYBR Green MasterMix to perform real-time quantitative PCR in ABI-7500 system using gene-specific primers. The following cycling conditions were used for real-time qPCR: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s, 55 °C for 30 s, and 72 °C for 30 s. After the final extension, a melting curve analysis was performed to ensure the specificity of the products, and the fold changes in expression were determined using 2^ΔΔCt. 18s RNA and β-actin were simultaneously amplified in separate reactions and used for correcting the Ct value.

Enzyme-linked immunosorbent assay (ELISA)

We have measured TNFα and IL-6 cytokine levels in the cell culture medium of control and treated cells using mouse TNFα and IL-6 ELISA kits (BioLegend, USA) following the manufacturer’s instructions. ELISA was also performed to determine the CyPA level in zebrafish plasma, mouse serum, and 3T3-L1 adipocytes culture media following the procedure described previously [37]. Briefly, 100 ng of plasma proteins or cell secretory proteins was coated on the ELISA plate and incubated overnight at 4 °C. Each well was washed thoroughly with PBST (1X PBS, pH 7.4 containing 0.05% Tween-20) followed by the blocking with 5% BSA in PBST for 30 min at room temperature. After washing with PBST, an anti-CyPA antibody was added to each well at 1:50 dilution and incubated for 4 h. The wells were then washed and incubated with HRP-conjugated anti-rabbit secondary antibody for 2 h. On termination of incubation, wells were washed with PBST and TMB substrate was added and incubated further for 30 min in dark. The reaction was terminated by adding H₂SO₄ (2 M) and the absorbance was recorded at 492 nm. Different concentrations of CyPA protein were used for standard curve preparation to estimate the CyPA in samples.

Immunofluorescence analysis

Immunostaining was performed on cryosections of zebrafish and mouse visceral adipose tissue using specific antibodies. Briefly, adipose tissue samples were fixed in neutral buffer formalin (10%) overnight at 4 °C, then embedded in OCT (optimal cutting temperature compound) and frozen at −30 to −60 °C followed by cryosections using Cryotome (Leica CM 1860, Leica Biosystem, Wetzlar, Germany). Tissue cryosections (10 μm) were placed in gelatine-coated glass slides, fixed with ice-cold methanol for 5 min, blocked with 1% BSA containing blocking buffer, and incubated with specific primary antibodies for 1 h at room temperature. After washing, the signal was visualized by subsequent incubation with fluorescence-conjugated appropriate secondary antibodies and counterstained with an anti-fade mounting medium containing DAPI. Immunostaining was also performed on 3T3-L1 adipocytes. Briefly, control and treated adipocytes (1 × 10⁴ cells/well) were fixed with 4% paraformaldehyde for 10 min followed by blocking with 1% BSA in PBST for 1 h at room temperature. Cells were then incubated with anti-pNF-κB (S536) antibody at 1:50 dilution overnight at 4 °C. After washing, cells were incubated for 2 h with the Alexa Fluor 594 conjugated anti-mouse IgG antibody at 1:200 dilution. Cells were then washed with ice-cold PBS and mounted in an anti-fade mounting medium containing DAPI for nuclear counterstaining. Cellular images were taken by an inverted fluorescent microscope (Leica DMi8, Germany) using LAS X software.

Flow cytometric analysis

Visceral adipose tissue collected from mice was rinsed in sterile PBS, chopped into small pieces, and then digested in Hanks’ Balanced Salt Solution containing collagenase type II (2 mg/ml), glucose (5.5 mM), and 4% BSA (fatty acid-free) for 45 min at 37 °C water bath shaker. The enzymatic activity was then neutralized by the addition of serum and the digestion mixture was passed through a cell strainer (pore size: 70 μm, HiMedia, India). The isolated cell suspension was subjected to centrifugation at 130 g for 10 min at 4 °C. The cell pellets were re-suspended in FACS staining buffer (PBS containing 0.2% FBS and 0.09% NaNO₃) and blocked with TruStainFcX™ (Fcγ blocker, mouse anti-CD16/32 antibody, BioLegend) for 15 min at 4 °C. Cell pellet was washed using ice-cold PBS and the cells were incubated with fluorochrome tagged F4/80 and CD11b antibody for the staining of adipose tissue macrophages (ATMs) in ice for 1 h. Cells were then washed twice with chilled PBS, re-suspended in FACS staining buffer, and analyzed in a Flow Cytometer (BD Accuri C6 + , BD Biosciences, San Jose, CA) using FlowJo™ v10.6.1 software. The cell surface markers CD80 and CD206 expression in control or treated RAW264.7 macrophages were also assessed through flow cytometry. Briefly, RAW264.7 cells (1 × 10⁶ cells/well) were treated without or with CyPA for 8 h in the absence or presence of TMN355 and on termination of incubations, cells were re-suspended in FACS staining buffer, and blocked with TruStainFcX™ for 15 min at 4 °C. Cells were then stained with fluorochrome-labeled antibodies against mouse CD80 and CD206 for 60 min at 4 °C followed by two steps of washing. Data was collected in BD Accuri C6 + and analyzed using FlowJo™ Software.

Cell viability assay

3T3-L1 cells were seeded in a 96-well plate and differentiated into mature adipocytes and treated with varied concentrations of CyPA or TMN355 for 24 h. MTT reagent (Cat. No. TC191; HiMedia Laboratories, Mumbai, India) was added to each well and incubated for 4 h at 37 °C. On termination of incubations, formazan crystals formed in cells were dissolved in acidic isopropanol and incubated further for 30 min at 37 °C. Cytotoxicity was measured spectrophotometrically at 570 nm with Multiskan GO Microplate Spectrophotometer (Thermo Scientific, Finland). Absorbance values were blanked against acidic isopropanol and the absorbance of cells exposed to medium only (without any treatment) was taken as 100% cell viability (control).

Monocyte migration assay

Monocyte migration assay was performed using transwell inserts containing 5.0 μm pores (Cat. No. #3421, Corning, NY). The serum-starved THP-1 monocytes (0.5 × 10⁶ cells) were placed in the upper chamber of 5.0 μm transwell inserts and recombinant CyPA protein was added to the media placed in the lower chamber of 24-well plates and allowed for its migration for 6 h. Media containing 10% FBS was used as a positive control. The upper surface of the membrane was gently scrubbed with a cotton swab, and the cells that migrated to the lower membrane surface were fixed with 2.5% glutaraldehyde for 10 min and stained with 0.5% crystal violet solution for 2 h. The bright-field images of migrated cells were captured by a microscope (Leica DMi8, Germany). The crystal violet staining was measured by a spectrophotometer at 570 nm.

Glut4 translocation assay

GFP-Glut 4 transfection was carried out according to our earlier published method [38]. Briefly, 3T3-L1 preadipocytes were cultured and differentiated on sterile Millicell® EZ-Slide, and transfected with GFP-Glut 4 (2 μg) plasmid using Lipofectamine 3000 Transfection Reagent in accordance with the manufacturer’s protocol. After 48 h of transfection, cells were incubated without or with insulin (100 nM) in the absence or presence of FFA (Palmitate, 0.75 mM) or CyPA (10 ng/ml) for 4 h. On termination of the incubation, cells were fixed in 4% paraformaldehyde and mounted in Vectashield anti-fade mounting medium containing DAPI for nuclear staining. Cellular images were taken by an inverted fluorescent microscope (Leica DMi8, Germany) using LAS X software.

Site directed mutagenesis

A pCMV-BSG (CD147) construct containing 621 bp human CD147 (Plasmid #53146) was obtained from Addgene. We used this plasmid as a template for the generation of CD147 mutant plasmids (P309A and G310A) using a QuickChange Lightning Multi Site-Directed Mutagenesis kit following the manufacture’s protocol. Primers used to mutate P309A and G310A sites of CD147 were designed with the help of QuikChange Primer Design Program available online at www.agilent.com/genomics/qcpd. Forward and reverse primer sequences used for mutated CD147 plasmid construction are listed in Supplementary Table 2.

Statistical analysis

All data were derived from at least three independent experiments and statistical analyses were conducted using Sigma Plot 10.0 software. Densitometric analysis of RT-PCR and Western blot data was performed using ImageJ software (1.48v, NIH, USA). Data were analyzed by unpaired Student’s t test, where the p value indicated significance. All values were means ± SEM. A level of p < 0.05 was considered significant.

Results

CyPA-induces impairment of insulin sensitivity and glucose tolerance in obese zebrafish

To investigate the role of CyPA on insulin resistance in vivo, we prepared diet-induced obesity (DIO) in zebrafish by overfeeding (120 mg/fish/day) of commercially available feed for 4 weeks (Fig. 1A). We have found that overfeeding significantly increased body weight, and BMI in zebrafish (Fig. 1B,C) along with the enhancement of blood glucose and plasma CyPA level (Fig. 1D,E) compared to normal feeding (20 mg/fish/day). We then examined whether inhibition of CyPA by its pharmacological inhibitor TMN355 is able to mitigate obesity-induced impairment of adipose tissue dysfunction and insulin sensitivity in vivo, as it will be an ideal model to understand the underlying mechanism of CyPA action in implementing lipid-induced insulin resistance. To check the specificity of TMN355 action on zebrafish CyPA, we explored the protein sequence alignment of CyPA in different organisms which showed higher sequence similarity with conservation of its functional domains (Supplementary Fig. 1A). Administration of TMN355 on overfed zebrafish notably decreased blood glucose level (Fig. 1D), along with the reduction of plasma CyPA level (Fig. 1E). Importantly, TMN355 administration rescued zebrafish from overfeeding-induced insulin resistance as evident from the glucose tolerance test (GTT), and insulin tolerance test (ITT) (Fig. 1F, G). Obesity-induced impairment of insulin-signaling pathway markedly associated with the induction of NF-κB-dependent inflammation that caused inhibitory serine phosphorylation, instead of tyrosine phosphorylation, of IRS-1 disrupting Akt activation [6]. Delivery of TMN355 significantly attenuated DIO-associated inhibitory serine phosphorylation in IRS-1 and Akt inactivation in zebrafish (Fig. 1H). Moreover, DIO-associated increased activation of NF-κB was strikingly inhibited by TMN355 treatment in zebrafish adipocytes (Fig. 1I, J and Supplementary Fig. 1B) indicating the significance of CyPA effect on adipose tissue inflammation. All these results implicated that DIO caused increased production of CyPA which is involved in adipose tissue inflammation and insulin resistance in zebrafish.

Fig. 1.

CyPA-induces insulin resistance and type 2 diabetes in obese zebrafish. A–C Photographic images (A), body weight (B), BMI (C) value of lean and diet-induced obese (DIO) zebrafish (n = 4). D, E Blood glucose level (D), and ELISA of plasma CyPA level (E) of lean- or DIO- or DIO plus TMN355 administrated zebrafish (n = 3). F–H Insulin resistance was determined by measuring oral glucose tolerance test (F), intraperitoneal insulin tolerance test (G), and western blot analyses of insulin-signaling pathway molecules activation and inactivation (H) in these zebrafish (n = 12). I, J Immunohistofluorescence images of pNF-κB (I) and its quantification (J) in the adipose tissue from lean- or DIO- or DIO plus TMN355 administrated zebrafish (n = 3). Scale bar, 100 µm. Each value represent as mean ± SEM, ***p < 0.001, **p < 0.01, *p < 0.05 vs lean; ###p < 0.001, ##p < 0.01, #p < 0.05 vs DIO

Lipid incubation stimulates macrophage CyPA expression that promotes insulin resistance in adipocytes

It is now well established that the increased number of adipose tissue macrophages and their pro-inflammatory polarization state in obese subjects are primary inducers of chronic adipose tissue inflammation that is negatively correlated with insulin sensitivity [9–18]. We, therefore, embarked to ask the question of whether an increased lipid microenvironment in obese adipose tissue may induce CyPA expression in the adipose tissue macrophages and that attenuates adipocytes insulin sensitivity. To investigate this, we first performed a glucose uptake assay in 3T3-L1 adipocytes incubated with the RAW264.7 macrophages treated without or with palmitate, a saturated free-fatty acid. A significant attenuation of insulin-stimulated 2-NBDG uptake was observed in adipocytes when cocultured with palmitate incubated macrophages (Fig. 2A), suggesting the possible involvement of lipid-induced macrophage secretory mediator(s) in the impairment of adipocytes insulin sensitivity. Previous reports have highlighted that increased plasma levels of cyclophilin-A (CyPA) in type 2 diabetes (T2D) patients are mainly contributed by the macrophages [22, 29]. We found that palmitate incubation significantly increased CyPA promoter luciferase activity in RAW264.7 and THP-1 macrophages, however, it could be interesting to note that CyPA reporter activity and CyPA gene expression were not altered in 3T3-L1 adipocytes in response to palmitate treatment (Supplementary Fig. 2A, B and Fig. 2B) indicating lipid specifically induces CyPA expression in macrophages. To confirm the macrophage originated CyPA associated with the impairment of adipocytes insulin sensitivity, CyPA-silenced RAW264.7 cells (Supplementary Fig. 2C) were treated without or with palmitate and exposed to coculture with 3T3-L1 adipocytes for analyzing its effect on cellular glucose uptake. Interestingly, CyPA-silenced macrophages treated with palmitate failed to inhibit insulin-stimulated 2-NBDG uptake by 3T3-L1 adipocytes (Fig. 2C) suggesting the participation of macrophage-derived CyPA in the attenuation of adipocytes insulin sensitivity. We performed a dose and time kinetics study of palmitate on CyPA gene expression in RAW264.7 macrophages and found a dose- and time-dependent induction of macrophage CyPA gene expression in response to palmitate incubation (Fig. 2D). We have also found a striking increase in CyPA secretion in the culture media and the intracellular CyPA level in THP-1 macrophages in response to palmitate incubation (Fig. 2E, F). To have direct evidence in support of the role of CyPA on adipocytes insulin resistance, we incubated 3T3-L1 adipocytes with the different concentrations of CyPA for varied time periods. CyPA incubation caused dose- and time-dependent impairment of 2-NBDG uptake (Fig. 2G) and attenuates insulin-stimulated Glut-4 migration (Supplementary Fig. 2D) in 3T3-L1 adipocytes. Further, CyPA-induced impairment of glucose uptake and insulin-signaling pathway proteins activation was markedly inhibited by CyPA inhibitor TMN355. (Fig. 2H, I). Since JNK activation plays a key role on inhibitory IRS-1 serine phosphorylation that attenuates insulin sensitivity, we measured pJNK level and found that CyPA incubation strikingly increased JNK activation (Supplementary Fig. 2E). Moreover, inhibition of JNK activation by its pharmacological inhibitor SP600125 significantly attenuates CyPA-induced JNK activation and IRS-1 inhibitory serine phosphorylation (Supplementary Fig. 2F). MTT assay clearly indicates that CyPA and TMN355 incubations did not produce any toxic effects on 3T3-L1 adipocytes in response to the concentrations used in this study (Supplementary Fig. 2G). In obese individuals, adipose tissue macrophages (ATM) are pro-inflammatory in nature showing a classically activated M1 phenotype whereas ATMs of lean individuals exhibit an anti-inflammatory signature representing an alternatively activated M2 state [16, 17]. To examine the effect of CyPA on RAW264.7 macrophage polarization, we observed a significant induction of macrophage pro-inflammatory state in response to CyPA as indicated by the enhanced level of CD80, however, such effect was diminished by TMN355 along with the increase of anti-inflammatory marker CD206 (Fig. 2J and Supplementary Fig. 2H). Collectively, these results illustrated that lipid-induced upregulation of macrophage CyPA significantly promotes macrophage M1 polarization and attenuate adipocytes insulin sensitivity.

Fig. 2.

Lipid incubation stimulates macrophage CyPA expression which induces insulin resistance in 3T3-L1 adipocytes. A Determination of insulin-stimulated 2-NBDG uptake by 3T3-L1 adipocytes in response to the conditional media of RAW264.7 macrophage treated without or with a saturated free-fatty acid palmitate (FFA, 0.75 mM) for 8 h, ***p < 0.001 vs Mϕ-BSA-Con, ##p < 0.01 vs Mϕ-BSA-Ins. B CyPA promoter activity was measured in RAW264.7 macrophages, THP-1 macrophages, and 3T3-L1 adipocytes transfected with CyPA promoter luciferase reporter plasmid followed by the incubation of palmitate (0.75 mM) for 8 h, **p < 0.01, *p < 0.05 vs Con, ns  non-significant. C Analysis of insulin-stimulated 2-NBDG uptake by 3T3-L1 adipocytes in response to the conditional media obtained from control siRNA or CyPA siRNA-transfected cells treated without or with palmitate (0.75 mM) for 8 h. ***p < 0.001 vs Mϕ-BSA-siCon, ##p < 0.05 vs Mϕ-FFA-siCon. D RT-qPCR analysis of CyPA gene expression in RAW264.7 macrophages treated with different concentrations of palmitate (0, 0.25, 0.50, and 0.75 mM) for 8 h or 0.75 mM of palmitate for varied time periods (0, 2, 4, 8 h). 18 s RNA was used as loading control, ***p < 0.001, **p < 0.01 *p < 0.05 vs Con. E ELISA showing CyPA secretion from THP-1 macrophages treated without or with palmitate (0.75 mM) for 8 h, **p < 0.01 vs Con. F Western blot (upper) and its quantification (lower) showing abundance of CyPA in THP-1 macrophages in response to palmitate (0.75 mM) incubation for 8 h. β-Actin was used as loading control, **p < 0.01 vs Con. G Dose- and time-dependent effect of CyPA on insulin-stimulated 2-NBDG uptake by 3T3-L1 adipocytes, ***p < 0.001 vs Con; ###p < 0.001, ##p < 0.01, #p < 0.05 vs Ins. H Western blot (left) and its quantification (right) showing pAkt (S473) and pIRS1 (S636) abundance in 3T3-L1 adipocytes treated without or with CyPA (10 ng/ml) in the absence or presence of CyPA inhibitor TMN355 (1 μM) for 8 h. β-Actin was used as loading control, **p < 0.01, *p < 0.05 vs Con; ##p < 0.01 vs CyPA. I Determination of insulin-stimulated 2-NBDG uptake by 3T3-L1 adipocytes in response to CyPA (10 ng/ml) treated without or with TMN355 (1 μM) for 8 h, ***p < 0.001 vs Con, ##p < 0.01 vs Ins, ^†p < 0.05 vs Ins + CyPA. J Flow cytometric analysis of CD80 and CD206 levels in RAW264.7 macrophages treated without or with CyPA (10 ng/ml) in the absence or presence of TMN355 (1 μM) for 8 h. All experiments were performed in triplicate. Each value is the mean ± SEM of three independent experiments

CyPA fueled adipocyte inflammation and macrophage infiltration

Several studies implicated the role of CyPA on cellular inflammation [20–22, 39] and since adipose tissue inflammation plays a key role in adipocyte dysfunction and insulin resistance [40–42], we decided to decipher the role of CyPA on adipocyte inflammation and its function. CyPA incubation remarkably enhanced adipocyte inflammation as exhibited by the increased expression of various pro-inflammatory cytokines genes (iNOS, TNF-α, IL-1β, and IL-6) (Fig. 3A) without any significant impairment of anti-inflammatory gene expression (Supplementary Fig. 3A). Notably, suppression of CyPA by TMN355 caused inhibition of pro-inflammatory cytokines gene expressions in response to CyPA (Fig. 3A). Activation of NF-κB transcription factor by phosphorylation and its separation from IκBα controls the upregulation of various pro-inflammatory cytokines expression governing cellular inflammation [43]. CyPA-induced activation of NF-κB (Fig. 3B, C and Supplementary Fig. 3B), κB-luciferase reporter activity (Fig. 3D), and NF-κB regulated TNF-α and IFN-γ protein expression (Fig. 3E) in 3T3-L1 adipocytes were inhibited by TMN355. Massive accumulation of infiltrated macrophages in the obese adipose tissue [9–11] and the involvement of extracellular CyPA in macrophage chemotaxis [44] have been shown to be correlated with the chronic inflammatory states. We made a similar observation as incubation of CyPA displayed an enhancement of THP-1 cells migration in the transwell chamber (Supplementary Fig. 3C).

Fig. 3.

CyPA stimulates adipocyte inflammation. A RT-PCR analysis (left) and its quantification (right) of iNOS, TNF-α, IL-1β and IL-6 gene expressions in 3T3-L1 adipocytes treated without or with CyPA (10 ng/ml) in the absence or presence of TMN355 (1 μM). LPS (100 ng/ml) was used as positive control. β-Actin serves as loading control. B Western blot (upper) and its quantification (lower) showing pNF-κB abundance in 3T3-L1 adipocytes incubated without or with CyPA (10 ng/ml) in the absence or presence of TMN355 (1 μM) for 8 h. β-Actin was used as loading control. C Immunofluorescence images showing pNF-κB level in 3T3-L1 adipocytes incubated without or with CyPA (10 ng/ml) in the absence or presence of TMN355 (1 μM) for 8 h. Scale bar, 20 µm. D 3T3-L1 adipocytes transfected with κB promoter luciferase plasmid was incubated without or with CyPA (10 ng/ml) in the absence or presence of TMN355 (1 μM) for 8 h. On termination of incubations, cells were lysed and luciferase activity was measured by multimode reader. E Western blots (left) and their quantifications (right) showing TNF-α, and IFN-γ abundance in 3T3-L1 adipocytes incubated without or with CyPA (10 ng/ml) in the absence or presence of TMN355 (1 μM) for 8 h. β-Actin was serves as loading control. All experiments were performed in triplicate. Each value is the mean ± SEM of three independent experiments, ***p < 0.001, **p < 0.01, *p < 0.05 vs Con; ###p < 0.001, ##p < 0.01, #p < 0.05 vs CyPA

CyPA incubation attenuates adipocyte differentiation

Adipocyte function is mainly governed by the maintenance of intricate homeostasis of preadipocyte to adipocyte differentiation or adipogenesis [45]. To examine the role of CyPA on adipogenesis, we incubated 3T3-L1 preadipocytes with CyPA for 7 days in an adipocyte differentiation medium (ADM). Treatment of CyPA in 3T3-L1 preadipocytes affected adipogenesis as indicated by the Oil-Red O staining of lipid accumulation and C16 BODIPY fluorescence, however, such attribute was prevented when co-treated with TMN355 (Fig. 4A, B). Since PPAR-γ and C/EBP-β function as key transcriptional activators regulating adipocyte differentiation through the induction of different adipogenic gene expressions such as FABP-4, and Glut-4 [45], we evaluated the expression profile of these markers in response to CyPA. A striking reduction of PPAR-γ, C/EBP-β, and FABP-4 gene expression in CyPA incubated 3T3-L1 cells were notably rescued by TMN355 treatment (Fig. 4C). In addition, the subdued level of FABP-4 and Glut-4 proteins in 3T3-L1 cells in response to CyPA was significantly inhibited by TMN355 incubation (Fig. 4D). There was no change in de novo lipogenesis as indicated by triglyceride release from adipocytes (Fig. 4E) and the lipogenic markers gene expression such as Acetyl-CoA carboxylase (ACC), and sterol regulatory element-binding protein-1c (SREBP-1c) (Fig. 4F), although a considerable downregulation of lipoprotein lipase (LPL) gene expression was observed in CyPA-treated cells (Fig. 4F). Together, these experiments demonstrated that CyPA-induced impairment of adipogenesis and lipid uptake may be responsible for adipocyte dysfunction.

Fig. 4.

CyPA-induced adipocyte dysfunction was prevented by TMN355. A, B Oil-Red O staining images (left) and the estimation of lipid contents (right) (A), and C16 BODIPY fluorescence images (left) and its quantifications (right) (B) representing 3T3-L1 adipocyte differentiation in response to CyPA (10 ng/ml) incubation without or with TMN355 (1 μM) for 7 days in the presence of adipocyte differentiation medium (ADM). Scale bar, 100 µm (A) and 250 µm (B). C,D RT-PCR analysis (left) and its quantification (right) of PPAR-γ, C/EBP-β, and FABP-4 gene expressions (C) and Western blot images (upper) and its quantifications (lower) showing abundance of FABP-4 and Glut-4 proteins (D) in 3T3-L1 cells treated without or with CyPA (10 ng/ml) in the absence or presence of TMN355 (1 μM) for 7 days in ADM. β-Actin was serves as loading control for RT-PCR and Western blotting. E Lipogenesis was measured by the estimation of triglyceride release into the cell culture medium of 3T3-L1 cells treated without or with CyPA (10 ng/ml) in the absence or presence of TMN355 (1 μM) for 7 days in ADM. F RT-PCR analysis (left) and its quantification (right) showing abundance of ACC, SREBP1c, and LPL mRNA levels in 3T3-L1 cells treated without or with CyPA (10 ng/ml) in the absence or presence of TMN355 (1 μM) for 7 days in ADM. β-Actin was used as loading control. All experiments were performed in triplicate. Each value is the mean ± SEM of three independent experiments, ***p < 0.001, **p < 0.01 vs Con; ###p < 0.001, ##p < 0.01, #p < 0.05 vs CyPA; ns  non-significant

CyPA–CD147 signaling involves in promoting adipocyte inflammation and impairment of adipogenesis in vitro

Accumulating evidence indicated CD147 as a cell surface signaling receptor of extracellular CyPA which mediates its chemotactic activity toward a variety of immune cells [26, 27, 46]. A report in this direction implicated that Pro³⁰⁹ and Gly³¹⁰ residues in CD147 are critical for CyPA-mediated signaling [27]. We, therefore, investigated the role of CyPA–CD147 signaling on adipogenesis and adipocyte inflammation. For this, wild-type CD147 or mutant CD147 (P309A and G310A) plasmid (Supplementary Fig. 4) was cotransfected with either PPRE-luc or κB-luc plasmid followed by the incubation of CyPA in 3T3-L1 preadipocytes for 7 days in ADM. CyPA-induced impairment of PPRE-luciferase activity and the induction of κB-luciferase activity were significantly waived in 3T3-L1 cells expressing mutated CD147 (P309A and G310A) (Fig. 5A, B). This was further validated by gene expression analysis of adipogenic and inflammatory markers, as CyPA-induced inhibition of adipogenic PPAR-γ and C/EBP-β gene expression and the induction of pro-inflammatory cytokines TNF-α and IL-1β gene expression were markedly prevented in 3T3-L1 cells expressing mutated CD147 (P309A and G310A) (Fig. 5C, D). These data strongly suggest the participation of P309/G310 residues of CD147 in CyPA-induced impairment of adipogenesis and induction of adipocyte inflammation. However, to explore the underlying mechanism of CyPA action downstream of CD147, we used pharmacological inhibitors of NF-κB pathway (Bay 11-7082) and Wnt signaling pathway (IWR-1-endo) as these signaling cascades were involved in cellular inflammation [43] and adipocyte differentiation [47], respectively. Although treatment of NF-κB pathway inhibitor significantly diminished CyPA effected upregulation of pro-inflammatory cytokines gene expression and downregulation of adipogenic markers gene expression (Fig. 6A), however, inhibition of Wnt signaling only rescued CyPA-mediated adipogenic markers expression without any notable changes in pro-inflammatory cytokines expression (Fig. 6B, C). In addition, while treatment of NF-κB inhibitor prevents CyPA-induced insulin sensitivity, the effect of Wnt pathway inhibitor on insulin sensitivity was comparatively lowered than NF-κB inhibitor-treated cells (Supplementary Fig. 5). Depending on cell types and disease pathogenicity, both positive and negative regulation of Wnt/β-catenin by NF-κB was reported previously [48]. It has been reported that NF-κB indirectly regulates the Wnt/β-catenin pathway either through leucine zipper tumor suppressor 2 (LZTS2) or by SMAD ubiquitination regulatory factor 1 (Smurf1) and Smurf2 that interact with β-catenin and regulates its stability or degradation affecting transcriptional activity [49, 50]. Investigating this aspect, we successfully demonstrated that CyPA incubation significantly downregulates LZTS2 gene expression without any noticeable changes in Smurf1 and Smurf2 gene expression. By contrast, subdued expression of LZTS2 in response to CyPA incubation was considerably prevented by Bay 11-7082 treatment in 3T3-L1 adipocytes (Fig. 6D). We conclude that CyPA–CD147 signaling-mediated NF-κB activation directly promotes adipocyte inflammation and indirectly attenuates adipogenesis through the downregulation of LZTS2.

Fig. 5.

CD147 mediates CyPA-induced adipocyte inflammation and its dysfunction. A, B Wild-type or mutated CD147 (basigin, BSG) plasmids (P309A and G310A) were cotransfected with PPRE promoter luciferase plasmid (A) or κB promoter luciferase plasmid (B) in 3T3-L1 adipocytes and incubated without or with CyPA (10 ng/ml) for 8 h. On termination of incubations, cells were lysed and luciferase activity was measured by a multimode reader. C, D RT-PCR analysis (C) and its quantification (D) of PPAR-γ, C/EBP-β, TNF-α, and IL-1β gene expressions in wild-type or mutated CD147 plasmid transfected 3T3-L1 adipocytes treated without or with CyPA (10 ng/ml) for 8 h. β-Actin was used as loading control. All experiments were performed in triplicate. Each value is the mean ± SEM of three independent experiments, ***p < 0.001, **p < 0.01 vs BSG, ##p < 0.01, #p < 0.05 vs BSG + CyPA

Fig. 6.

CyPA–CD147-mediated NF-κB activation impairs adipogenesis through β-catenin stabilization. A Real-time quantitative PCR analysis showing TNF-α, IL-1β, PPAR-γ, and C/EBP-β mRNA level in 3T3-L1 adipocytes treated without or with CyPA (10 ng/ml) in the absence or presence of NF-κB inhibitor Bay 11-7085 (20 μM) for 8 h. β-Actin served as an internal control for normalization. B RT-PCR analysis (left) and its quantification (right) of PPAR-γ, C/EBP-β, TNF-α, and IL-1β gene expressions in 3T3-L1 adipocytes treated without or with CyPA (10 ng/ml) in the absence or presence of Wnt signaling inhibitor IWR-1-endo (15 μM) for 8 h. β-Actin was used as loading control. C Western blot images (upper) and its quantifications (lower) showing C/EBP-β and TNF-α abundance in 3T3-L1 adipocytes treated without or with CyPA (10 ng/ml) in the absence or presence of IWR-1-endo (15 μM) for 8 h. D RT-PCR analysis (left) and its quantification (right) of LZTS2, Smurf1, and Smurf2 gene expressions in 3T3-L1 adipocytes treated without or with CyPA (10 ng/ml) in the absence or presence of Bay 11-7082 (20 μM) or IWR-1-endo (15 μM) for 8 h. β-Actin was used as loading control. All experiments were performed in triplicate. Each value is the mean ± SEM of three independent experiments, ***p < 0.001, **p < 0.01, *p < 0.05 vs Con; ###p < 0.001, ##p < 0.01, #p < 0.05 vs CyPA

Macrophage–adipocyte cross-talk connecting CyPA–CD147 signaling augments adipose tissue inflammation and insulin resistance in vivo

To examine the role of palmitate-induced CyPA on adipose tissue inflammation and insulin resistance in vivo, we infused palmitate intraperitoneally in wild-type BL/6 mice followed by the intraperitoneal administration of CyPA inhibitor TMN355. Palmitate infusion markedly augments serum CyPA level which was significantly waived by TMN355 (Fig. 7A). Increased accumulation of adipose tissue macrophages (ATMs) in response to palmitate infusion was notably rescued by TMN355 administration as indicated by flow cytometric analysis of F4/80 + CD11b + cells in the stromal vascular fraction of adipose tissue (Fig. 7B) and immunohistochemical analyses of F4/80 level in the visceral adipose tissue (Fig. 7C and Supplementary Fig. 6A). Moreover, TMN355 delivery significantly relinquished palmitate-induced enhancement of TNFα and IL-6 pro-inflammatory cytokines levels (Fig. 7D). Inhibition of palmitate-mediated adipose tissue inflammation by TMN355 was coincided with the amelioration of glucose intolerance and insulin insensitivity as evident from the glucose tolerance test (Fig. 7E), homeostasis model assessment–insulin resistance (HOMA-IR) scores (Fig. 7F), and the inhibitory or stimulatory phosphorylation of the insulin-signaling molecules IRS-1 and Akt, respectively (Fig. 7G). All these results indicate that palmitate infusion in mice notably upregulates CyPA expression which is associated with adipose tissue inflammation and insulin resistance. For the in vivo validation of the participation of CD147 in mediating the CyPA effect on adipocyte dysfunction and insulin resistance, we infused CyPA in mice and then delivered anti-CD147 antibody (CD147-Ab) through intraperitoneal route. Similar to the TMN355 effect on palmitate-induced ATMs accumulation, we have found that CD147-Ab delivery significantly prevented CyPA-induced macrophage migration as evident from the F4/80 + CD11b + cell population in the stromal vascular fraction of adipose tissue (Fig. 8A) and the F4/80 level in the visceral adipose tissue (Fig. 8B and Supplementary Fig. 6B). However, it could be interesting to note that the CyPA effect on adipose tissue macrophage migration was more pronounced than palmitate infusion. Moreover, delivery of CD147-Ab notably waived CyPA-induced upregulation of serum level of TNFα and IL-6 pro-inflammatory mediators (Fig. 8C). Since we have found that CyPA–CD147 signaling impairs the adipogenesis process in the in vitro condition, we explored the gene expression profile of two key adipogenesis regulators PPARγ and C/EBPβ in mice infused with CyPA without or with CD147-Ab. Delivery of CD147-Ab in mice significantly restored CyPA-induced attenuation of PPARγ and C/EBPβ gene expression (Fig. 8D). Furthermore, targeted blocking of CD147 by delivering CD147-Ab in mice rescued from CyPA-induced glucose intolerance (Fig. 8E), insulin sensitivity as indicated by the HOMA-IR scores (Fig. 8F), and insulin-signaling pathway molecules IRS-1 and Akt inactivation and activation, respectively (Fig. 8G). These results provide clear evidence that CyPA is directly associated with the impairment of adipocyte functions and insulin sensitivity in the in vivo condition. In summary, the CyPA–CD147 signaling pathway involved in macrophage–adipocyte cross-talk in obese adipose tissue implementing insulin resistance is presented in a schematic diagram (Fig. 9).

Fig. 7.

Delivery of CyPA inhibitor TMN355 alleviates adipose tissue inflammation and insulin resistance in palmitate-infused mice model. A ELISA of serum CyPA level of saline (Con) or palmitate (PAL) or PAL plus TMN355 administrated mice (n = 3). B Flow cytometric analyses for F4/80 + CD11b + positive cells in the adipose tissue stromal vascular fraction of above-mentioned mice (n = 3). C Representative images of visceral adipose tissue sections showing hematoxylin–eosin staining along with the immunohistochemical analyses of F4/80 in saline (Con), palmitate (PAL) or PAL plus TMN355 administrated mice (n = 3). Scale bar, 100 μm. D Quantification of serum TNFα and IL-6 level in the above mice (n = 3). E, F Glucose tolerance test (E) (n = 3), and HOMA-IR analyses (F) (n = 3) were performed in these mice. G Western blot images (upper) and its quantifications (lower) showing pIRS-1 (S636) and pAkt (S473) abundance in the adipose tissue of the above-mentioned mice (n = 3). IRS-1 and Akt were used as loading control for normalization. ***p < 0.001, **p < 0.01, *p < 0.05 vs Con; ###p < 0.001, ##p < 0.01, #p < 0.05 vs PAL

Fig. 8.

Anti-CD147 antibody administration rescued CyPA-induced adipose tissue inflammation and insulin resistance in mice model. A Flow cytometric analyses for F4/80 + CD11b + positive cells in the adipose tissue stromal vascular fraction of saline (Con) or CyPA or CyPA plus anti-CD147 antibody (CD147-Ab) administrated mice (n = 3). B Representative images of visceral adipose tissue sections showing hematoxylin–eosin staining along with the immunohistochemical analyses of F4/80 in Con or CyPA or CD147-Ab administrated mice (n = 3). Scale bar, 100 μm. C Quantification of serum TNFα and IL-6 level in the above-mentioned mice (n = 3). D Real-time quantitative PCR analysis showing PPAR-γ, and C/EBP-β mRNA level in the visceral adipose tissue of these mice. β-Actin served as an internal control for normalization. E, F Glucose tolerance test (E), and HOMA-IR (F) analyses were performed in these mice (n = 3). G Western blot images (left) and its quantifications (right) showing pIRS-1 (S636) and pAkt (S473) abundance in the adipose tissue of the above-mentioned mice (n = 3). IRS-1 and Akt were used as loading control for normalization. ***p < 0.001, **p < 0.01, *p < 0.05 vs Con; ##p < 0.01, #p < 0.05 vs CyPA

Fig. 9.

Proposed model deciphering involvement of lipid-induced monokine CyPA on adipocyte dysfunction in obesity-induced macrophage–adipocyte cross-talk implementing insulin resistance

Discussion

In this study, we identified a novel lipid-induced monokine cyclophilin-A (CyPA) that attenuates adipocyte function and insulin sensitivity. CyPA is most abundant among all cyclophilins [21] that involved in different biological functions such as protein folding, trafficking, and immune cell activation [20–25]. Although CyPA primarily is intracellular in nature, but also secreted in response to inflammatory stimuli such as hyperglycemia, hypoxia, infection, and oxidative stress [23–25] and thus involved in intercellular communications associated with inflammation and chemotaxis [21–24, 44]. Many lines of evidence also suggested its involvement in different disease pathologies [20–23, 28–30]. To investigate the role of CyPA in adipose tissue dysfunction and insulin resistance in vivo, we established a diet-induced obese T2D model in zebrafish by overfeeding commercially available fish feed for 4 weeks. The zebrafish (Danio rerio) has been increasingly used to study various metabolic diseases including obesity, hepatosteatosis, atherosclerosis, and diabetes due to the similarities in structure and functions of organs, metabolic parameters, and energy homeostasis between zebrafish and humans [51, 52]. However, due to the limited information and the differences in findings, the zebrafish model-based study requires cautious evaluation for possible application in clinical practice. Although several zebrafish models of T2D have emerged but studies have postulated that diet-induced obesity (DIO) provides an excellent zebrafish model system to investigate T2D disease biology as it shares common pathological features of obesity with humans [31, 53]. We have found that overfeeding for 4 weeks caused DIO in zebrafish as indicated by the significant increase in body weight, body-mass index (BMI), and fasting blood glucose level. The remarkable growth of zebrafish for a 4-week period of diet manipulation is more than what was reported by Zang et al. [31], which could possibly be due to the differences in strain, age, and health of zebrafish and the feed composition. Investigating glucose tolerance and insulin sensitivity, we noticed that intraperitoneal administration of non-immunosuppressant CyPA inhibitor TMN355 in the DIO group caused a notable reduction of fasting blood glucose level indicating the association of CyPA in T2D development in DIO zebrafish. This was further confirmed by the results of the glucose tolerance test (GTT) and insulin tolerance test (ITT) analysis. Moreover, inhibition of CyPA by TMN355 also waived DIO-associated enhancement of adipose tissue inflammation.

Among the different cyclophilin inhibitors, cyclic undecapeptide cyclosporins (CsA) are the most widely studied and well-characterized immunosuppressant which revolutionized solid organ transplantation after its approval in 1983 for preventing graft rejection. The CsA inflicted its immunosuppressive activity through its binding with CyPA forming CsA-CyPA dimer that inhibits lymphocyte-activating phosphatase, calcineurin, preventing activation of nuclear factor of activated T cells (NFAT) and its downstream signaling [54, 55]. Although CsA is a potent cyclophilin inhibitor, however, the immunosuppressive activity of CsA largely limits its therapeutic application as a cyclophilin inhibitor. To address this limitation, many compounds have been designed and synthesized that antagonize cyclophilins without poising any significant immunosuppressive activity. Several non-immunosuppressive CsA analogs have been developed such as valspodar, NIM811, EDP-546, SCY635, MM284, and alisporivir (Debio-025) [56]. Among these, alisporivir advanced to Phase 3 clinical trials exhibited most potent antiviral activity toward hepatitis C virus, as hepatitis C virus (HCV) replication is dependent on interactions with host cell cyclophilins [57]. The alisporivir was also shown to disrupt the interaction between CyPA and CrkII and reduces tumorigenicity and metastasis of breast cancer [58]. The most recently developed cyclophilin inhibitor is a CsA analog CRV431 which is currently in Phase 1 clinical trials. In a recent study with the cyclophilin inhibitor CRV431 demonstrated its potential as an effective drug candidate for chronic liver diseases [59]. Several other small molecule cyclophilin inhibitors have also been developed such as immunosuppressive macrolide derivative sanglifehrin A and non-immunosuppressive sangamide derivatives such as NV556. Despite all the different cyclophilin inhibitors developed, none of them advanced from clinical development to the market stage, and therefore, development of potent cyclophilin inhibitors that could overcome the clinical challenges are need of the hours.

Previous reports showed that increased circulatory level of CyPA in patients with type 2 diabetes (T2D) is mainly due to the aberrant expression and secretion of CyPA from the monocytes [22, 29]. However, the underlying mechanism of increased expression of CyPA in the monocytes of T2D patients and its role in adipocyte dysfunction and insulin insensitivity has not been explored. Since an enhanced level of circulatory saturated free-fatty acids is an important prognostic indicator of obesity-induced insulin resistance in T2D patients [60], we investigated the effect of saturated free-fatty acid palmitate on CyPA expression in macrophages and adipocytes. Intriguingly, we found that lipid incubation did not alter the CyPA expression in 3T3-L1 adipocytes while its expression was strikingly increased both in THP1 and RAW264.7 macrophages exhibiting unique cell-type specific phenomena of lipid-induced macrophage CyPA expression. Future study in this direction to explore the differential effect of palmitate on CyPA expression in macrophage and adipocyte is needed in order to understand the mechanistic aspects of CyPA’s role in pathophysiological settings. To investigate the role of macrophage-derived CyPA on adipocytes insulin sensitivity, we performed a macrophage–adipocyte coculture experiment. Control siRNA or CyPA siRNA-transfected macrophages were treated with palmitate and then cocultured with adipocytes for analyzing adipocytes glucose uptake. Impairment of adipocytes insulin sensitivity in response to the palmitate treated macrophages was significantly prevented when we used CyPA-silenced macrophages. Moreover, the application of TMN355 markedly rescued CyPA-induced impairment of insulin sensitivity. These results clearly suggest the involvement of macrophage-derived CyPA on macrophage–adipocyte cross-talk in obese adipose tissue microenvironment and that participated in adipocytes insulin resistance.

A healthy and functional white adipose tissue is detrimental to whole-body metabolism and responsible for the maintenance of energy homeostasis in human beings [1, 45]. The homeostatic balance of adipocyte formation and lipid storage/utilization is mainly responsible for maintaining normal adipocyte functions in an organism [1, 45]. In the process of adipogenesis, early induction and activation of C/EBP-β along with a complex network of different transcription factors and coactivators lead to the activation of a master regulator of adipogenesis PPAR-γ that promotes a later stage of adipogenic differentiation [45]. Activated C/EBP-β and PPAR-γ markedly induce and maintain the expression of various adipogenic genes, such as FABP-4, Glut-4, and adiponectin, which are essential for adipocyte functions including insulin sensitivity [45]. Investigating the effect of CyPA on adipogenesis, we noticed a striking reduction of adipocyte differentiation in response to CyPA which corroborated with the subdued expression of both C/EBP-β and PPAR-γ. CyPA-mediated attenuation of adipogenesis was significantly prevented by TMN355 incubation indicating a detrimental effect of CyPA on adipogenesis. Thus, inhibition of lipid-induced CyPA synthesis in macrophages could be beneficial for the maintenance of normal adipocyte differentiation along with the prevention of hypertrophied adipocyte formation.

It has been well established that obesity-associated chronic low-grade inflammation in adipose tissue is accountable for the development of insulin resistance and T2D [2–5]. The causal relationship between adipose tissue inflammation and insulin resistance was first reported by Hotamisligil et al. [61] showing an increase of TNFα concentration in obese adipose tissue and the neutralization of TNFα improves insulin sensitivity in obese rodents. Previous studies have reported the striking increase of macrophage population in the adipose tissue of obese individuals [9–11] and suppression of macrophage infiltration into the adipose tissue significantly ameliorate insulin sensitivity in the rodent model of obesity [14, 15]. It was also observed that the extracellular CyPA has chemotactic ability on various immune cells including macrophages [44] and that responsible to elicit inflammatory responses [39]. Since we observed an increased expression and secretion of CyPA from lipid-incubated macrophages, the massive infiltration of macrophages in obese adipose tissue could be a major source of CyPA. Although macrophages play an essential role in innate immunity, the clinical and experimental studies also support their involvement in a broad spectrum of chronic inflammatory diseases including T2D [62, 63]. Contingent to environmental cues, adipose tissue macrophages (ATMs) exhibit different activation states continuum from classically activated pro-inflammatory M1 to various alternatively activated anti-inflammatory M2 states [19, 64]. Moreover, studies have also demonstrated that ATMs of lean mice expressed M2 phenotypic genes, while in diet-induced obesity, overexpression of various pro-inflammatory genes characteristic of M1 phenotype was observed [9–12, 16–18]. Recent studies highlighted the existence of a diverse phenotypic population of ATMs in the obese patients where several classical M1-M2 gene signatures were observed, although, the prevalence of M1 population is higher than M2-like cells [19]. Obesity displayed a macrophage phenotypic skewing from an anti-inflammatory M2 to a pro-inflammatory M1 polarization state in the adipose tissue that contributes to insulin resistance [16–18]. However, it could be noted here that macrophage polarization states generated in vitro by the inducers of M1 (LPS) or M2 (IL-4/IL-13) does not necessarily reflect the in vivo skewing of macrophage polarization in the obese adipose tissue [19]. Moreover, it has been revealed that ATMs adopt a metabolically activated (MMe) phenotype that could be associated with the pathophysiological changes during the progression of obesity [65]. It has also been observed that in obese adipose tissue, CD9 + ATMs population are lipid-laden and localized to the Crown-like structures (CLS) that mainly responsible for the adipose tissue inflammation. The adoptive transfer of this ATMs population notably promoted obesity-associated inflammation in lean mice [66]. Recently, a lipid-associated macrophage (LAM) phenotype has been identified that are predominant ATMs subset in the adipose tissue of different mouse models of obesity [67]. Interestingly, we have found that CyPA incubation notably altered the macrophage polarization as indicated by the increased expression of M1 marker CD80 with the reduction of M2 phenotypic marker CD206. In addition, the adipocytes incubation with CyPA markedly enhanced the adipocyte inflammation as evident from both pro-inflammatory gene and protein expression profiles. Application of CyPA inhibitor TMN355 revered the CyPA effect on macrophage polarization and adipocyte inflammation and thus suggesting CyPA's role on the induction of inflammatory cascade in the obese adipose tissue. Genetic ablation of different inflammatory genes has been shown to influence inflammatory response in adipose tissue and systemic glucose tolerance and insulin sensitivity [16]. Thus, the interplay between metabolic and immune systems critically linked with obesity-induced T2D pathogenesis providing indisputable evidence of adipose tissue inflammation on insulin resistance.

CD147 functions as a principal signaling receptor for extracellular cyclophilins [26, 27]. Human CD147 is a type I integral membrane glycoprotein of 269 amino acids originally discovered as a tumor cell-derived factor that involved in the production of matrix metalloproteinase from the fibroblasts, and therefore, it was widely known as ‘extracellular matrix metalloproteinase inducer’ (EMMPRIN) [68]. Subsequent studies have shown that aberrant expression of CD147 is implicated in a variety of pathological conditions such as rheumatoid arthritis, systemic lupus erythematosus, chronic liver disease, atherosclerosis, and cardiovascular disease [26]. Increased levels of extracellular cyclophilins have also been detected in inflammatory diseases [28–30] and studies have demonstrated the critical role of cyclophilin–CD147 interactions in the regulation of inflammatory responses and the recruitment of immune cells to sites of inflammation [39, 44]. It has been shown that Pro309 and Gly310 residues of CD147 play a critical role in CyPA-mediated signaling [27]. We also found that mutations of these residues completely abrogated CyPA-mediated CD147 signaling as evident from the attenuation of inflammation and induction of adipogenesis. To explore the consistency of our in vitro observation to in vivo condition, we developed palmitate and CyPA infusion models in mice. Similar to in vitro experimental data, we have found that both palmitate and CyPA infusion for 7 days significantly upregulates macrophage migration into the adipose tissue promoting adipose tissue inflammation, its dysfunction and insulin insensitivity. These attributes were notably reversed in TMN355 and anti-CD147 antibody administered mice.

Over the last decade, an abundance of evidence has emerged demonstrating cross-talk between inflammatory macrophages and adipocytes within the obese adipose tissue microenvironment that play a critical role in the pathogenesis of obesity-induced insulin resistance and T2D. However, the specific macrophage mediator that involved in such cross-talk is missing and that created an important lacuna in our understanding about the nature of macrophage–adipocyte interaction in T2D pathophysiology. Our findings demonstrated that a lipid-induced monokine CyPA, by interacting with its receptor CD147 on adipocytes, mediates obesity-induced macrophage–adipocyte cross-talk implementing adipose tissue dysfunction and insulin resistance. However, the present study also has several limitations. The study particularly focused on the involvement of a saturated free-fatty acid palmitate on CyPA expression in macrophages and, therefore, future studies in this direction with different saturated and unsaturated fatty acids will uncover the regulation of CyPA synthesis in physiological and pathophysiological settings. It is not clear from this study about the nature of differential effect of palmitate on CyPA expression in macrophages and adipocytes. Moreover, present study did not include the measure of hypertrophy and fat lipolysis and as such, it is not clear whether the increased level of CyPA has any effect on adipocytes hypertrophy and lipolysis. The detailed signaling cascade that linked CyPA–CD147 interaction with NF-κB signaling requires further study in this direction. Furthermore, future study with inclusion of macrophage-specific knockout of CyPA and adipocyte-specific ablation of CD147 will strengthen our findings about the involvement of CyPA in obesity-induced insulin resistance and T2D.

Based on the evidence of this study, it can be concluded that lipid-induced monokine CyPA through its interaction with CD147 promotes adipose tissue dysfunction and insulin resistance in zebrafish and mice models of obesity. Therefore, targeting CyPA–CD147 pathway could provide a novel therapeutic option for the management of obesity-induced insulin resistance and T2D.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

SDG, DP, and DB conceived and designed the experiments. DB, DeP, AS, SR, RP, RS performed the experiments. SDG, DP, KT, DB, and DeP analyzed the data. RS, RD, and SKB developed obese diabetic zebrafish model and helped in experiments. RP and KT developed mice models and helped in experiments. SDG, DP, and DB wrote the manuscript. SDG and DP supervised the study. All authors read and approved the final manuscript. SDG is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Funding

This work was supported by the “DBT-Twinning Grant” from the Department of Biotechnology (DBT), New Delhi (Grant number: BT/PR24700/NER/95/819/2017)” to S.D.G. (Tezpur University) and D.P. (IIT Ropar). The SERB-Early Career Research Grant from the Science and Engineering Research Board (SERB), New Delhi (Grant number: ECR/2017/000892) to D.P. also supported this work.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

No potential conflicts of interest relevant to this article were reported.