SHP2 inhibitor PHPS1 regulates macrophage-adipocyte interaction to alleviate inflammation and adipocyte insulin resistance, via NF-κB suppression and IRS-1/GLUT-4 restoration

SHP2 inhibitor PHPS1 regulates macrophage-adipocyte interaction to alleviate inflammation and adipocyte insulin resistance, via NF-κB suppression and IRS-1/GLUT-4 restoration | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article SHP2 inhibitor PHPS1 regulates macrophage-adipocyte interaction to alleviate inflammation and adipocyte insulin resistance, via NF-κB suppression and IRS-1/GLUT-4 restoration Xinxin Yue, Xiaoyan Yin, Yang Fu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8391295/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract This study explored the anti-inflammatory and insulin resistance-regulating effects of SHP2 inhibitor PHPS1 using a Transwell co-culture system of adipocytes and macrophages to mimic the in vivo adipose microenvironment. Two models were established: TNF-α-stimulated 3T3-L1 adipocytes (single culture) and LPS-induced insulin-resistant 3T3-L1/RAW 264.7 co-cultures. After 24 h of 10 μM PHPS1 pretreatment, ELISA, qRT-PCR, and Western blotting were used to detect inflammatory factors, gene expression, and protein phosphorylation, respectively. In TNF-α-stimulated adipocytes, PHPS1 reduced pro-inflammatory factors (IL-6: 62.35%, IL-1β: 54.10%, MCP-1: 49.19%), increased IL-10 (103.98%), and downregulated iNOS/COX-2 mRNA. In LPS-induced co-cultures, PHPS1 decreased IL-6 (68.73%), IL-1β (63.01%), MCP-1 (47.79%), upregulated IL-10 (167.49%), and inhibited iNOS/COX-2 mRNA. Mechanistically, PHPS1 suppressed NF-κB pathway phosphorylation (pp65, pIkkα) in both models, reversed LPS-induced pIRS-1 Ser307 phosphorylation (61.12% reduction), and upregulated GLUT-4 (198.0%). Thus, PHPS1 alleviates inflammation via NF-κB inhibition and improves insulin resistance by restoring the IRS-1/GLUT-4 axis, making it a potential candidate for insulin resistance-related metabolic diseases. SHP2 inhibitor PHPS1 macrophages adipocyte inflammatory factors NF-κB Insulin resistance Figures Figure 1 Figure 2 Figure 3 Introduction Obesity, a highly prevalent chronic metabolic disorder globally, is defined by abnormal accumulation and dysregulated distribution of adipose tissue. Despite decades of research, its multifactorial etiology, encompassing genetic, dietary, and environmental factors, remains incompletely understood, posing a major barrier to effective intervention¹. Epidemiological data consistently highlight a rising global burden of obesity, which is tightly linked to the escalating prevalence of type 2 diabetes mellitus (T2DM) and insulin resistance (IR)—two interrelated conditions that impose substantial clinical and socioeconomic costs². Adipose tissue, long regarded merely as an energy storage organ, is now recognized as a key endocrine and immunometabolic hub. It secretes a spectrum of adipokines and cytokines that regulate systemic energy homeostasis; however, in obese states, adipose tissue undergoes pathological remodeling, characterized by increased infiltration of adipose tissue macrophages (ATMs)³. These ATMs, together with dysfunctional adipocytes, drive the production of pro-inflammatory mediators including interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α), initiating a state of chronic low-grade metabolic inflammation—the central driver of obesity-related IR⁴. Under pathological stimuli including high-fat diet (HFD) or lipopolysaccharide (LPS) exposure, cross-activation between adipocytes and macrophages amplifies inflammatory signaling: pro-inflammatory factors activate the nuclear factor-κB (NF-κB) pathway, which in turn induces abnormal phosphorylation of insulin receptor substrate-1 (IRS-1, Ser307) and downregulates glucose transporter type 4 (GLUT-4) expression. This disrupts insulin-mediated glucose uptake and metabolism, ultimately leading to IR and subsequent T2DM⁵. Unraveling the regulatory mechanisms of key molecules in this "inflammation-IR" axis is therefore critical for developing targeted therapies to mitigate T2DM and its complications. The Src homology 2 domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 gene, is a ubiquitously expressed non-receptor protein tyrosine phosphatase with pivotal roles in immune regulation and metabolic homeostasis. It modulates multiple signaling cascades in immune and metabolic tissues, including mitogen-activated protein kinase (MAPK), Toll-like receptors (TLRs), and NF-κB—pathways closely tied to inflammatory responses and IR⁶. Aberrant SHP2 activation exacerbates inflammation by promoting the secretion of pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6, and contributes to tissue damage in organs such as the liver, kidneys, and adipose tissue⁷. Phenylhydrazono pyrazolone sulfonate 1 (PHPS1), a specific small-molecule inhibitor of SHP2, blocks SHP2 phosphorylation and its downstream pro-inflammatory effects. Previous studies have validated PHPS1’s efficacy in ameliorating inflammatory diseases including atherosclerosis and acute kidney injury, but its role in regulating adipose tissue inflammation and improving adipocyte IR—key pathological features of T2DM—remains unaddressed 8,9 . To fill this gap, we established a Transwell co-culture system of 3T3-L1 adipocytes and RAW 264.7 macrophages to mimic the in vivo adipose tissue microenvironment—a model that better recapitulates the cell-cell interactions driving obesity-related IR than single-cell cultures¹⁰. Using this system, we investigated whether PHPS1 exerts anti-inflammatory effects by targeting the NF-κB pathway and explored its potential to improve adipocyte IR via restoring the IRS-1/GLUT-4 insulin signaling axis. This study aims to clarify the role of SHP2 in metabolic inflammation and provide preclinical evidence for PHPS1 as a potential therapeutic candidate for T2DM and obesity-related IR. Materials and Methods Cell culture The 3T3-L1 pre-adipocyte and RAW 264.7 macrophage cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). RAW 264.7 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were incubated at 37 °C in a humidified atmosphere containing 5% carbon dioxide (CO₂) and 95% air, with the culture medium refreshed every 48 hours. Differentiation of 3T3-L1 pre-adipocytes was induced as follows: on day 0, pre-adipocytes were treated with induction medium (DMEM containing 10% FBS, 0.5 mmol/L isobutylmethylxanthine, 1 μmol/L dexamethasone, and 10 μg/mL insulin). After 48 hours (day 2), the medium was replaced with maintenance medium (DMEM containing 10% FBS and 10 μg/mL insulin). The maintenance medium was refreshed every 48 hours until days 7–8, when cells were fully differentiated into mature adipocytes. Mature 3T3-L1 adipocytes were randomly divided into three groups: Control group: treated with DMEM only; TNF-α group: treated with 10 ng/mL TNF-α (R&D Systems, Minneapolis, MN, USA); TNF-α + PHPS1 group: pre-treated with 10μM PHPS1 (MedChemExpress, Monmouth Junction, NJ, USA; purity ≥98%) for 30 minutes, followed by co-treatment with 10 ng/mL TNF-α. All groups were incubated for an additional 24 hours before subsequent experiments. Co-culture of adipocytes and macrophages A Transwell co-culture system with a 0.4 μm porous membrane (Corning, Corning, NY, USA) was used to establish direct contact between adipocytes and macrophages. Fully differentiated 3T3-L1 adipocytes (1×10⁵ cells/well) were seeded in the lower chamber of 6-well Transwell plates, while RAW 264.7 macrophages (5×10⁴ cells/well) were seeded in the upper chamber inserts. After 24 hours of co-culture to allow cell adaptation, the co-cultures were divided into three groups: Co-culture control group: treated with DMEM only: LPS group: treated with 1 μg/mL LPS (Escherichia coli serotype O111:B4; Sigma-Aldrich, St. Louis, MO, USA); LPS + PHPS1 group: pre-treated with 10μM PHPS1 for 30 minutes, followed by co-treatment with 1 μg/mL LPS. All groups were incubated for another 24 hours before sample collection. Measurement of IL-6, IL-10, IL-1β and MCP-1 Levels of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α)and anti-inflammatory cytokine (IL-10) were measured using commercial mouse enzyme-linked immunosorbent assay (ELISA) kits (BioLegend, San Diego, CA, USA; catalog numbers: IL-6: 431304, IL-1β: 432604, TNF-α: 430904, IL-10: 431404). The level of monocyte chemoattractant protein-1 (MCP-1/CCL2) was detected using a mouse CCL2 ELISA kit (Invitrogen, Waltham, MA, USA; catalog number: BMS6002). All assays were performed strictly according to the manufacturers' instructions, and cytokine concentrations were calculated based on standard curves. Quantitative real-time polymerase chain reaction Total RNA was extracted from 3T3-L1 adipocytes (single-cell culture) and co-cultured cells using TRIzol reagent (Invitrogen), and its purity and concentration were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the ImProm-II Reverse Transcription System (Promega, Madison, WI, USA) according to the manufacturer's protocol. qRT-PCR was performed on an Applied Biosystems 7900 Detection System (Thermo Fisher Scientific) using Fast SYBR Master Mix (Applied Biosystems, Foster City, CA, USA). The primer sequences were as follows: Inducible nitric oxide synthase (iNOS): Forward 5’-TCACGCTTGGGTCTTGTTCA-3’, Reverse 5’-CCTTTTCCTCTTTCAGGTCACTT-3’; Cyclooxygenase-2 (COX-2): Forward 5’-CTGCCAATAGAACTTCCAATCC-3’, Reverse 5’-CGGTTTGATGTTACTGTTGCTT-3’; Glyceraldehyde 3-phosphate dehydrogenase (GAPDH): Forward 5’-GACAACTTTGGCATTGTG-3’, Reverse 5’-ATGCAGGGATGATGTTCTG-3’. The reaction conditions were: 95 °C for 10 minutes, followed by 40 cycles of 95 °C for 15 seconds and 60 °C for 1 minute. The relative mRNA expression levels were calculated using the 2⁻ΔΔCt method, with GAPDH as the internal reference gene. Western blot analysis In-cell western blotting was used for protein quantification, as it combines the specificity of traditional western blotting with the quantitative accuracy of ELISA. RAW 264.7 macrophages were seeded in 96-well plates at a density of 5×10⁴ cells/mL and cultured until reaching 70% confluence. Cells were divided into three groups: Control group; LPS group (treated with 1 μg/mL LPS); LPS + PHPS1 group (pre-treated with 10μM PHPS1 for 30 minutes, then 1 μg/mL LPS). After 24 hours of treatment, cells were fixed with 8% paraformaldehyde (100 μL/well) for 15 minutes at room temperature, washed three times with phosphate-buffered saline (PBS), and permeabilized with 0.1% Triton X-100 for 10 minutes. After blocking with 5% non-fat milk for 1 hour at room temperature, cells were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-phospho-p65 (Cell Signaling Technologies, Danvers, MA, USA; catalog number: 3033), rabbit anti-phospho-Iκκα (Santa Cruz Biotechnology, Dallas, TX, USA; catalog number: sc-16677-R), rabbit anti-Iκκα (Santa Cruz Biotechnology; catalog number: sc-374353), rabbit anti-phospho-IRS-1 (Ser 307) (Cell Signaling Technologies; catalog number: 2381), rabbit anti-GLUT-4 (Santa Cruz Biotechnology; catalog number: sc-166864), and rabbit anti-β-actin (Cell Signaling Technologies; catalog number: 4970). The next day, cells were washed three times with PBS and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Cell Signaling Technologies; catalog number: 7074) for 2 hours at room temperature. After three additional PBS washes, 100 μL of HRP substrate (Thermo Fisher Scientific) was added to each well, and the absorbance was measured at 450 nm using a microplate reader (BioTek, Winooski, VT, USA). Protein expression levels were normalized to β-actin (for single-cell samples) or Janus Green stain (for co-culture samples, Abcam, Cambridge, UK) to correct for cell number differences. Statistical analysis All experiments were independently repeated three times, with each group measured in triplicate. Data are presented as the mean ± standard error (SE). Statistical analysis was performed using GraphPad PRISM 8.0 software (GraphPad Software, San Diego, CA, USA). Prior to statistical testing, all data were verified to conform to a normal distribution using the Shapiro-Wilk test (p>0.05) and to have homogeneous variance using Levene’s test ( p >0.05), ensuring compliance with the assumptions of analysis of variance (ANOVA). Comparisons between multiple groups were performed using one-way ANOVA followed by Tukey’s post hoc test. A p -value <0.05 was considered statistically significant. Results PHPS1 (10μM) exerts anti-inflammatory effects in TNF-α-stimulated mature 3T3-L1 adipocytes To evaluate the anti-inflammatory activity of PHPS1, we first detected the secretion of inflammatory cytokines and chemokines in TNF-α-stimulated mature 3T3-L1 adipocytes, with key data summarized in Table 1A-B. Compared with the TNF-α(−)+PHPS1(−) control group, stimulation with TNF-α [in the TNF-α(+)+PHPS1(−) group] significantly increased the secretion of pro-inflammatory cytokines IL-6 ( p <0.0001, q =120) and IL-1β ( p <0.0001 ,q =103), as well as the chemokine MCP-1 (p<0.0001,q=118.1), while significantly decreasing the secretion of the anti-inflammatory cytokine IL-10 ( p <0.0001, q =29.21). Pretreatment with PHPS1 (10 μM) [in the TNF-α(+)+PHPS1(+) group] significantly reversed these TNF-α-induced changes. As precisely determined by our quantitative analysis, compared with the TNF-α(+)+PHPS1(−) group, the TNF-α(+)+PHPS1(+) group showed a remarkable 62.35% ± 2.08 reduction in IL-6 secretion ( p <0.0001, q =76.89), a 54.10% ± 1.85 reduction in IL-1β secretion ( p <0.0001, q =58.19), and a 49.19% ± 2.03 reduction in MCP-1 secretion ( p <0.0001, q =61.84). Meanwhile, IL-10 secretion was increased by 103.98% ± 13.79 in the TNF-α(+)+PHPS1(+) group compared with the TNF-α(+)+PHPS1(−) group ( p <0.0001, q =21.49). These highly significant and precisely quantified results (Table 1A-B) strongly indicate that PHPS1 (10 μM) effectively inhibits TNF-α-induced inflammatory responses in mature 3T3-L1 adipocytes. PHPS1 Alleviates LPS - Induced Inflammation in Adipocyte - Macrophage Co - Cultures In the LPS-stimulated adipocyte-macrophage co-culture system, with key data summarized in Table 1A-B, stimulation with LPS [in the LPS(+)+PHPS1(−) group] significantly elevated the levels of pro-inflammatory cytokines IL-6 and IL-1β, as well as the chemokine MCP-1, while reducing the secretion of anti-inflammatory cytokine IL-10, compared with the unstimulated control co-culture [LPS(−)+PHPS1(−) group]. Specifically, when comparing the LPS(+)+PHPS1(−) group with the LPS(−)+PHPS1(−) group, IL-6 secretion increased from an average of 31.53 pg/mL (SE =4.02) to 725.62 pg/mL (SE = 6.61), IL-1β secretion rose from an average of 25.58 pg/mL (SE = 3.52) to 704.01 pg/mL (SE = 12.95), MCP-1 secretion increased from an average of 34.95 pg/mL (SE =1.30) to 587.63 pg/mL (SE = 5.75), and IL-10 secretion decreased from an average of 307.48 pg/mL (SE = 10.12) to 104.15 pg/mL (SE = 6.30). All these LPS-induced changes were statistically significant ( F= 4047, F= 875.9, F= 1593, F= 117.8). Pretreatment with PHPS1 (10 μM) [in the LPS(+)+PHPS1(+) group] significantly suppressed the LPS-induced upregulation of pro-inflammatory factors and reversed the downregulation of the anti-inflammatory cytokine. Compared with the LPS(+)+PHPS1(−) group, the LPS(+)+PHPS1(+) group showed a 68.73% ± 1.16 reduction in IL-6 secretion ( p <0.0001, q =96.43), a 63.01% ± 3.44 reduction in IL-1β secretion ( p <0.0001, q =41.35), and a 47.79% ± 2.42 reduction in MCP-1 secretion ( p <0.0001, q =42.54). Meanwhile, IL-10 secretion was increased by 167.49% ± 23.78 in the LPS(+)+PHPS1(+) group compared with the LPS(+)+PHPS1(−) group ( p <0.0001, q =19.11). These results (Table 1A-B) indicate that PHPS1 mitigates LPS-induced inflammation in the adipocyte-macrophage interaction microenvironment. Place Table 1A-B here PHPS1 Inhibits TNF - α - Induced iNOS and COX - 2 mRNA Expression in Mature Adipocytes iNOS and COX - 2 are key enzymes mediating inflammatory mediator synthesis. As shown in Table 2A-B, TNF - α stimulation significantly upregulated iNOS and COX - 2 mRNA expression compared with the control group. For iNOS, the control group (TNF - α⁻PHPS1⁻) mRNA expression levels were relatively stable with an average value of 4.73 (SE = 0.47). In contrast, TNF - α stimulation (TNF - α⁺PHPS1⁻) led to a marked increase in iNOS mRNA expression, with an average level of 32.35 (SE = 1.59). This represents an upregulation of approximately 5.89 - fold (32.35 / 4.73). However, pretreatment with PHPS1 (10 μM) (TNF - α⁺PHPS1⁺) effectively downregulated the TNF - α - induced iNOS mRNA expression. The average aexpression level in the TNF - α + PHPS1 group was 11.64 (SE = 0.51). Compared with the TNF - α group, PHPS1 pretreatment reduced iNOS mRNA expression by approximately 63.99% [(32.35 - 11.64) / 32.35 × 100%], and this reduction was statistically significant ( p <0.0001, q =23.42). Regarding COX - 2, the control group (TNF - α⁻PHPS1⁻) had an average mRNA expression level of 4.53 (SE = 0.59). When stimulated with TNF - α (TNF - α⁺PHPS1⁻), COX - 2 mRNA expression increased significantly to an average of 15.69 (SE = 0.60). This indicates an upregulation of about 2.47 - fold (15.69 / 4.53). Nevertheless, PHPS1 pretreatment (TNF - α⁺PHPS1⁺) notably decreased the TNF - α - induced COX - 2 mRNA expression. The average expression level in the TNF - α + PHPS1 group was 7.20 (SE = 0.36). Compared with the TNF - α group, PHPS1 pretreatment led to a reduction of approximately 54.11% [(15.69 - 7.20) / 15.69×100%]in COX - 2 mRNA expression, and this was also statistically significant ( p <0.0001, q =15.44). These findings further validate PHPS1’s anti - inflammatory activity at the transcriptional level, as it effectively suppresses the upregulation of iNOS and COX - 2 PHPS1 Reduces LPS - Induced iNOS and COX - 2 Transcription in Co - Cultures In LPS - stimulated co - cultures ( Table 2A-B), LPS significantly increased the mRNA expression of iNOS and COX - 2, two crucial enzymes involved in the inflammatory response, compared with the control co - culture. Our data clearly demonstrate the impact of LPS and the subsequent protective effect of PHPS1. For iNOS, in the control group (LPS⁻PHPS1⁻), the average mRNA expression level was 2.93 (SE = 0.28). Upon LPS stimulation (LPS⁺PHPS1⁻), iNOS mRNA expression soared to an average of 37.69 (SE = 0.42). This represents an upregulation of approximately 11.87 - fold (37.69 / 2.93). However, pretreatment with PHPS1 (10 μM) (LPS⁺PHPS1⁺) notably reduced the LPS - induced iNOS mRNA expression. The average expression level in the LPS + PHPS1 group was 17.28 (SE = 0.31). Compared with the LPS group, PHPS1 pretreatment decreased iNOS mRNA expression by approximately 54.15% [(37.69 - 17.28) / 37.69× 100%], and this reduction was highly statistically significant ( p <0.0001, q =64.62). Regarding COX - 2(the control group (LPS⁻PHPS1⁻) had an average mRNA expression level of 4.06 (SD = 0.53), obtained from 3.43, 4.23, and 4.52. LPS stimulation (LPS⁺PHPS1⁻) led to a significant increase in COX - 2 mRNA expression to an average of 16.61 (SD = 0.66), based on 16.82, 15.71, and 17.31. This indicates an upregulation of about 3.84 - fold (16.61 / 4.06). Nevertheless, PHPS1 pretreatment (LPS⁺PHPS1⁺) effectively mitigated the LPS - induced increase. The average expression level in the LPS + PHPS1 group was 9.28 (SD = 0.31), calculated from 8.93, 9.4, and 9.51. Compared with the LPS group, PHPS1 reduced COX - 2 mRNA expression by approximately 44.13% [(16.61 - 9.28) / 16.61×100%], and this was also statistically significant ( p <0.0001, q =18.02). These findings indicate that PHPS1 inhibits inflammatory gene transcription in the macrophage - adipocyte interaction context, highlighting its potential as an anti - inflammatory agent in co - culture systems. Place Table 2A-B here Anti‑infammatory activity of PHPS1 is mediated through inhibition of NF‑κB activity The NF - κB signaling pathway plays a central and well - established role in the regulation of inflammatory responses. Activation of this pathway leads to the upregulation of numerous pro - inflammatory genes, making it a key target for anti - inflammatory therapeutic strategies. Here, we investigated the impact of PHPS1 on the NF - κB pathway under different inflammatory stimuli (TNF - α and LPS) in relevant cellular models to elucidate its anti - inflammatory mechanism. In the context of TNF - α stimulation, as shown in our study (Figure 1,Table 3), TNF - α led to a marked activation of the NF - κB pathway, as evidenced by the increased phosphorylation levels of key pathway components p-65 and p-IKKβ. In the group treated with TNF-α alone (TNF - α⁺PHPS1⁻), the mean phosphorylation level of p-65 was 2.347. However, when adipocytes were pretreated with PHPS1 (TNF - α⁺PHPS1⁺), the mean phosphorylation level of p-65 decreased to 1.233. This represents a significant reduction of approximately 47.46% [(2.347 - 1.233) / 2.347×100%]. An unpaired two tailed t-test confirmed that this difference was statistically significant (t = 5.070, df = 4, p = 0.0071). Similarly, for p-IKKβ, the mean phosphorylation level in the TNF - α alone group (TNF - α⁺PHPS1⁻) was 2.347, while in the PHPS1 - pretreated group (TNF- α⁺PHPS1⁺), it dropped to 1.247, showing a decrease of about 46.87% [(2.347 - 1.247) / 2.347 ×100%]. An unpaired two tailed t-test revealed a highly significant difference (t = 5.291, df = 4, p = 0.0061). These results clearly indicate that PHPS1 effectively suppresses TNF - α - induced NF - κB pathway activation by reducing the phosphorylation of both p - 65 and p - IKKβ. Place Figure 1 here Place Table 3 here As depicted in Figure 2 and Table 3, LPS stimulation also robustly activated the NF - κB pathway. In the LPS - only group (LPS⁺PHPS1⁻), the mean phosphorylation level of p65 was 1.627. Upon pretreatment with PHPS1 (LPS⁺PHPS1⁺), the mean p65 phosphorylation level decreased to 0.8767, resulting in a remarkable reduction of approximately 46.12% [(1.627 - 0.8767) / 1.627 ×100%]. The unpaired two tailed t-test demonstrated a highly significant difference (t = 13.42, df = 4, p = 0.0002). Place Figure 2 here PHPS1 improve insulin resistance may via the insulin receptor substrate 1(IRS-1)/glucose transporter type 4 isoform(GLUT-4) pathway Insulin resistance is a key pathophysiological feature of metabolic disorders, tightly linked to chronic inflammation. The IRS-1/GLUT-4 pathway is critical for insulin-mediated glucose uptake, and its disruption is a hallmark of IR. Here, we validated PHPS1’s role in restoring this pathway using LPS-induced adipocyte-macrophage co-cultures (a model that recapitulates in vivo adipose microenvironment interactions). As shown in Figure 3 and Table 3, LPS stimulation (LPS⁺PHPS1⁻ group) significantly disrupted insulin signaling: it increased aberrant phosphorylation of IRS-1 (Ser307)—a marker of IRS-1 inactivation—to a mean level of 2.937 (vs. 1.000 in LPS⁻PHPS1⁻ controls), while reducing GLUT-4 (a key glucose transporter) to a mean level of 1.093 (vs. 2.100 in controls). These changes are consistent with prior reports that LPS-induced inflammation impairs insulin signaling via IRS-1 hyperphosphorylation 11 . Pretreatment with 10 μM PHPS1 (LPS⁺PHPS1⁺ group) reversed these defects: IRS-1 (Ser307) phosphorylation was reduced by 61.12% (from 2.937 to 1.14), with statistical significance (t=8.431, df=4, p=0.0011). This reduction restores IRS-1’s ability to transduce insulin signals, as hyperphosphorylation of Ser307 blocks IRS-1 binding to insulin receptors 12 . GLUT-4 levels were increased by 198.0% (from 1.093 to 3.257), with high significance (t=12.03, df=4, p=0.0003). This elevation enhances glucose uptake capacity, directly counteracting IR 13 . Place Figure 3 here Notably, these effects of PHPS1 on IRS-1/GLUT-4 were accompanied by concurrent NF-κB inhibition (46.12% reduction in p-p65, Figure 3), suggesting a causal link: PHPS1 reduces inflammation via NF-κB suppression, which in turn relieves inflammatory-mediated damage to the IRS-1/GLUT-4 axis 14 . This is supported by our cytokine data (68.73% reduction in IL-6, Table 1A-B), as pro-inflammatory cytokines like IL-6 directly promote IRS-1 phosphorylation. Discussion In the present study, we systematically investigated the regulatory effects of the SHP2 inhibitor PHPS1 on inflammation and insulin resistance (IR) using two experimental models: TNF-α-stimulated mature 3T3-L1 adipocytes (single-cell culture) and LPS-induced adipocyte-macrophage Transwell co-cultures. We confirmed that PHPS1 exerts anti-inflammatory effects and improves adipocyte IR primarily by inhibiting the NF-κB signaling pathway and restoring the IRS-1/GLUT-4 insulin signaling axis—two mechanisms closely tied to the pathogenesis of type 2 diabetes mellitus (T2DM), the core focus of metabolic disease research. The NF-κB pathway is a well-recognized core regulator of chronic metabolic inflammation, and its abnormal activation is strongly associated with obesity-related IR and T2DM development⁵. Previous studies have reported that SHP2 inhibitors suppress NF-κB activation by reducing the phosphorylation of key pathway components (p-IKK and p-p65), thereby decreasing pro-inflammatory cytokine secretion 7,8 . Our findings align with this research: in TNF-α-stimulated adipocytes, 10 μM PHPS1 reduced the secretion of pro-inflammatory cytokines IL-6 (62.35% ± 2.08 reduction), IL-1β (54.10% ± 1.85 reduction), and chemokine MCP-1 (49.19% ± 2.03 reduction), while increasing the anti-inflammatory cytokine IL-10 (103.98% ± 13.79 increase). Mechanistically, this was attributed to PHPS1-mediated inhibition of NF-κB: the phosphorylation levels of p-p65 decreased by 47.46% (from 2.347 to 1.233, p=0.0071) and p-IKK by 46.87% (from 2.347 to 1.247, p=0.0061). Kunz HE et al. confirmed that the transcription of IL-6 and IL-1β is directly regulated by the NF-κB pathway, and our cytokine data further validate that PHPS1’s suppression of NF-κB activation translates to effective regulation of cytokine secretion, reinforcing its role in alleviating adipose tissue inflammation⁹. In LPS-induced adipocyte-macrophage co-cultures— a model that better mimics the in vivo adipose microenvironment—PHPS1 exerted similar anti-inflammatory effects: it reduced IL-6 secretion by 68.73% ± 1.16, IL-1β by 63.01% ± 3.44, and MCP-1 by 47.79% ± 2.42, while increasing IL-10 by 167.49% ± 23.78. Concurrently, PHPS1 inhibited LPS-induced NF-κB activation, with p-p65 phosphorylation reduced by 46.12% (from 1.627 to 0.8767, p=0.0002) and p-IKK by 41.33% (from 1.337 to 0.7833, p<0.0001). This is consistent with research emphasizing that LPS-induced cross-talk between adipocytes and macrophages relies on NF-κB activation, and inhibiting this pathway can break the inflammatory cycle in adipose tissue— a critical step in mitigating obesity-related IR¹⁰. Notably, our study addresses key limitations of previous research. Most prior studies on SHP2 inhibitors focused on single-cell models (e.g., hepatocytes or macrophages alone) 15-17 , whereas our Transwell co-culture system recapitulates the complex cellular interactions of the in vivo adipose microenvironment 18 , providing more physiologically relevant evidence for PHPS1’s role in targeting adipose tissue inflammation. Additionally, while previous studies reported changes in cytokine secretion¹⁵, our data further confirm that PHPS1 suppresses the transcription of NF-κB-dependent inflammatory genes (iNOS and COX-2): in TNF-α-stimulated adipocytes, PHPS1 reduced iNOS mRNA expression by 63.99% (from 32.35 to 11.64, p<0.0001) and COX-2 by 54.11% (from 15.69 to 7.20, p<0.0001); in LPS-stimulated co-cultures, iNOS transcription decreased by 54.15% (from 37.69 to 17.28, p<0.0001) and COX-2 by 44.13% (from 16.61 to 9.28, p<0.0001). This indicates PHPS1 acts upstream of gene transcription—likely by blocking NF-κB nuclear translocation20—rather than merely affecting mRNA stability, offering a more comprehensive understanding of SHP2 inhibitor-mediated anti-inflammatory effects. Chronic inflammation disrupts insulin signaling, and aberrant phosphorylation of IRS-1 (Ser307) combined with downregulation of GLUT-4 are key events in IR development 21 . Wen L et al. reported that SHP2 inhibitors improve IR in high-fat diet-fed mice, but the specific molecular mechanism—especially the role of the IRS-1/GLUT-4 axis—remained unclear 22 . Our data fill this gap: LPS stimulation increased p-IRS-1 (Ser307) levels to 2.937 (vs. control) and reduced GLUT-4 levels to 1.093, consistent with research showing LPS-induced inflammation promotes IRS-1 hyperphosphorylation to impair insulin signaling 23 . Pretreatment with PHPS1 reversed these defects: p-IRS-1 (Ser307) was reduced by 61.12% (to 1.14, p=0.0011), and GLUT-4 levels increased by 198.0% (to 3.257, p=0.0003). This directly links SHP2 inhibition to the restoration of the IRS-1/GLUT-4 axis, a mechanism not previously identified in prior studies 23 . This study has two key innovations: first, the combined use of single adipocyte and adipocyte-macrophage co-culture models addresses the limitations of single-cell studies and better reflects the in vivo adipose tissue microenvironment 24,25 ; second, we integrated data from cytokine detection, pathway phosphorylation, gene transcription, and insulin signaling molecules to establish a complete regulatory cascade: “PHPS1 → NF-κB inhibition → inflammation reduction → IRS-1/GLUT-4 restoration”. This study also has limitations that require consideration. First, we used a single PHPS1 concentration (10 μM) based on prior research validating SHP2 inhibitor efficacy across multiple concentrations 7,26 ; future studies should include dose-response curves to determine the EC50 and rule out off-target effects. Second, our in vitro findings need confirmation in animal models; we plan to further test whether PHPS1 reduces adipose tissue macrophage infiltration (via MCP-1 inhibition) and improves glucose tolerance (via GLUT-4 upregulation) in vivo, to provide stronger preclinical evidence for its translational potential. Conclusion In summary, the SHP2 inhibitor PHPS1 alleviates inflammation in both TNF-α-stimulated 3T3-L1 adipocytes and LPS-induced adipocyte-macrophage co-cultures by suppressing the phosphorylation of the NF-κB signaling pathway. Mechanistically, PHPS1 reduces the secretion of pro-inflammatory cytokines (IL-6, IL-1β, MCP-1), upregulates the anti-inflammatory cytokine IL-10, and inhibits the transcription of NF-κB-dependent inflammatory genes (iNOS, COX-2). More importantly, PHPS1 improves adipocyte insulin resistance by restoring the IRS-1/GLUT-4 insulin signaling axis—reducing aberrant phosphorylation of IRS-1 (Ser307) and increasing GLUT-4 expression to enhance glucose uptake. These findings clarify the role of SHP2 in metabolic inflammation and IR, and highlight PHPS1 as a potential therapeutic candidate for insulin resistance-related metabolic diseases, including type 2 diabetes mellitus. Declarations Author Contributions: Writing the original draft preparation, YUE Xin-xin, FU Yang. Writing review and editing, YUE Xin-xin and Yin Xiaoyan. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the National Natural Science Foundation Committee of China (grant number:72171042), and the “Scientific Research Funding Project of the Education Department of Liaoning Province” (grant number: LJKMZ20221981). Acknowledgments: We thank all past and present members of the Clinical College Institute for fruitful discussions and daily support and our collaborators for their inspiring inputs. We apologize to those authors whose work we could not cite directly owing to space constraints. Conflicts of Interest: The authors declare no conflict of interest. Data Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. References Benjamin CL, Amanda YL, Soo LC, et al. The impact of obesity: a narrative review[J].Singapore medical journal,2023,64(3): 163-171. Stafeev I , Yudaeva A D , Michurina S ,et al.The interactions between inflammation and insulin resistance: prospects of immunoregulation as a potential approach for the type 2 diabetes mellitus treatment[J].Diabetes mellitus, 2023.26(2):192-202. Dorenkamp M, Nasiry M ,Koch S ,et al. Inflammatory and diabetic conditions trigger SHP2 tyrosine phosphatase expression and subsequent aberrant activation of primary human monocytes[J].European Heart Journal,2022,43:136-148. Liu Y, Yang X, Wang Y et al. Targeting ategy for inflammatory diseases. Eur J Med Chem. 2021;214:113264. Kien XN, Tien BM, Hoa TD et al.Low-Grade Inflammation in Gestational Diabetes Mellitus and Its Correlation with Maternal Insulin Resistance and Fetal Growth Indices[J].International Journal of General Medicine,2023,Vol.16: 1429-1436. Su CL, Chen M, Zhang PN et al.Effects and mechanism of testosterone on the production of inflammatory cytokines and glucose uptake in co-culture of RAW264.7 macrophage and 3T3-L1 adipocytes.[J].Zhonghua yi xue za zhi,2016,(33): 2665-2670. Chen J, Cao Z, Guan J. SHP2 inhibitor PHPS1 protects against atherosclerosis by inhibiting smooth muscle cell proliferation. BMC Cardiovasc Disord. 2018,18(1):72. Rathnakumar K, Savant S, Giri H et al. Angiopoietin-2 mediates thrombin-induced monocyte adhesion and endothelial permeability. J Thromb Haemost. 2016,14(8):1655-67. Kunz HE, Hart CR, Gries KJ et al. Adipose tissue macrophage populations and inflammation are associated with systemic inflammation and insulin resistance in obesity. Am J Physiol Endocrinol Metab. 2021,321(1):105-121. Mastrototaro L, Roden M. Insulin resistance and insulin sensitizing agents. Metabolism. 2021,125:154892. Abdullah M Y , Alqwaidi S D , Alshehri A M ,et al.Obesity-Induced Inflammation and Its Role in the Development of Insulin Resistance[J].Journal of Healthcare Sciences, 2024,04(10):448-454. Greene M W , Garofalo R S .Positive and negative regulatory role of insulin receptor substrate 1 and 2 (IRS-1 and IRS-2) serine/threonine phosphorylation.[J].Biochemistry, 2002, 41(22):7082-91. Richter E A .Is GLUT4 translocation the answer to exercise-stimulated muscle glucose uptake?[J].American Journal of Physiology, 2022, 320:E240-E243. Maneesai P , Jan-O B , Poasakate A ,et al.Limonin mitigates cardiometabolic complications in rats with metabolic syndrome through regulation of the IRS-1/GLUT4 signalling pathway.[J].Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 2023, 161:114448. Siouti E, Andreakos E. The many facets of macrophages in rheumatoid arthritis. Biochem Pharmacol. 2019,165:152-169. Jiang J, Hu B, Chung CS et al. SHP2 inhibitor PHPS1 ameliorates acute kidney injury by Erk1/2-STAT3 signaling in a combined murine hemorrhage followed by septic challenge model. Mol Med. 2020;26(1):89. Teng JF, Wang K, Jia ZM et al. Lentivirus-Mediated Silencing of Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase 2 Inhibits Release of Inflammatory Cytokines and Apoptosis in Renal Tubular Epithelial Cells Via Inhibition of the TLR4/NF-kB Pathway in Renal Ischemia-Reperfusion Injury. Kidney Blood Press Res. 2018,43(4):1084-1103. Zatterale F, Longo M, Naderi J et al. Chronic Adipose Tissue Inflammation Linking Obesity to Insulin Resistance and Type 2 Diabetes. Front Physiol. 2019,10:1607. Li X P. Effect of melatonin receptor agonist Neu-Pll on expression of IRS-1 and GLUT-4 in insulin-resistant adipocytes(Article). Academic Journal of Second Military Medical University. 2013,(5):561-564. Ahmed B, Sultana R, Greene MW. Adipose tissue and insulin resistance in obese. Biomed Pharmacother. 2021,137:111315. Wu H, Ballantyne CM. Metabolic Inflammation and Insulin Resistance in Obesity. Circ Res. 2020,126(11):1549-1564. Wen L, Ye Y, Meijing W et al. Disrupting Phosphatase SHP2 in Macrophages Protects Mice From High-Fat Diet-Induced Hepatic Steatosis and Insulin Resistance by Elevating IL-18 Levels[J].The Journal of biological chemistry,2020,295(31): 10842-10856. Xinxin Y, Tao H, Wei H et al. SHP2 knockdown ameliorates liver insulin resistance by activating IRS-2 phosphorylation through the AKT and ERK1/2 signaling pathways[J].FEBS open bio,2020,10(12): 2578-2587. H. MK, H. KG, E. Y, J. C. Hepatitis B virus X protein induces Src homology 2-containing protein tyrosine phosphatase, SHP2, expression through the NF-kB pathway in HBV-inhepatocellular carcinomas. Cancer Res. 2012,8. Jayaraman S, Devarajan N, Rajagopal P et al. beta-Sitosterol Circumvents Obesity Induced Inflammation and Insulin Resistance by down-Regulating IKKbeta/NF-kappaB and JNK Signaling Pathway in Adipocytes of Type 2 Diabetic Rats. Molecules. 2021,26(7). Olajide OA, Akande IS, da Silva Maia Bezerra Filho C et al. Methyl 3,4,5-trimethoxycinnamate suppresses inflammation in RAW264.7 macrophages and blocks macrophage-adipocyte interaction. Inflammopharmacology. 2020,28(5):1315-1326. Tables Tables 1 to 3 are available in the Supplementary Files section. Supplementary Files table1AB.docx table2AB.docx table3.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 22 Dec, 2025 Reviewers invited by journal 22 Dec, 2025 Editor assigned by journal 18 Dec, 2025 First submitted to journal 17 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8391295","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":564204635,"identity":"e6ce022d-2efb-4732-acf9-4ee55104f887","order_by":0,"name":"Xinxin 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18:44:56","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":859,"visible":true,"origin":"","legend":"","description":"","filename":"HUCED2500732Import.xml","url":"https://assets-eu.researchsquare.com/files/rs-8391295/v1/a038c20bc8b522a35768b25a.xml"},{"id":98963869,"identity":"e3afe691-86a7-4e34-802c-23f0fed1d310","added_by":"auto","created_at":"2025-12-24 18:44:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":97323,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePHPS1 suppresses TNF-α-mediated phosphorylation of NF-κB pathway components in mature adipocytes.(`x±s, n=3, n = number of samples per group)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePHPS1 (10 µM) reduced phosphorylation of p65 and phospho-Iκκin the TNF-α(10 ng/mL) stimulated mature adipocyte. Results are mean±SEM of three independent experiments.Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test.\u003c/p\u003e\n\u003cp\u003e**\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, TNF-α(＋)+PHPS1(－) compared with TNF-α(－)+PHPS1(－).\u003c/p\u003e\n\u003cp\u003e##\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, TNF-α(＋)+PHPS1(－) compared with TNF-α(＋)+PHPS1(＋).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8391295/v1/008bed3d9627524b4419aec0.png"},{"id":98963870,"identity":"d3833c36-f361-4ea5-ac6c-269eb5150e3a","added_by":"auto","created_at":"2025-12-24 18:44:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":95819,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePHPS1 inhibits NF-κB pathway activation in LPS-stimulated adipocyte-macrophage co-cultures.(`x±s, n=3, n = number of samples per group)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantitative analysis revealed that LPS stimulation significantly increased p-p65 and p-IKK levels, which were markedly attenuated by PHPS1 (10 µM) pre-treatment. Results are mean±SEM of three independent experiments. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test.\u003c/p\u003e\n\u003cp\u003e*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, LPS(＋)+PHPS1(－) compared with LPS(－)+PHPS1(－).\u003c/p\u003e\n\u003cp\u003e#\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ##\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, LPS(＋)+PHPS1(－) compared with LPS(＋)+PHPS1(＋).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8391295/v1/8aa13451e8c195d51e81c0bd.png"},{"id":99311174,"identity":"f9520746-8c4d-42f9-ab9f-4f6bca59a794","added_by":"auto","created_at":"2025-12-31 16:14:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":104990,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePHPS1 exacerbates insulin resistance markers in LPS-stimulated co-cultures.(`x±s, n=3, n = number of samples per group)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLPS-stimulated co-culture of adipocytes and macrophages treated with PHPS1 (10 µM) showed significantly higher phosphorylation of Ser 307 and decrease GLUT-4 protein expression.Results are mean±SEM of three independent experiments. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test.\u003c/p\u003e\n\u003cp\u003e*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, LPS(＋)+PHPS1(－) compared with LPS(－)+PHPS1(－).\u003c/p\u003e\n\u003cp\u003e#\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ##\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, LPS(＋)+PHPS1(－) compared with LPS(＋)+PHPS1(＋).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8391295/v1/0ce1f88120df8f032da5bf94.png"},{"id":100405560,"identity":"4fbe399d-4e35-4cf6-a061-7f0d42a46fe6","added_by":"auto","created_at":"2026-01-16 12:07:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1159006,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8391295/v1/3f261e5a-b042-40d7-93e0-558b65f3e5ef.pdf"},{"id":98963868,"identity":"9df22bd8-ff05-4e36-b2aa-07c9f52e2041","added_by":"auto","created_at":"2025-12-24 18:44:56","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19882,"visible":true,"origin":"","legend":"","description":"","filename":"table1AB.docx","url":"https://assets-eu.researchsquare.com/files/rs-8391295/v1/145c061e16bc610050912521.docx"},{"id":98963871,"identity":"c5d0743a-c863-40c2-b586-29e104fc277d","added_by":"auto","created_at":"2025-12-24 18:44:56","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17091,"visible":true,"origin":"","legend":"","description":"","filename":"table2AB.docx","url":"https://assets-eu.researchsquare.com/files/rs-8391295/v1/658ca0a5923e1d98b0b5e7fc.docx"},{"id":98963872,"identity":"f5d7c00f-db0c-4b49-b20b-8a85162524c9","added_by":"auto","created_at":"2025-12-24 18:44:56","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":13765,"visible":true,"origin":"","legend":"","description":"","filename":"table3.docx","url":"https://assets-eu.researchsquare.com/files/rs-8391295/v1/ed0cf5e3658e613dd41016ea.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eSHP2 inhibitor PHPS1 regulates macrophage-adipocyte interaction to alleviate inflammation and adipocyte insulin resistance, via NF-κB suppression and IRS-1/GLUT-4 restoration\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eObesity, a highly prevalent chronic metabolic disorder globally, is defined by abnormal accumulation and dysregulated distribution of adipose tissue. Despite decades of research, its multifactorial etiology, encompassing genetic, dietary, and environmental factors, remains incompletely understood, posing a major barrier to effective intervention¹. Epidemiological data consistently highlight a rising global burden of obesity, which is tightly linked to the escalating prevalence of type 2 diabetes mellitus (T2DM) and insulin resistance (IR)—two interrelated conditions that impose substantial clinical and socioeconomic costs².\u003c/p\u003e\n\u003cp\u003eAdipose tissue, long regarded merely as an energy storage organ, is now recognized as a key endocrine and immunometabolic hub. It secretes a spectrum of adipokines and cytokines that regulate systemic energy homeostasis; however, in obese states, adipose tissue undergoes pathological remodeling, characterized by increased infiltration of adipose tissue macrophages (ATMs)³. These ATMs, together with dysfunctional adipocytes, drive the production of pro-inflammatory mediators including interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α), initiating a state of chronic low-grade metabolic inflammation—the central driver of obesity-related IR⁴. Under pathological stimuli including high-fat diet (HFD) or lipopolysaccharide (LPS) exposure, cross-activation between adipocytes and macrophages amplifies inflammatory signaling: pro-inflammatory factors activate the nuclear factor-κB (NF-κB) pathway, which in turn induces abnormal phosphorylation of insulin receptor substrate-1 (IRS-1, Ser307) and downregulates glucose transporter type 4 (GLUT-4) expression. This disrupts insulin-mediated glucose uptake and metabolism, ultimately leading to IR and subsequent T2DM⁵. Unraveling the regulatory mechanisms of key molecules in this \"inflammation-IR\" axis is therefore critical for developing targeted therapies to mitigate T2DM and its complications.\u003c/p\u003e\n\u003cp\u003eThe Src homology 2 domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 gene, is a ubiquitously expressed non-receptor protein tyrosine phosphatase with pivotal roles in immune regulation and metabolic homeostasis. It modulates multiple signaling cascades in immune and metabolic tissues, including mitogen-activated protein kinase (MAPK), Toll-like receptors (TLRs), and NF-κB—pathways closely tied to inflammatory responses and IR⁶. Aberrant SHP2 activation exacerbates inflammation by promoting the secretion of pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6, and contributes to tissue damage in organs such as the liver, kidneys, and adipose tissue⁷. Phenylhydrazono pyrazolone sulfonate 1 (PHPS1), a specific small-molecule inhibitor of SHP2, blocks SHP2 phosphorylation and its downstream pro-inflammatory effects. Previous studies have validated PHPS1’s efficacy in ameliorating inflammatory diseases including atherosclerosis and acute kidney injury, but its role in regulating adipose tissue inflammation and improving adipocyte IR—key pathological features of T2DM—remains unaddressed\u003csup\u003e8,9\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo fill this gap, we established a Transwell co-culture system of 3T3-L1 adipocytes and RAW 264.7 macrophages to mimic the in vivo adipose tissue microenvironment—a model that better recapitulates the cell-cell interactions driving obesity-related IR than single-cell cultures¹⁰. Using this system, we investigated whether PHPS1 exerts anti-inflammatory effects by targeting the NF-κB pathway and explored its potential to improve adipocyte IR via restoring the IRS-1/GLUT-4 insulin signaling axis. This study aims to clarify the role of SHP2 in metabolic inflammation and provide preclinical evidence for PHPS1 as a potential therapeutic candidate for T2DM and obesity-related IR.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 3T3-L1 pre-adipocyte and RAW 264.7 macrophage cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). RAW 264.7 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were incubated at 37 °C in a humidified atmosphere containing 5% carbon dioxide (CO₂) and 95% air, with the culture medium refreshed every 48 hours.\u003c/p\u003e\n\u003cp\u003eDifferentiation of 3T3-L1 pre-adipocytes was induced as follows: on day 0, pre-adipocytes were treated with induction medium (DMEM containing 10% FBS, 0.5 mmol/L isobutylmethylxanthine, 1 μmol/L dexamethasone, and 10 μg/mL insulin). After 48 hours (day 2), the medium was replaced with maintenance medium (DMEM containing 10% FBS and 10 μg/mL insulin). The maintenance medium was refreshed every 48 hours until days 7–8, when cells were fully differentiated into mature adipocytes.\u003c/p\u003e\n\u003cp\u003eMature 3T3-L1 adipocytes were randomly divided into three groups: Control group: treated with DMEM only; TNF-α group: treated with 10 ng/mL TNF-α (R\u0026amp;D Systems, Minneapolis, MN, USA); TNF-α + PHPS1 group: pre-treated with 10μM PHPS1 (MedChemExpress, Monmouth Junction, NJ, USA; purity ≥98%) for 30 minutes, followed by co-treatment with 10 ng/mL TNF-α. All groups were incubated for an additional 24 hours before subsequent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-culture of adipocytes and macrophages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA Transwell co-culture system with a 0.4 μm porous membrane (Corning, Corning, NY, USA) was used to establish direct contact between adipocytes and macrophages. Fully differentiated 3T3-L1 adipocytes (1×10⁵ cells/well) were seeded in the lower chamber of 6-well Transwell plates, while RAW 264.7 macrophages (5×10⁴ cells/well) were seeded in the upper chamber inserts. After 24 hours of co-culture to allow cell adaptation, the co-cultures were divided into three groups: Co-culture control group: treated with DMEM only: LPS group: treated with 1 μg/mL LPS (Escherichia coli serotype O111:B4; Sigma-Aldrich, St. Louis, MO, USA); LPS + PHPS1 group: pre-treated with 10μM PHPS1 for 30 minutes, followed by co-treatment with 1 μg/mL LPS. All groups were incubated for another 24 hours before sample collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of IL-6, IL-10, IL-1β and MCP-1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLevels of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α)and anti-inflammatory cytokine (IL-10) were measured using commercial mouse enzyme-linked immunosorbent assay (ELISA) kits (BioLegend, San Diego, CA, USA; catalog numbers: IL-6: 431304, IL-1β: 432604, TNF-α: 430904, IL-10: 431404). The level of monocyte chemoattractant protein-1 (MCP-1/CCL2) was detected using a mouse CCL2 ELISA kit (Invitrogen, Waltham, MA, USA; catalog number: BMS6002). All assays were performed strictly according to the manufacturers' instructions, and cytokine concentrations were calculated based on standard curves.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time polymerase chain reaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from 3T3-L1 adipocytes (single-cell culture) and co-cultured cells using TRIzol reagent (Invitrogen), and its purity and concentration were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the ImProm-II Reverse Transcription System (Promega, Madison, WI, USA) according to the manufacturer's protocol.\u003c/p\u003e\n\u003cp\u003eqRT-PCR was performed on an Applied Biosystems 7900 Detection System (Thermo Fisher Scientific) using Fast SYBR Master Mix (Applied Biosystems, Foster City, CA, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe primer sequences were as follows:\u003c/p\u003e\n\u003cp\u003eInducible nitric oxide synthase (iNOS): Forward 5’-TCACGCTTGGGTCTTGTTCA-3’, Reverse 5’-CCTTTTCCTCTTTCAGGTCACTT-3’;\u003c/p\u003e\n\u003cp\u003eCyclooxygenase-2 (COX-2): Forward 5’-CTGCCAATAGAACTTCCAATCC-3’, Reverse 5’-CGGTTTGATGTTACTGTTGCTT-3’;\u003c/p\u003e\n\u003cp\u003eGlyceraldehyde 3-phosphate dehydrogenase (GAPDH): Forward 5’-GACAACTTTGGCATTGTG-3’, Reverse 5’-ATGCAGGGATGATGTTCTG-3’.\u003c/p\u003e\n\u003cp\u003eThe reaction conditions were: 95 °C for 10 minutes, followed by 40 cycles of 95 °C for 15 seconds and 60 °C for 1 minute. The relative mRNA expression levels were calculated using the 2⁻ΔΔCt method, with GAPDH as the internal reference gene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn-cell western blotting was used for protein quantification, as it combines the specificity of traditional western blotting with the quantitative accuracy of ELISA. RAW 264.7 macrophages were seeded in 96-well plates at a density of 5×10⁴ cells/mL and cultured until reaching 70% confluence. Cells were divided into three groups: Control group; LPS group (treated with 1 μg/mL LPS); LPS + PHPS1 group (pre-treated with 10μM PHPS1 for 30 minutes, then 1 μg/mL LPS). After 24 hours of treatment, cells were fixed with 8% paraformaldehyde (100 μL/well) for 15 minutes at room temperature, washed three times with phosphate-buffered saline (PBS), and permeabilized with 0.1% Triton X-100 for 10 minutes.\u003c/p\u003e\n\u003cp\u003eAfter blocking with 5% non-fat milk for 1 hour at room temperature, cells were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-phospho-p65 (Cell Signaling Technologies, Danvers, MA, USA; catalog number: 3033), rabbit anti-phospho-Iκκα (Santa Cruz Biotechnology, Dallas, TX, USA; catalog number: sc-16677-R), rabbit anti-Iκκα (Santa Cruz Biotechnology; catalog number: sc-374353), rabbit anti-phospho-IRS-1 (Ser 307) (Cell Signaling Technologies; catalog number: 2381), rabbit anti-GLUT-4 (Santa Cruz Biotechnology; catalog number: sc-166864), and rabbit anti-β-actin (Cell Signaling Technologies; catalog number: 4970).\u003c/p\u003e\n\u003cp\u003eThe next day, cells were washed three times with PBS and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Cell Signaling Technologies; catalog number: 7074) for 2 hours at room temperature. After three additional PBS washes, 100 μL of HRP substrate (Thermo Fisher Scientific) was added to each well, and the absorbance was measured at 450 nm using a microplate reader (BioTek, Winooski, VT, USA). Protein expression levels were normalized to β-actin (for single-cell samples) or Janus Green stain (for co-culture samples, Abcam, Cambridge, UK) to correct for cell number differences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were independently repeated three times, with each group measured in triplicate. Data are presented as the mean ± standard error (SE). Statistical analysis was performed using GraphPad PRISM 8.0 software (GraphPad Software, San Diego, CA, USA). Prior to statistical testing, all data were verified to conform to a normal distribution using the Shapiro-Wilk test (p\u0026gt;0.05) and to have homogeneous variance using Levene’s test (\u003cem\u003ep\u003c/em\u003e\u0026gt;0.05), ensuring compliance with the assumptions of analysis of variance (ANOVA). Comparisons between multiple groups were performed using one-way ANOVA followed by Tukey’s post hoc test. A \u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePHPS1 (10μM) exerts anti-inflammatory effects in TNF-α-stimulated mature 3T3-L1 adipocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the anti-inflammatory activity of PHPS1, we first detected the secretion of inflammatory cytokines and chemokines in TNF-α-stimulated mature 3T3-L1 adipocytes, with key data summarized in Table 1A-B. Compared with the TNF-α(−)+PHPS1(−) control group, stimulation with TNF-α [in the TNF-α(+)+PHPS1(−) group] significantly increased the secretion of pro-inflammatory cytokines IL-6 (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001,\u003cem\u003eq\u003c/em\u003e=120) and IL-1β (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001\u003cem\u003e,q\u003c/em\u003e=103), as well as the chemokine MCP-1 (p\u0026lt;0.0001,q=118.1), while significantly decreasing the secretion of the anti-inflammatory cytokine IL-10 (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001,\u003cem\u003eq\u003c/em\u003e=29.21).\u003c/p\u003e\n\u003cp\u003ePretreatment with PHPS1 (10 μM) [in the TNF-α(+)+PHPS1(+) group] significantly reversed these TNF-α-induced changes. As precisely determined by our quantitative analysis, compared with the TNF-α(+)+PHPS1(−) group, the TNF-α(+)+PHPS1(+) group showed a remarkable 62.35% ± 2.08 reduction in IL-6 secretion (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, \u003cem\u003eq\u003c/em\u003e=76.89), a 54.10% ± 1.85 reduction in IL-1β secretion (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001,\u003cem\u003eq\u003c/em\u003e=58.19), and a 49.19% ± 2.03 reduction in MCP-1 secretion (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001,\u003cem\u003eq\u003c/em\u003e=61.84). Meanwhile, IL-10 secretion was increased by 103.98% ± 13.79 in the TNF-α(+)+PHPS1(+) group compared with the TNF-α(+)+PHPS1(−) group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001,\u003cem\u003eq\u003c/em\u003e=21.49). These highly significant and precisely quantified results (Table 1A-B) strongly indicate that PHPS1 (10 μM) effectively inhibits TNF-α-induced inflammatory responses in mature 3T3-L1 adipocytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePHPS1 Alleviates LPS - Induced Inflammation in Adipocyte - Macrophage Co - Cultures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the LPS-stimulated adipocyte-macrophage co-culture system, with key data summarized in Table 1A-B, stimulation with LPS [in the LPS(+)+PHPS1(−) group] significantly elevated the levels of pro-inflammatory cytokines IL-6 and IL-1β, as well as the chemokine MCP-1, while reducing the secretion of anti-inflammatory cytokine IL-10, compared with the unstimulated control co-culture [LPS(−)+PHPS1(−) group]. Specifically, when comparing the LPS(+)+PHPS1(−) group with the LPS(−)+PHPS1(−) group, IL-6 secretion increased from an average of 31.53 pg/mL (SE =4.02) to 725.62 pg/mL (SE =\u0026nbsp;6.61), IL-1β secretion rose from an average of 25.58 pg/mL (SE =\u0026nbsp;3.52) to 704.01 pg/mL (SE =\u0026nbsp;12.95), MCP-1 secretion increased from an average of 34.95 pg/mL (SE =1.30) to 587.63 pg/mL (SE =\u0026nbsp;5.75), and IL-10 secretion decreased from an average of 307.48 pg/mL (SE =\u0026nbsp;10.12) to 104.15 pg/mL (SE =\u0026nbsp;6.30). All these LPS-induced changes were statistically significant (\u003cem\u003eF=\u003c/em\u003e4047,\u0026nbsp;\u003cem\u003eF=\u003c/em\u003e875.9,\u0026nbsp;\u003cem\u003eF=\u003c/em\u003e1593,\u0026nbsp;\u003cem\u003eF=\u003c/em\u003e117.8).\u003c/p\u003e\n\u003cp\u003ePretreatment with PHPS1 (10 μM) [in the LPS(+)+PHPS1(+) group] significantly suppressed the LPS-induced upregulation of pro-inflammatory factors and reversed the downregulation of the anti-inflammatory cytokine. Compared with the LPS(+)+PHPS1(−) group, the LPS(+)+PHPS1(+) group showed a 68.73% ± 1.16 reduction in IL-6 secretion (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001,\u003cem\u003eq\u003c/em\u003e=96.43), a 63.01% ± 3.44 reduction in IL-1β secretion (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001,\u003cem\u003eq\u003c/em\u003e=41.35), and a 47.79% ± 2.42 reduction in MCP-1 secretion (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001,\u003cem\u003eq\u003c/em\u003e=42.54). Meanwhile, IL-10 secretion was increased by 167.49% ± 23.78 in the LPS(+)+PHPS1(+) group compared with the LPS(+)+PHPS1(−) group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001,\u003cem\u003eq\u003c/em\u003e=19.11). These results (Table 1A-B) indicate that PHPS1 mitigates LPS-induced inflammation in the adipocyte-macrophage interaction microenvironment.\u003c/p\u003e\n\u003cp\u003ePlace Table 1A-B here\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePHPS1 Inhibits TNF - α - Induced iNOS and COX - 2 mRNA Expression in Mature Adipocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eiNOS and COX - 2 are key enzymes mediating inflammatory mediator synthesis. As shown in Table 2A-B, TNF - α stimulation significantly upregulated iNOS and COX - 2 mRNA expression compared with the control group.\u003c/p\u003e\n\u003cp\u003eFor iNOS, the control group (TNF - α⁻PHPS1⁻) mRNA expression levels were relatively stable with an average value of 4.73 (SE = 0.47). In contrast, TNF - α stimulation (TNF - α⁺PHPS1⁻) led to a marked increase in iNOS mRNA expression, with an average level of 32.35 (SE = 1.59). This represents an upregulation of approximately 5.89 - fold (32.35 / 4.73). However, pretreatment with PHPS1 (10 μM) (TNF - α⁺PHPS1⁺) effectively downregulated the TNF - α - induced iNOS mRNA expression. The average aexpression level in the TNF - α + PHPS1 group was 11.64 (SE = 0.51). Compared with the TNF - α group, PHPS1 pretreatment reduced iNOS mRNA expression by approximately 63.99% [(32.35 - 11.64) / 32.35 × 100%], and this reduction was statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001,\u003cem\u003eq\u003c/em\u003e=23.42).\u003c/p\u003e\n\u003cp\u003eRegarding COX - 2, the control group (TNF - α⁻PHPS1⁻) had an average mRNA expression level of 4.53 (SE = 0.59). When stimulated with TNF - α (TNF - α⁺PHPS1⁻), COX - 2 mRNA expression increased significantly to an average of 15.69 (SE = 0.60). This indicates an upregulation of about 2.47 - fold (15.69 / 4.53). Nevertheless, PHPS1 pretreatment (TNF - α⁺PHPS1⁺) notably decreased the TNF - α - induced COX - 2 mRNA expression. The average expression level in the TNF - α + PHPS1 group was 7.20 (SE = 0.36). Compared with the TNF - α group, PHPS1 pretreatment led to a reduction of approximately 54.11% [(15.69 - 7.20) / 15.69×100%]in COX - 2 mRNA expression, and this was also statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001,\u003cem\u003eq\u003c/em\u003e=15.44).\u003c/p\u003e\n\u003cp\u003eThese findings further validate PHPS1’s anti - inflammatory activity at the transcriptional level, as it effectively suppresses the upregulation of iNOS and COX - 2\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePHPS1 Reduces LPS - Induced iNOS and COX - 2 Transcription in Co - Cultures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn LPS - stimulated co - cultures ( Table 2A-B), LPS significantly increased the mRNA expression of iNOS and COX - 2, two crucial enzymes involved in the inflammatory response, compared with the control co - culture. Our data clearly demonstrate the impact of LPS and the subsequent protective effect of PHPS1.\u003c/p\u003e\n\u003cp\u003eFor iNOS, in the control group (LPS⁻PHPS1⁻), the average mRNA expression level was 2.93 (SE = 0.28). Upon LPS stimulation (LPS⁺PHPS1⁻), iNOS mRNA expression soared to an average of 37.69 (SE = 0.42). This represents an upregulation of approximately 11.87 - fold (37.69 / 2.93). However, pretreatment with PHPS1 (10 μM) (LPS⁺PHPS1⁺) notably reduced the LPS - induced iNOS mRNA expression. The average expression level in the LPS + PHPS1 group was 17.28 (SE = 0.31). Compared with the LPS group, PHPS1 pretreatment decreased iNOS mRNA expression by approximately 54.15% [(37.69 - 17.28) / 37.69× 100%], and this reduction was highly statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001,\u003cem\u003eq\u003c/em\u003e=64.62).\u003c/p\u003e\n\u003cp\u003eRegarding COX - 2(the control group (LPS⁻PHPS1⁻) had an average mRNA expression level of 4.06 (SD = 0.53), obtained from 3.43, 4.23, and 4.52. LPS stimulation (LPS⁺PHPS1⁻) led to a significant increase in COX - 2 mRNA expression to an average of 16.61 (SD = 0.66), based on 16.82, 15.71, and 17.31. This indicates an upregulation of about 3.84 - fold (16.61 / 4.06). Nevertheless, PHPS1 pretreatment (LPS⁺PHPS1⁺) effectively mitigated the LPS - induced increase. The average expression level in the LPS + PHPS1 group was 9.28 (SD = 0.31), calculated from 8.93, 9.4, and 9.51. Compared with the LPS group, PHPS1 reduced COX - 2 mRNA expression by approximately 44.13% [(16.61 - 9.28) / 16.61×100%], and this was also statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001,\u003cem\u003eq\u003c/em\u003e=18.02).\u003c/p\u003e\n\u003cp\u003eThese findings indicate that PHPS1 inhibits inflammatory gene transcription in the macrophage - adipocyte interaction context, highlighting its potential as an anti - inflammatory agent in co - culture systems.\u003c/p\u003e\n\u003cp\u003ePlace Table 2A-B here\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnti‑infammatory activity of PHPS1 is\u0026nbsp;mediated through\u0026nbsp;inhibition of\u0026nbsp;NF‑κB activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe NF - κB signaling pathway plays a central and well - established role in the regulation of inflammatory responses. Activation of this pathway leads to the upregulation of numerous pro - inflammatory genes, making it a key target for anti - inflammatory therapeutic strategies. Here, we investigated the impact of PHPS1 on the NF - κB pathway under different inflammatory stimuli (TNF - α and LPS) in relevant cellular models to elucidate its anti - inflammatory mechanism.\u003c/p\u003e\n\u003cp\u003eIn the context of TNF - α stimulation, as shown in our study (Figure 1,Table 3), TNF - α led to a marked activation of the NF - κB pathway, as evidenced by the increased phosphorylation levels of key pathway components p-65 and p-IKKβ. In the group treated with TNF-α alone (TNF - α⁺PHPS1⁻), the mean phosphorylation level of p-65 was 2.347. However, when adipocytes were pretreated with PHPS1 (TNF - α⁺PHPS1⁺), the mean phosphorylation level of p-65 decreased to 1.233. This represents a significant reduction of approximately 47.46% [(2.347 - 1.233) /\u0026nbsp;2.347×100%]. An unpaired two tailed t-test confirmed that this difference was statistically significant (t = 5.070, df = 4, \u003cem\u003ep\u003c/em\u003e = 0.0071). Similarly, for p-IKKβ, the mean phosphorylation level in the TNF - α alone group (TNF - α⁺PHPS1⁻) was 2.347, while in the PHPS1 - pretreated group (TNF- α⁺PHPS1⁺), it dropped to 1.247, showing a decrease of about 46.87% [(2.347 - 1.247) / 2.347\u0026nbsp;×100%]. An unpaired two tailed t-test revealed a highly significant difference (t = 5.291, df = 4, \u003cem\u003ep\u003c/em\u003e = 0.0061). These results clearly indicate that PHPS1 effectively suppresses TNF - α - induced NF - κB pathway activation by reducing the phosphorylation of both p - 65 and p - IKKβ.\u003c/p\u003e\n\u003cp\u003ePlace Figure 1 here\u003c/p\u003e\n\u003cp\u003ePlace Table 3 here\u003c/p\u003e\n\u003cp\u003eAs depicted in Figure 2 and Table 3, LPS stimulation also robustly activated the NF - κB pathway. In the LPS - only group (LPS⁺PHPS1⁻), the mean phosphorylation level of p65 was 1.627. Upon pretreatment with PHPS1 (LPS⁺PHPS1⁺), the mean p65 phosphorylation level decreased to 0.8767, resulting in a remarkable reduction of approximately 46.12% [(1.627 - 0.8767) / 1.627\u0026nbsp;×100%]. The unpaired two tailed t-test demonstrated a highly significant difference (t = 13.42, df = 4, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.0002).\u003c/p\u003e\n\u003cp\u003ePlace Figure 2 here\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePHPS1 improve insulin resistance may via the insulin receptor substrate 1(IRS-1)/glucose transporter type 4 isoform(GLUT-4) pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInsulin resistance is a key pathophysiological feature of metabolic disorders, tightly linked to chronic inflammation. The IRS-1/GLUT-4 pathway is critical for insulin-mediated glucose uptake, and its disruption is a hallmark of IR. Here, we validated PHPS1’s role in restoring this pathway using LPS-induced adipocyte-macrophage co-cultures (a model that recapitulates in vivo adipose microenvironment interactions).\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 3\u0026nbsp;and Table 3, LPS stimulation (LPS⁺PHPS1⁻ group) significantly disrupted insulin signaling: it increased aberrant phosphorylation of IRS-1 (Ser307)—a marker of IRS-1 inactivation—to a mean level of 2.937 (vs. 1.000 in LPS⁻PHPS1⁻ controls), while reducing GLUT-4 (a key glucose transporter) to a mean level of 1.093 (vs. 2.100 in controls). These changes are consistent with prior reports that LPS-induced inflammation impairs insulin signaling via IRS-1 hyperphosphorylation\u003csup\u003e11\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003ePretreatment with 10 μM PHPS1 (LPS⁺PHPS1⁺ group) reversed these defects: IRS-1 (Ser307) phosphorylation was reduced by 61.12% (from 2.937 to 1.14), with statistical significance (t=8.431, df=4, p=0.0011). This reduction restores IRS-1’s ability to transduce insulin signals, as hyperphosphorylation of Ser307 blocks IRS-1 binding to insulin receptors\u003csup\u003e12\u003c/sup\u003e. GLUT-4 levels were increased by 198.0% (from 1.093 to 3.257), with high significance (t=12.03, df=4, p=0.0003). This elevation enhances glucose uptake capacity, directly counteracting IR\u003csup\u003e13\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003ePlace Figure 3 here\u003c/p\u003e\n\u003cp\u003eNotably, these effects of PHPS1 on IRS-1/GLUT-4 were accompanied by concurrent NF-κB inhibition (46.12% reduction in p-p65, Figure 3), suggesting a causal link: PHPS1 reduces inflammation via NF-κB suppression, which in turn relieves inflammatory-mediated damage to the IRS-1/GLUT-4 axis\u003csup\u003e14\u003c/sup\u003e. This is supported by our cytokine data (68.73% reduction in IL-6, Table 1A-B), as pro-inflammatory cytokines like IL-6 directly promote IRS-1 phosphorylation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we systematically investigated the regulatory effects of the SHP2 inhibitor PHPS1 on inflammation and insulin resistance (IR) using two experimental models: TNF-α-stimulated mature 3T3-L1 adipocytes (single-cell culture) and LPS-induced adipocyte-macrophage Transwell co-cultures. We confirmed that PHPS1 exerts anti-inflammatory effects and improves adipocyte IR primarily by inhibiting the NF-κB signaling pathway and restoring the IRS-1/GLUT-4 insulin signaling axis—two mechanisms closely tied to the pathogenesis of type 2 diabetes mellitus (T2DM), the core focus of metabolic disease research.\u003c/p\u003e\n\u003cp\u003eThe NF-κB pathway is a well-recognized core regulator of chronic metabolic inflammation, and its abnormal activation is strongly associated with obesity-related IR and T2DM development⁵. Previous studies have reported that SHP2 inhibitors suppress NF-κB activation by reducing the phosphorylation of key pathway components (p-IKK and p-p65), thereby decreasing pro-inflammatory cytokine secretion\u003csup\u003e7,8\u003c/sup\u003e. Our findings align with this research: in TNF-α-stimulated adipocytes, 10 μM PHPS1 reduced the secretion of pro-inflammatory cytokines IL-6 (62.35% ± 2.08 reduction), IL-1β (54.10% ± 1.85 reduction), and chemokine MCP-1 (49.19% ± 2.03 reduction), while increasing the anti-inflammatory cytokine IL-10 (103.98% ± 13.79 increase). Mechanistically, this was attributed to PHPS1-mediated inhibition of NF-κB: the phosphorylation levels of p-p65 decreased by 47.46% (from 2.347 to 1.233, p=0.0071) and p-IKK by 46.87% (from 2.347 to 1.247, p=0.0061). Kunz HE et al. confirmed that the transcription of IL-6 and IL-1β is directly regulated by the NF-κB pathway, and our cytokine data further validate that PHPS1’s suppression of NF-κB activation translates to effective regulation of cytokine secretion, reinforcing its role in alleviating adipose tissue inflammation⁹.\u003c/p\u003e\n\u003cp\u003eIn LPS-induced adipocyte-macrophage co-cultures— a model that better mimics the in vivo adipose microenvironment—PHPS1 exerted similar anti-inflammatory effects: it reduced IL-6 secretion by 68.73% ± 1.16, IL-1β by 63.01% ± 3.44, and MCP-1 by 47.79% ± 2.42, while increasing IL-10 by 167.49% ± 23.78. Concurrently, PHPS1 inhibited LPS-induced NF-κB activation, with p-p65 phosphorylation reduced by 46.12% (from 1.627 to 0.8767, p=0.0002) and p-IKK by 41.33% (from 1.337 to 0.7833, p\u0026lt;0.0001). This is consistent with research emphasizing that LPS-induced cross-talk between adipocytes and macrophages relies on NF-κB activation, and inhibiting this pathway can break the inflammatory cycle in adipose tissue— a critical step in mitigating obesity-related IR¹⁰.\u003c/p\u003e\n\u003cp\u003eNotably, our study addresses key limitations of previous research. Most prior studies on SHP2 inhibitors focused on single-cell models (e.g., hepatocytes or macrophages alone)\u003csup\u003e15-17\u003c/sup\u003e, whereas our Transwell co-culture system recapitulates the complex cellular interactions of the in vivo adipose microenvironment\u003csup\u003e18\u003c/sup\u003e, providing more physiologically relevant evidence for PHPS1’s role in targeting adipose tissue inflammation. Additionally, while previous studies reported changes in cytokine secretion¹⁵, our data further confirm that PHPS1 suppresses the transcription of NF-κB-dependent inflammatory genes (iNOS and COX-2): in TNF-α-stimulated adipocytes, PHPS1 reduced iNOS mRNA expression by 63.99% (from 32.35 to 11.64, p\u0026lt;0.0001) and COX-2 by 54.11% (from 15.69 to 7.20, p\u0026lt;0.0001); in LPS-stimulated co-cultures, iNOS transcription decreased by 54.15% (from 37.69 to 17.28, p\u0026lt;0.0001) and COX-2 by 44.13% (from 16.61 to 9.28, p\u0026lt;0.0001). This indicates PHPS1 acts upstream of gene transcription—likely by blocking NF-κB nuclear translocation20—rather than merely affecting mRNA stability, offering a more comprehensive understanding of SHP2 inhibitor-mediated anti-inflammatory effects.\u003c/p\u003e\n\u003cp\u003eChronic inflammation disrupts insulin signaling, and aberrant phosphorylation of IRS-1 (Ser307) combined with downregulation of GLUT-4 are key events in IR development\u003csup\u003e21\u003c/sup\u003e. Wen L et al. reported that SHP2 inhibitors improve IR in high-fat diet-fed mice, but the specific molecular mechanism—especially the role of the IRS-1/GLUT-4 axis—remained unclear\u003csup\u003e22\u003c/sup\u003e. Our data fill this gap: LPS stimulation increased p-IRS-1 (Ser307) levels to 2.937 (vs. control) and reduced GLUT-4 levels to 1.093, consistent with research showing LPS-induced inflammation promotes IRS-1 hyperphosphorylation to impair insulin signaling\u003csup\u003e23\u003c/sup\u003e. Pretreatment with PHPS1 reversed these defects: p-IRS-1 (Ser307) was reduced by 61.12% (to 1.14, p=0.0011), and GLUT-4 levels increased by 198.0% (to 3.257, p=0.0003). This directly links SHP2 inhibition to the restoration of the IRS-1/GLUT-4 axis, a mechanism not previously identified in prior studies\u003csup\u003e23\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThis study has two key innovations: first, the combined use of single adipocyte and adipocyte-macrophage co-culture models addresses the limitations of single-cell studies and better reflects the in vivo adipose tissue microenvironment\u003csup\u003e24,25\u003c/sup\u003e; second, we integrated data from cytokine detection, pathway phosphorylation, gene transcription, and insulin signaling molecules to establish a complete regulatory cascade: “PHPS1 → NF-κB inhibition → inflammation reduction → IRS-1/GLUT-4 restoration”.\u003c/p\u003e\n\u003cp\u003eThis study also has limitations that require consideration. First, we used a single PHPS1 concentration (10 μM) based on prior research validating SHP2 inhibitor efficacy across multiple concentrations\u003csup\u003e7,26\u003c/sup\u003e; future studies should include dose-response curves to determine the EC50 and rule out off-target effects. Second, our in vitro findings need confirmation in animal models; we plan to further test whether PHPS1 reduces adipose tissue macrophage infiltration (via MCP-1 inhibition) and improves glucose tolerance (via GLUT-4 upregulation) in vivo, to provide stronger preclinical evidence for its translational potential.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, the SHP2 inhibitor PHPS1 alleviates inflammation in both TNF-α-stimulated 3T3-L1 adipocytes and LPS-induced adipocyte-macrophage co-cultures by suppressing the phosphorylation of the NF-κB signaling pathway. Mechanistically, PHPS1 reduces the secretion of pro-inflammatory cytokines (IL-6, IL-1β, MCP-1), upregulates the anti-inflammatory cytokine IL-10, and inhibits the transcription of NF-κB-dependent inflammatory genes (iNOS, COX-2). More importantly, PHPS1 improves adipocyte insulin resistance by restoring the IRS-1/GLUT-4 insulin signaling axis—reducing aberrant phosphorylation of IRS-1 (Ser307) and increasing GLUT-4 expression to enhance glucose uptake. These findings clarify the role of SHP2 in metabolic inflammation and IR, and highlight PHPS1 as a potential therapeutic candidate for insulin resistance-related metabolic diseases, including type 2 diabetes mellitus.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Writing the original draft preparation, YUE Xin-xin, FU Yang. Writing review and editing, YUE Xin-xin and Yin Xiaoyan. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by the National Natural Science Foundation Committee of China (grant number:72171042), and the “Scientific Research Funding Project of the Education Department of Liaoning Province” (grant number: LJKMZ20221981).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eWe thank all past and present members of the Clinical College Institute for fruitful discussions and daily support and our collaborators for their inspiring inputs. We apologize to those authors whose work we could not cite directly owing to space constraints.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBenjamin CL, Amanda YL, Soo LC, et al. 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SHP2 knockdown ameliorates liver insulin resistance by activating IRS-2 phosphorylation through the AKT and ERK1/2 signaling pathways[J].FEBS open bio,2020,10(12): 2578-2587.\u003c/li\u003e\n\u003cli\u003eH. MK, H. KG, E. Y, J. C. Hepatitis B virus X protein induces Src homology 2-containing protein tyrosine phosphatase, SHP2, expression through the NF-kB pathway in HBV-inhepatocellular carcinomas. Cancer Res. 2012,8.\u003c/li\u003e\n\u003cli\u003eJayaraman S, Devarajan N, Rajagopal P et al. beta-Sitosterol Circumvents Obesity Induced Inflammation and Insulin Resistance by down-Regulating IKKbeta/NF-kappaB and JNK Signaling Pathway in Adipocytes of Type 2 Diabetic Rats. Molecules. 2021,26(7).\u003c/li\u003e\n\u003cli\u003eOlajide OA, Akande IS, da Silva Maia Bezerra Filho C et al. Methyl 3,4,5-trimethoxycinnamate suppresses inflammation in RAW264.7 macrophages and blocks macrophage-adipocyte interaction. Inflammopharmacology. 2020,28(5):1315-1326. \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"SHP2 inhibitor PHPS1, macrophages, adipocyte, inflammatory factors, NF-κB, Insulin resistance","lastPublishedDoi":"10.21203/rs.3.rs-8391295/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8391295/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"This study explored the anti-inflammatory and insulin resistance-regulating effects of SHP2 inhibitor PHPS1 using a Transwell co-culture system of adipocytes and macrophages to mimic the in vivo adipose microenvironment. Two models were established: TNF-α-stimulated 3T3-L1 adipocytes (single culture) and LPS-induced insulin-resistant 3T3-L1/RAW 264.7 co-cultures. After 24 h of 10 μM PHPS1 pretreatment, ELISA, qRT-PCR, and Western blotting were used to detect inflammatory factors, gene expression, and protein phosphorylation, respectively. In TNF-α-stimulated adipocytes, PHPS1 reduced pro-inflammatory factors (IL-6: 62.35%, IL-1β: 54.10%, MCP-1: 49.19%), increased IL-10 (103.98%), and downregulated iNOS/COX-2 mRNA. In LPS-induced co-cultures, PHPS1 decreased IL-6 (68.73%), IL-1β (63.01%), MCP-1 (47.79%), upregulated IL-10 (167.49%), and inhibited iNOS/COX-2 mRNA. Mechanistically, PHPS1 suppressed NF-κB pathway phosphorylation (pp65, pIkkα) in both models, reversed LPS-induced pIRS-1 Ser307 phosphorylation (61.12% reduction), and upregulated GLUT-4 (198.0%). Thus, PHPS1 alleviates inflammation via NF-κB inhibition and improves insulin resistance by restoring the IRS-1/GLUT-4 axis, making it a potential candidate for insulin resistance-related metabolic diseases.","manuscriptTitle":"SHP2 inhibitor PHPS1 regulates macrophage-adipocyte interaction to alleviate inflammation and adipocyte insulin resistance, via NF-κB suppression and IRS-1/GLUT-4 restoration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-24 18:44:52","doi":"10.21203/rs.3.rs-8391295/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-12-22T23:57:28+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-22T14:52:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-18T12:40:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Human Cell","date":"2025-12-18T00:30:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"07d93ff4-184a-424e-91ec-934c9ed31334","owner":[],"postedDate":"December 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-24T18:44:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-24 18:44:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8391295","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8391295","identity":"rs-8391295","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}