The biological actions of insulin are transmitted through a receptor tyrosine kinase, the insulin receptor, which upon ligand binding undergoes autophosphorylation and recruits the insulin receptor substrate (IRS) family of docking proteins, principally IRS-1 and IRS-2 [1]. Tyrosine-phosphorylated IRS proteins activate phosphatidylinositol 3-kinase (PI3K), generating the lipid second messenger PIP3, which recruits and activates the serine/threonine kinase Akt (protein kinase B) [2]. Akt occupies a central nodal position in insulin signalling: it drives GLUT4 translocation and glucose uptake via phosphorylation of AS160, promotes glycogen synthesis by inhibiting glycogen synthase kinase-3, and suppresses hepatic gluconeogenesis via exclusion of FOXO1 from the nucleus [1,3]. The fidelity of signal flux through this receptor–IRS-1–PI3K–Akt axis is therefore the molecular determinant of cellular insulin sensitivity, and its impairment is now recognized as the unifying biochemical lesion of insulin resistance [4]. Insulin resistance does not arise from a single defect but from the convergence of multiple molecular perturbations that attenuate this signalling axis. Central among these is inhibitory serine/threonine phosphorylation of IRS-1: whereas tyrosine phosphorylation propagates the insulin signal, phosphorylation at specific serine residues — exemplified by Ser307 in rodent IRS-1 — impairs its coupling to the receptor and PI3K and promotes its proteasomal degradation [5,6]. This inhibitory phosphorylation is driven by stress- and inflammation-activated kinases. The demonstration that adipose tissue TNF-α over-expression contributes directly to obesity-linked insulin resistance [7], and that TNF-α induces inhibitory IRS-1 serine phosphorylation [8], established a direct biochemical link between inflammation and impaired insulin signalling. Subsequent work identified c-Jun N-terminal kinase (JNK) as playing a central role in obesity-associated insulin resistance [9], while genetic and pharmacological disruption of IκB kinase-β (IKKβ)/nuclear factor-κB (NF-κB) signalling was shown to reverse diet-induced insulin resistance [10], establishing both stress-kinase pathways as validated therapeutic and mechanistic targets. Beyond direct serine kinase activity, two further negative regulators are consistently implicated. The suppressor of cytokine signalling protein SOCS3, induced by inflammatory cytokine signalling, binds the insulin receptor and IRS-1 and targets them for ubiquitin-mediated proteasomal degradation [11]. Protein tyrosine phosphatase 1B (PTP1B), localized to the endoplasmic reticulum, directly dephosphorylates the activated insulin receptor and IRS-1; mice lacking PTP1B show increased insulin sensitivity and resistance to diet-induced obesity, establishing it as a validated node for therapeutic modulation [12]. The balance between activating tyrosine phosphorylation and inhibitory serine phosphorylation/dephosphorylation at IRS-1 is thus the proximate determinant of downstream PI3K–Akt signal strength [1,4]. A persistent obstacle in translating this mechanistic literature — derived predominantly from rodent models and from liver, muscle, and adipose tissue studies — into human clinical research has been the difficulty of obtaining serial tissue samples from classical insulin target organs. Peripheral blood mononuclear cells (PBMCs) express the core insulin-signalling machinery, including the insulin receptor, IRS-1, PI3K, and Akt, and have been proposed as an accessible, minimally invasive surrogate tissue capable of reflecting systemic insulin signalling status in human subjects [13]. However, relatively few studies have applied a comprehensive, multi-level approach — combining transcript, protein, and functional ex vivo analysis — to characterize this cascade in PBMCs of insulin-resistant individuals in a hospital-based Indian population, where the burden of insulin resistance and type 2 diabetes is exceptionally high and the underlying metabolic phenotype may differ from Western cohorts. This study was therefore designed to characterize, in PBMCs of insulin-resistant subjects and matched controls attending a tertiary care teaching centre, the expression and activation status of the insulin receptor–IRS-1–PI3K–Akt–GLUT4 axis, the inflammatory/stress kinases JNK and NF-κB, and the negative regulators SOCS3 and PTP1B; to assess functional insulin responsiveness by ex vivo stimulation; and to determine which molecular and clinical variables independently predict the severity of insulin resistance.

Study design and ethics: This case-control study was conducted in the Department of Physiology at a tertiary care teaching hospital, approved by the Institutional Ethics Committee, and conducted per the Declaration of Helsinki and ICMR National Ethical Guidelines (2017), with written informed consent obtained from all participants. Participants: Sixty insulin-resistant subjects (HOMA-IR ≥2.5, aged 18–60 years) and 60 age- and sex-matched insulin-sensitive controls were recruited from the outpatient department and screening clinics, using the inclusion/exclusion criteria described in the companion clinical analysis of this cohort (established diabetes on insulin/insulin-sensitizers, pregnancy, acute/chronic infection, malignancy, autoimmune disease, secondary causes of insulin resistance, and drugs affecting insulin sensitivity within 3 months were exclusionary). PBMC isolation: PBMCs were isolated from EDTA-anticoagulated venous blood by density-gradient centrifugation using Ficoll-Paque (400×g, 30 min, room temperature, no brake), washed twice in PBS, and either used immediately for ex vivo stimulation or cryopreserved (10% DMSO in FBS, liquid nitrogen) for batch processing. Cell viability was confirmed by trypan blue exclusion (>90% required for inclusion). Ex vivo insulin stimulation: Freshly isolated PBMCs were serum-starved for 2 hours in serum-free RPMI-1640, then stimulated with 100 nM insulin for 10–15 minutes at 37°C alongside an unstimulated control aliquot, to assess IRS-1 tyrosine phosphorylation and Akt Ser473 phosphorylation response by Western blot. RNA isolation and quantitative real-time PCR: Total RNA was isolated from PBMCs using TRIzol reagent or a column-based kit, with quantity/purity assessed spectrophotometrically (A260/A280 1.8–2.0) and integrity confirmed by gel electrophoresis. cDNA was synthesized from 500 ng–1 µg RNA. Quantitative real-time PCR was performed using SYBR Green chemistry for IRS-1, PI3K (p85α), GLUT4, TNF-α, IL-6, SOCS3, and PTP1B, normalized to β-actin/GAPDH/18S rRNA, with relative expression calculated by the 2^(−ΔΔCt) method in accordance with MIQE guidelines. Protein extraction and Western blot: Protein was extracted using RIPA lysis buffer with protease/phosphatase inhibitors; concentration was determined by the BCA method. Equal protein amounts (20–50 µg) were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies against total and phosphorylated IRS-1 (Ser307/Tyr612), PI3K p85, Akt (Ser473), GSK-3β, JNK (Thr183/Tyr185), IKKβ, NF-κB p65 (Ser536), PTP1B, and SOCS3, followed by HRP-conjugated secondary antibody and chemiluminescent detection. Densitometry was performed using ImageJ, with phosphoproteins normalized to total protein and total proteins to β-actin/GAPDH. Statistical analysis: Data were analysed using SPSS v26.0/GraphPad Prism. Continuous variables were tested for normality (Shapiro-Wilk) and compared by independent-samples t-test/Mann-Whitney U test or paired t-test/Wilcoxon signed-rank test (basal vs stimulated). Correlations between molecular markers and clinical/biochemical indices were assessed by Pearson's/Spearman's coefficients. Multivariable linear regression, adjusted for age and sex, identified independent predictors of HOMA-IR. A two-tailed p<0.05 was considered statistically significant.

Table 1. mRNA Expression of Insulin-Signalling Genes in PBMCs (qRT-PCR) Gene Control (n=60) Fold Change Insulin-Resistant (n=60) Fold Change p-value Direction IRS-1 1.00±0.18 0.52±0.14 <0.001 ↓ Down PI3K (p85α) 1.00±0.21 0.61±0.16 <0.001 ↓ Down GLUT4 1.00±0.16 0.48±0.12 <0.001 ↓ Down SOCS3 1.00±0.22 2.86±0.64 <0.001 ↑ Up PTP1B 1.00±0.19 2.42±0.58 <0.001 ↑ Up TNF-α 1.00±0.20 3.14±0.72 <0.001 ↑ Up Transcripts of the proximal signalling components IRS-1, PI3K, and GLUT4 were significantly downregulated, while the negative regulators SOCS3 and PTP1B, along with TNF-α, were significantly upregulated in insulin-resistant subjects. This transcriptional signature reproduces the classical molecular paradigm of insulin resistance in an accessible, circulating cell compartment. Table 2. Protein Expression by Western Blot (PBMCs) Protein (Relative Density, A.U.) Control (n=60) Insulin-Resistant (n=60) p-value p-IRS-1(Tyr612) / Total IRS-1 0.82±0.12 0.34±0.09 <0.001 p-Akt(Ser473) / Total Akt 0.76±0.11 0.31±0.08 <0.001 p-JNK(Thr183/Tyr185) / Total JNK 0.28±0.07 0.74±0.15 <0.001 p-NF-κB p65(Ser536) / Total p65 0.24±0.06 0.68±0.14 <0.001 PTP1B (relative density) 0.42±0.09 0.96±0.18 <0.001 SOCS3 (relative density) 0.38±0.08 0.88±0.17 <0.001 Insulin-resistant subjects showed markedly reduced activating phosphorylation of IRS-1 (Tyr612) and Akt (Ser473), with concurrently elevated phosphorylation of JNK and NF-κB p65 and elevated PTP1B/SOCS3 protein — providing protein-level confirmation of the transcriptional findings and direct evidence of inflammatory/stress-kinase pathway activation. Table 3. Ex Vivo Insulin-Stimulated PBMC Signalling Response Condition Control (n=60) p-Akt/Akt (A.U.) Insulin-Resistant (n=60) p-Akt/Akt (A.U.) p-value Basal (unstimulated) 0.31±0.07 0.29±0.08 0.184 (NS) Insulin-stimulated (100 nM, 15 min) 0.79±0.13 0.46±0.10 <0.001 Fold-change (stimulated/basal) 2.6±0.4 1.6±0.3 <0.001 Basal p-Akt/Akt did not differ between groups, but the increment in Akt phosphorylation following acute insulin stimulation was markedly blunted in insulin-resistant subjects (1.6-fold vs 2.6-fold), directly demonstrating a functional defect in insulin responsiveness at the cellular signalling level. Table 4. Correlation of Signalling Markers with Clinical and Biochemical Indices Variable Pair r p-value HOMA-IR vs p-Akt/Akt ratio −0.71 <0.001 HOMA-IR vs SOCS3 mRNA +0.64 <0.001 HOMA-IR vs PTP1B protein +0.68 <0.001 TNF-α vs p-JNK/JNK +0.62 <0.001 HOMA-IR correlated strongly and inversely with PBMC p-Akt/Akt, and positively with SOCS3 mRNA and PTP1B protein, indicating that whole-body insulin resistance severity tracks closely with proximal PBMC signalling status. TNF-α correlated positively with JNK activation, linking the inflammatory profile to stress-kinase engagement. Table 5. Multivariable Regression Analysis: Independent Predictors of Insulin Resistance Predictor Variable Standardized β 95% CI p-value BMI 0.21 0.06 to 0.36 0.006 Waist circumference 0.18 0.02 to 0.34 0.029 TNF-α 0.24 0.09 to 0.39 0.002 Adiponectin −0.27 −0.42 to −0.12 <0.001 SOCS3 mRNA 0.19 0.03 to 0.35 0.021 PTP1B protein 0.23 0.07 to 0.39 0.005 Model R²=0.58, adjusted R²=0.55 (F-test p<0.001; adjusted for age and sex). BMI, waist circumference, TNF-α, adiponectin, SOCS3 mRNA, and PTP1B protein were independent predictors of HOMA-IR, with adiponectin and TNF-α showing the strongest associations — indicating that molecular signalling markers contribute explanatory power beyond anthropometric measures alone.

This study demonstrates that insulin-resistant subjects exhibit a coordinated, multi-level derangement of the insulin-signalling cascade in PBMCs, spanning transcript downregulation of proximal effectors, protein-level reduction in activating phosphorylation, upregulation of negative regulators, activation of inflammatory kinases, and a functionally demonstrable blunting of insulin-stimulated Akt activation — findings that together recapitulate, in an accessible surrogate tissue, the molecular paradigm of insulin resistance established chiefly in liver, muscle, and adipose tissue. The coordinated downregulation of IRS-1, PI3K, and GLUT4 transcripts, alongside upregulation of SOCS3 and PTP1B, is mechanistically coherent: SOCS3, induced by inflammatory cytokine signalling, binds IRS-1 and targets it for proteasomal degradation, directly reducing its transcript and protein stability [11], while PTP1B dephosphorylates the activated receptor and IRS-1, terminating signal transduction [12]. That both negative regulators were concordantly elevated at transcript and protein level (Tables 1 and 2) indicates that the defect in this cohort involves coordinated induction of multiple, mechanistically distinct inhibitory mechanisms rather than a single point of failure. The reduced p-IRS-1(Tyr612)/total IRS-1 ratio is explicable as the convergent consequence of increased inhibitory kinase activity, increased phosphatase-mediated dephosphorylation, and increased degradative turnover acting in concert — consistent with the paradigm first established by the demonstration that Ser307 phosphorylation of IRS-1 by JNK disrupts its coupling to the insulin receptor [5,6]. The elevated phosphorylation of JNK and NF-κB p65 observed at the protein level provides direct evidence that the stress- and inflammation-activated kinase pathways implicated by prior rodent and cell-culture work [7-10] are active in human PBMCs in vivo. JNK has been shown, using genetic knockout models, to play a central causal role in obesity-associated insulin resistance [9], and disruption of IKKβ/NF-κB signalling reverses diet- and obesity-induced insulin resistance in mice [10]; the concordant activation of both pathways in this human cohort strengthens the translational relevance of these previously largely rodent-derived mechanisms. The most functionally direct finding is the blunted increment in Akt phosphorylation following acute ex vivo insulin stimulation in insulin-resistant subjects, despite comparable basal phosphorylation between groups. This distinguishes a genuine impairment in dynamic insulin responsiveness — the functionally decisive parameter for glucose disposal in vivo — from a static difference in resting signalling tone, and directly validates PBMCs as a minimally invasive, functionally interrogable surrogate tissue for human insulin-signalling research, addressing a long-standing practical obstacle of serial tissue biopsy [13]. The strong inverse correlation between HOMA-IR and PBMC p-Akt/Akt (r=−0.71) is particularly notable given that HOMA-IR is derived exclusively from fasting glucose and insulin and contains no direct molecular information, lending substantial weight to the biological relevance of the PBMC signalling readout. The multivariable regression findings extend this interpretation: SOCS3 mRNA and PTP1B protein remained independent predictors of HOMA-IR after adjustment for BMI, waist circumference, TNF-α, and adiponectin, indicating that the molecular signalling derangements documented here contribute explanatory power to insulin resistance beyond what anthropometric and cytokine measures capture alone. The prominence of adiponectin and TNF-α as the strongest predictors in this model is consistent with their proposed roles as, respectively, a protective, insulin-sensitising adipokine and a primary driver of JNK/NF-κB pathway activation [7,10]. Limitations: The cross-sectional design precludes causal inference; PBMCs, while informative, are not classical insulin target tissues and their quantitative concordance with hepatic or muscle signalling was not directly assessed; and PBMC subpopulation heterogeneity was not resolved by cell-subtype-specific analysis.

Insulin-resistant subjects show a coordinated, multi-level derangement of the insulin-signalling cascade in PBMCs — encompassing transcriptional and protein-level suppression of IRS-1, PI3K, and GLUT4, upregulation of the negative regulators SOCS3 and PTP1B, activation of the inflammatory/stress kinases JNK and NF-κB, and a functionally demonstrable blunting of the acute insulin-stimulated Akt response — that correlates strongly with whole-body HOMA-IR and remains partly independent of anthropometric predictors. These findings support PBMC-based signalling assessment as a translational tool for mechanistic biomarker development and identify the adipokine-cytokine-kinase axis characterized here as a rational target for future therapeutic and preventive strategies in insulin resistance.

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