Plasma fetuin-A/α2-HS-glycoprotein correlates negatively with inflammatory cytokines, chemokines and activation biomarkers in individuals with type-2 diabetes

Abstract

Background: Fetuin-A/AHSH is a novel hepatokine that acts as a vascular calcification inhibitor and as an endogenous TLR-4 ligand. Fetuin-A may act as a positive or negative acute phase protein (APP) in disease conditions. The relationship between circulatory fetuin-A and inflammatory biomarkers in type-2 diabetes (T2D) remains controversial. Therefore, the purpose of this study was to determine the plasma fetuin-A levels in 53 T2D (BMI = 29.7 ± 4.5 kg/m²) and 72 non-diabetic individuals (BMI = 28.2 ± 5.8 kg/m²) using premixed 38-plex MAP human cytokine/chemokine magnetic bead immunoassays and the data (mean ± SEM) were statistically analyzed to determine Pearson's correlation (r) between fetuin-A and detected analytes; P-values ≤0.05 were considered significant.

Results: The data show that plasma fetuin-A levels were comparable in both groups (P = 0.27) and in T2D individuals, fetuin-A associated negatively (P ≤ 0.05) with a large number of proinflammatory cytokines/chemokines and activation biomarkers including TNF-α, IFN-α2, IFN-γ, IL-1α, IL-1β, IL-1RA, IL-3, IL-4, IL-7, IL-9, IL-12p40/p70, IL-15, CCL-2, CCL-4, CCL-11, CCL-22, CXCL-8, CX3CL-1, EFF-2, EGF, G-CSF, GM-CSF, GRO, sCD40L, and VEGF. In non-diabetics, fetuin-A also correlated positively with certain T_H2 cytokines (IL-5, IL-13) and chemokines (CCL-3, CCL-5, CCL-7). Notably, in vitro fetuin-A production was significantly suppressed in HepG2 cells treated with TNF-α, IL-1β, and IFN-γ which supported the clinical findings of a negative association between fetuin A and inflammatory mediators.

Conclusions: The negative association between circulatory fetuin-A and systemic inflammatory mediators in T2D patients suggests that plasma fetuin-A may have predictive significance as a negative APP in metabolic disease.

Keywords: Chemokines; Fetuin-A; Metabolic inflammation; Proinflammatory cytokines; Type-2 diabetes; α2-HS-glycoprotein.

Fig. 1

Fetuin A in supernatants of HepG2 cell cultures treated with proinflammatory cytokines. Human hepatocellular carcinoma HepG2 cells were cultured at 37 °C with 5 % CO₂ in high-glucose DMEM medium containing 10 % fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin in 6-well plates at a density of 0.25 × 10⁶ cells/mL until about 70 % confluence and old medium was replaced with fresh medium. Cell monolayers were then treated with rhTNF-α (50 ng/mL), rhIL-1β (10 ng/mL), rhIFN-γ (50 ng/mL), rhMCP-1 (40 ng/mL), and rhIL-6 (100 ng/mL) and incubated at 37 °C for 24 h. Cell supernatants were collected and fetuin-A levels were measured using sandwich high-sensitivity ELISA (Human fetuin-A PicoKine^TM ELISA kit, Boster Biological Technology, USA) following the manufacturer’s instructions as described in Patients and Methods. Fetuin-A production (mean ± SEM) was found to be significantly suppressed in HepG2 cells treated with TNF-α (82.05 ± 1.16 ng/mL, P = 0.002), IL-1β (82.73 ± 1.45 ng/mL, P = 0.003), and IFN-γ (85.70 ± 1.93 ng/mL, P = 0.02) as compared with untreated control (95.73 ± 1.43 ng/mL). However, fetuin-A production in cells treated with MCP-1 (84.74 ± 5.02 ng/mL, P = 0.10) and IL-6 (91.66 ± 2.55 ng/mL, P = 0.24) differed non-sidnificantly from control. The representative data from three independent determinations are shown