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

View Article: PubMed Central - PubMed

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 TH2 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.

Electronic supplementary material: The online version of this article (doi:10.1186/s12865-016-0171-y) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

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 PicoKineTM 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
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Fig1: 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 PicoKineTM 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

Mentions: Since metformin and angiotensin receptors blockers have been reported to have lowering effects on circulating fetuin-A levels [7–9], therefore, possible confounding (suppressive) effects of therapy in our study cohort may not be ruled out. We asked if the signature inflammatory cytokines, related with T2D pathogenesis, alone were able to suppress the production of fetuin-A in an in vitro HepG2 cell culture model. To address this, we treated HepG2 cell cultures (at 70–80 % confluence) with the typical proinflammatory cytokines and chemokine that are known to be upregulated in metabolic disease such as obesity and T2D and measured the expression of fetuin-A in supernatants for comparison with untreated controls. The treatments with these cytokines/chemokine were carried out using standard concentrations as available from the literature and these cytokines/chemokine concentrations also activated the respective signaling pathways in HepG2 cells (data not shown). Our data show that fetuin-A production was significantly reduced (P ≤ 0.05) in HepG2 cell cultures at 24 h following treatments with TNF-α, IL-1β, and IFN-γ while a non-significant suppression (P > 0.05) was observed after HepG2 cell treatment with MCP-1 and IL-6 as compared with controls (Fig. 1). In addition, time course experiments were also performed in which both the proinflammatory (IFN-γ and TNF-α) and antiinflammatory (IL-4 and IL-10) cytokines were used to treat HepG2 cells and fetuin-A was measured in culture supernatants at 1, 6, 12, 24, and 48 h. These data show (Additional file 1: Figure S1) that TNF-α treatment resulted in fetuin-A suppression at as early as 12 h and both IFN-γ and TNF-α suppressed fetuin-A at 24 h and 48 h while on the other hand, IL-10 induced the expression of fetuin A at these time points. As expected, no differences among treatments were observed at earlier (1 h and 6 h) time points since they may not represent the optimal time period required for fetuin-A synthesis and extracellular expression following the cytokine treatments used.Fig. 1


Plasma fetuin-A/ α 2-HS-glycoprotein correlates negatively with inflammatory cytokines, chemokines and activation biomarkers in individuals with type-2 diabetes
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 PicoKineTM 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
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Related In: Results  -  Collection

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Fig1: 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 PicoKineTM 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
Mentions: Since metformin and angiotensin receptors blockers have been reported to have lowering effects on circulating fetuin-A levels [7–9], therefore, possible confounding (suppressive) effects of therapy in our study cohort may not be ruled out. We asked if the signature inflammatory cytokines, related with T2D pathogenesis, alone were able to suppress the production of fetuin-A in an in vitro HepG2 cell culture model. To address this, we treated HepG2 cell cultures (at 70–80 % confluence) with the typical proinflammatory cytokines and chemokine that are known to be upregulated in metabolic disease such as obesity and T2D and measured the expression of fetuin-A in supernatants for comparison with untreated controls. The treatments with these cytokines/chemokine were carried out using standard concentrations as available from the literature and these cytokines/chemokine concentrations also activated the respective signaling pathways in HepG2 cells (data not shown). Our data show that fetuin-A production was significantly reduced (P ≤ 0.05) in HepG2 cell cultures at 24 h following treatments with TNF-α, IL-1β, and IFN-γ while a non-significant suppression (P > 0.05) was observed after HepG2 cell treatment with MCP-1 and IL-6 as compared with controls (Fig. 1). In addition, time course experiments were also performed in which both the proinflammatory (IFN-γ and TNF-α) and antiinflammatory (IL-4 and IL-10) cytokines were used to treat HepG2 cells and fetuin-A was measured in culture supernatants at 1, 6, 12, 24, and 48 h. These data show (Additional file 1: Figure S1) that TNF-α treatment resulted in fetuin-A suppression at as early as 12 h and both IFN-γ and TNF-α suppressed fetuin-A at 24 h and 48 h while on the other hand, IL-10 induced the expression of fetuin A at these time points. As expected, no differences among treatments were observed at earlier (1 h and 6 h) time points since they may not represent the optimal time period required for fetuin-A synthesis and extracellular expression following the cytokine treatments used.Fig. 1

View Article: PubMed Central - PubMed

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 TH2 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.

Electronic supplementary material: The online version of this article (doi:10.1186/s12865-016-0171-y) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus