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. 2007 Jul;293(1):E337-46.
doi: 10.1152/ajpendo.00718.2006. Epub 2007 Apr 10.

High glucose induces IL-1beta expression in human monocytes: mechanistic insights

Mohan R Dasu  1 , Sridevi Devaraj, Ishwarlal Jialal
Affiliations

High glucose induces IL-1beta expression in human monocytes: mechanistic insights

Mohan R Dasu et al. Am J Physiol Endocrinol Metab. 2007 Jul.

Abstract

Previously, IL-1beta secretion from Type 2 diabetic patients has been shown to be increased compared with controls. In this study, we aimed to delineate the mechanism of IL-1beta induction under high-glucose (HG) conditions in human monocytes. THP-1 cells cultured in normal glucose were treated with increasing concentrations of d-glucose (10-25 mM) for 6-72 h. IL-1beta and IL-1 receptor antagonist levels were measured by ELISA and Western blots, whereas mRNA was quantitated by RT-PCR. Specific inhibitors and small interfering RNAs of PKC, p38, ERK1/2, NF-kappaB, and NADPH oxidase were used to determine the mediators in parallel experiments under HG conditions. IL-1beta-secreted protein, cellular protein, and mRNA increase under HG conditions is time and dose dependent, with maximum increase at 15 mM (48 h; P < 0.05). IL-1 receptor antagonist release was time and dose dependent, similar to IL-1beta expression pattern; however, the molar ratio of IL-1beta to IL-1RA was increased. Data from inhibitor and small interfering RNA experiments indicate that IL-1beta release under HG is mediated by PKC-alpha, via phosphorylation of p38 MAPK, and ERK1/2 leading to NF-kappaB activation, resulting in increased mRNA and protein for IL-1beta. At the same time, it appears that NADPH oxidase via p47phox activates NF-kappaB, resulting in increased IL-1beta secretion. Data suggest that, under HG conditions, monocytes release significantly higher amounts of IL-1beta through multiple mechanisms, further compounding the disease progression. Targeting signaling pathways mediating IL-1beta release could result in the amelioration of inflammation and possibly diabetic vasculopathies.

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Figures

Fig. 1
Fig. 1
A: dose-dependent effect of high glucose on IL-1β release from THP-1 cells. Cells were challenged with increasing glucose concentration (10–25 mmol/l) for 48 h, and IL-1β release was measured as described in MATERIALS AND METHODS. As a control, 9.5 mmol/l mannitol was added with normal glucose (NG) in simultaneous wells (n = 5). *P < 0.05 vs. 5 mM glucose. B: time-dependent effect of high glucose on IL-1β release from THP-1 cells. Cells were challenged with 15 mmol/l glucose for 6–72 h, and IL-1β release was measured as described in MATERIALS AND METHODS. As a control, 9.5 mmol/l mannitol was added with NG in simultaneous wells (n = 5). *P < 0.05 vs. 6 h. C: effect of high glucose on cellular IL-1β protein. Total cell lysates of THP-1 cells were run on Tris-glycine gels and then blotted for IL-1β protein (n = 3).
Fig. 2
Fig. 2
A: dose-dependent effect of high glucose on IL-1β mRNA from THP-1 cells. Cells were challenged with increasing glucose concentration (10–25 mmol/l) for 48 h, and real-time RT-PCR analysis was performed to detect IL-1β and GAPDH mRNA, using sequence-specific primers as described in MATERIALS AND METHODS. As a control, 9.5 mmol/l mannitol was added with NG in simultaneous wells (n = 5). *P < 0.05 vs. 5 mM glucose. B: time-dependent effect of high glucose on IL-1β mRNA from THP-1 cells. Cells were challenged with 15 mmol/l glucose for 6–72 h, and real-time RT-PCR analysis was performed to detect IL-1β and GAPDH mRNA, using sequence-specific primers as described in MATERIALS AND METHODS. As a control, 9.5 mmol/l mannitol was added with NG in simultaneous wells (n = 5). *P < 0.05 vs. 5 mM glucose.
Fig. 3
Fig. 3
A: dose-dependent effect of high glucose on IL-1 receptor antagonist (IL-1ra) release from THP-1 cells. Cells were challenged with increasing glucose concentration (10–25 mmol/l) for 48 h, and IL-1ra release was measured as described in MATERIALS AND METHODS. As a control, 9.5 mmol/l mannitol was added with NG in simultaneous wells (n = 5). *P < 0.05 vs. 5 mM glucose B: time-dependent effect of high glucose on IL-1ra release from THP-1 cells. Cells were challenged with 15 mmol/l glucose for 6–72 h, and IL-1ra release was measured as described in MATERIALS AND METHODS. As a control, 9.5 mmol/l mannitol was added with NG in simultaneous wells (n = 5). *P < 0.05 vs. 6 h. C: effect of high glucose on IL-1β-to-IL-1ra molar ratio in THP-1 cells. Cells incubated with high-glucose concentration show a higher IL-1β-to-IL-1ra molar ratio than those incubated with NG (n = 5).
Fig. 4
Fig. 4
A: effect of PKC isoform inhibitors on IL-1β release from THP-1 cells under high glucose. Cells were cultured as described in Fig. 1 except for exposure to calphostin C (10 μmol/l), 2,2′,3,3′,4,4′-hexahydroxy-1,1′-biphenyl-6,6′-dimethanol dimethyl ether (HBDDE; 50 μmol/l), and LY-379196 (30 nmol/l) for 2 h followed by 15 mmol/l glucose challenge for 48 h. IL-1β release was then measured as described in MATERIALS AND METHODS (n = 5). *P < 0.05 vs. control (15 mmol/l). B: effect of small interfering RNAs (siRNAs) to PKC isoforms on IL-1β release from THP-1 cells under high glucose. Cells were transfected with PKC-α, PKC-β, and scrambled (S) siRNAs. After 48 h, cells were challenged with 15 mmol/l glucose. IL-1β release was then measured as described in MATERIALS AND METHODS (n = 5). *P < 0.05 vs. control (scrambled siRNA + 15 mmol/l glucose).
Fig. 5
Fig. 5
A and B: effect of high glucose on p38 MAPK and ERK phosphorylation in THP-1 cells. After cells were cultured with high glucose, cells were lysed, and cell lysates were blotted for total and phosphorylated p38 (pp38) and total and phosphorylated ERK (pERK) (n = 3). C and D: effect of inhibitors (SB-203580 and U-0126) on p38 and ERK phosphorylation in THP-1 cells. After cells were cultured with inhibitors and high glucose, cells were lysed, and cell lysates were blotted for total and phosphorylated p38 and total and phosphorylated ERK (n = 3).
Fig. 6
Fig. 6
A: effect of MAPK inhibitors on IL-1β release from THP-1 cells under high glucose. Cells were cultured as described in Fig. 1 except for exposure to SB-203580 (5 μmol/l), U-0126 (10 μmol/l), and LJNKI1 (JNK; 5 μmol/l) for 2 h followed by 15 mmol/l glucose challenge for 48 h. IL-1β release was then measured as described in MATERIALS AND METHODS (n =5). *P < 0.05 vs. control (15 mmol/l). B: effect of siRNAs to p38 and ERK1/2 on IL-1β release from THP-1 cells under high glucose. Cells were transfected with p38, ERK1/2, and scrambled siRNAs. After 48 h, cells were challenged with 15 mmol/l glucose. IL-1β release was then measured as described in MATERIALS AND METHODS (n = 5). *P < 0.05 vs. control (scrambled siRNA + 15 mmol/l glucose).
Fig. 7
Fig. 7
A: effect of NF-κB inhibitors on IL-1β release from THP-1 cells under high glucose. Cells were cultured as described in Fig. 1 except for exposure to caffeic acid phenethyl ester (CAPE; 5 μmol/l) and BAY-11-7085 (10 μmol/l) for 2 h followed by 15 mmol/l glucose challenge for 48 h. IL-1β release was then measured as described in MATERIALS AND METHODS (n = 5). *P < 0.05 vs. control (15 mmol/l). B: effect of siRNAs to NF-κB on IL-1β release from THP-1 cells under high glucose. Cells were transfected with NF-κB and scrambled siRNAs. After 48 h, cells were challenged with 15 mmol/l glucose. IL-1β release was then measured as described in MATERIALS AND METHODS (n = 5). *P < 0.05 vs. control (scrambled siRNA + 15 mmol/l glucose).
Fig. 8
Fig. 8
A: effect of NADPH oxidase inhibitors on IL-1β release from THP-1 cells under high glucose. Cells were cultured as described in Fig. 1 except for exposure to apocyanin (Apo; 30 μmol/l) and diphenyleneiodonium chloride (DPI; 10 μmol/l) for 2 h followed by 15 mmol/l glucose challenge for 48 h. IL-1β release was then measured as described in MATERIALS AND METHODS (n = 5). *P < 0.05 vs. control (15 mmol/l). B: effect of siRNA to p47phox on IL-1β release from THP-1 cells under high glucose. Cells were transfected with p47phox and scrambled siRNAs. After 48 h, cells were challenged with 15 mmol/l glucose. IL-1β release was then measured as described in MATERIALS AND METHODS (n = 5). *P < 0.05 vs. control (scrambled siRNA + 15 mmol/l glucose).
Fig. 9
Fig. 9
PKC-α is proximal mediator in IL-1β release from THP-1 cells under high glucose. A: cells were transfected with PKC-α, PKC-β, and scrambled siRNAs. After 48 h, cells were challenged with 15 mmol/l glucose. Cells were then lysed, and lysates were blotted for phosphorylated p38 and ERK (n = 3). B: cells were transfected with p38, ERK, p47phox, and scrambled siRNAs. After 48 h, cells were challenged with 15 mmol/l glucose. Cell lysates were then isolated and used for measuring PKC activity as described in MATERIALS AND METHODS (n = 5). *P < 0.05 vs. control (scrambled siRNA + 15 mmol/l glucose). C: cells were transfected with p38, ERK, p47phox, and scrambled siRNAs. After 48 h, cells were challenged with 15 mmol/l glucose. Nuclear extracts were then isolated and used for measuring NF-κB p65-dependent activity, as described in MATERIALS AND METHODS (n = 5). *P < 0.05 vs. control (scrambled siRNA + 15 mmol/l glucose).
Fig. 10
Fig. 10
Schema illustrating the mechanism of IL-1β release under high-glucose conditions in THP-1 cells. Inflammatory effects of high glucose are mediated through NADPH oxidase and activation of PKC-α. Activation of PKC-α results in the phosphorylation of p38 MAPK and ERK1/2 and culmination in NF-κB activation, leading to increased IL-1β secretion in monocytes. ROS, reactive oxygen species.
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