Lipopolysaccharides impair insulin gene expression in isolated islets of Langerhans via Toll-Like Receptor-4 and NF-κB signalling

Abstract

Background: Type 2 diabetes is characterized by pancreatic β-cell dysfunction and is associated with low-grade inflammation. Recent observations suggest that the signalling cascade activated by lipopolysaccharides (LPS) binding to Toll-Like Receptor 4 (TLR4) exerts deleterious effects on pancreatic β-cell function; however, the molecular mechanisms of these effects are incompletely understood. In this study, we tested the hypothesis that LPS alters insulin gene expression via TLR4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in islets.

Methodology/principal findings: A 24-h exposure of isolated human, rat and mouse islets of Langerhans to LPS dose-dependently reduced insulin gene expression. This was associated in mouse and rat islets with decreased mRNA expression of pancreas-duodenum homebox-1 (PDX-1) and mammalian homologue of avian MafA/l-Maf (MafA). Accordingly, LPS exposure also decreased glucose-induced insulin secretion. LPS repression of insulin, PDX-1 and MafA expression, as well as its inhibition of insulin secretion, were not observed in islets from TLR4-deficient mice. LPS inhibition of β-cell gene expression in rat islets was prevented by inhibition of the NF-κB pathway, but not the p38 mitogen-activated protein kinase (p38 MAPK) pathway.

Conclusions/significance: Our findings demonstrate that LPS inhibit β-cell gene expression in a TLR4-dependent manner and via NF-κB signaling in pancreatic islets, suggesting a novel mechanism by which the gut microbiota might affect pancreatic β-cell function.

Figure 1. Exposure to LPS dose-dependently represses insulin pre-mRNA expression in isolated rat and human islets.

Insulin pre-mRNA levels in response to increasing doses of LPS in isolated rat (A) and human (B) islets. Pre-mRNA levels were measured by RT-PCR and normalized to β-actin mRNA levels. Data are mean ± S.E.M. of 2–6 independent experiments; *p<0.05 vs 0 pg/mL.

Figure 2. Exposure to LPS decreases insulin, PDX-1 and MafA gene expression in isolated islets via TLR4.

(A) Insulin pre-mRNA expression (B) PDX-1 mRNA expression and (C) MafA mRNA expression in islets isolated from WT and TLR4-deficient mice exposed for 24 h to 2.8 (2.8 G) and 16.7 mM (16.7 G) glucose in the presence or absence of 0.5 mM palmitate (PA), 0.5 ng/mL IL-1β or 5 µg/mL LPS. mRNA levels were measured by RT-PCR and normalized to β-actin mRNA levels. Data are mean ± S.E.M. of 6 independent experiments; *p<0.05.

Figure 3. PDX-1 cellular localization is not altered in response to LPS.

HIT-T15 cells were transfected with a construct encoding a PDX-1-GFP fusion protein. PDX-1 localization (green) (A–F) was visualized by GFP fluorescence using a laser-scanning confocal microscope in cells cultured in 0.1 and 5 mM glucose with or without 0.5 mM palmitate or increasing doses of LPS. 4′,6-diamidino-2-phenylindole (DAPI) (blue) was used for nuclear staining (G–L). Images are representative of 3 replicate experiments.

Figure 4. Insulin secretion is reduced in response to LPS in WT but not TLR4-deficient mouse islets.

Insulin secretion (A), insulin content (B) and insulin secretion normalized by insulin content (C) as assessed in 1-h static incubations of isolated WT and TLR4-deficient islets at basal (2.8 mM) and stimulatory (16.7 mM) glucose following a 24-h exposure to 16.7 mM glucose in the presence or absence of 5 µg/mL LPS. Data are mean ± S.E.M. of 5 independent experiments; *p<0.05.

Figure 5. Inhibition of NF-κB, but not p38 MAPK, restores insulin, PDX-1, and MafA gene expression in islets exposed to LPS.

(A) Insulin pre-mRNA, (B) PDX-1 mRNA and (C) MafA mRNA expression in isolated rat islets exposed for 24 h to 16.7 mM (16.7 G) glucose in the presence or absence of 10 ng/mL LPS or 0.5 ng/mL IL-1β with or without SB202190 (10 µM) and IKK-2 Inh IV (10 µM). Data are mean ± S.E.M. of 3–4 independent experiments; *p<0.05.

Figure 6. Potential mechanism by which LPS repress insulin gene expression in isolated islets.

Exposure to LPS activates the NF-κB pathway in isolated islets and inhibits the expression of insulin, PDX-1 and MafA. The decrease in insulin expression might indirectly results from LPS inhibition of PDX-1 and MafA. NF-κB could also inhibit insulin gene expression by interacting with other proteins such as C/EBPβ and/or CREB, as observed in other cell types.