BACKGROUND

Diabetes mellitus is characterized by peripheral insulin resistance, hyperglycemia and defective insulin secretion. Insulin producing pancreatic β-cells are equipped with a highly developed endoplasmic reticulum (ER) and thus are affected by ER stress under hyperglycemic conditions. We have previously studied the influence of high glucose on cultured β-cells in vitro. Proteomic analysis revealed a number of proteins involved in glucose toxicity, while further biochemical analysis identified the endoplasmic reticulum protein ERp46 as a molecule with a possible role in insulin production at the post-translational level. In addition, the involvement of incretin hormone glucagon-like peptide 1 (GLP-1) in diabetes proposes that incretin-mimetic compounds may be among the optimal choices in future therapeutic interventions; therefore their effects on various aspects of the pathogenesis of diabetes mellitus should be explored in detail. Based on the above, we examined the possible involvement of ERp46 in insulin production and the effect of the GLP-1 analogue liraglutide on the expression of ERp46 in vitro, in β-cells cultured under high glucose conditions and in vivo, in the mouse db/db diabetic model, where pronounced hyperglycemia is a key characteristic.

RESULTS

Confocal microscopy revealed areas of co-localization of ERp46 and pro-insulin in pancreatic islets. In order to explore the possible interaction between ERp46 and insulin immunoprecipitation was used. In extracts from cultured β-cells, antibodies against pro-insulin co-precipitated ERp46 and antibodies against ERp46 co-precipitated pro-insulin, as shown by Western blotting. Furthermore, data from a proximity ligation assay positioned these two molecules closer than 30nm in distance. When pancreatic β-cells were cultured in high glucose conditions they exhibited a decrease in ERp46 expression, while treatment with the GLP-1 analogue liraglutide restored ERp46 levels, leading to a significant increase of ERp46 in comparison to hyperglycemic conditions. In the diabetic mouse model db(-)/db, ERp46 expression was reduced in pancreatic islets, as documented by morphological and biochemical techniques. This decrease was abolished after treatment with the GLP-1 analogue in a dose-dependent manner. In an attempt to understand the underlying mechanism, we examined the sequence of the promoter of ERp46 and found consensus motifs that can be recognized by transcription factors ATF6 and XBP1. Subsequently, we performed chromatin immunoprecipitation assay and demonstrated that treatment of β-TC-6 cells with 25mmol/L glucose decreases gradually the binding enrichment of ATF6 and XBP1 in ERp46 gene promoter.

CONCLUSIONS

We propose that since ERp46 is a member of the disulfide isomerases family, it is likely to play a key role in insulin biosynthesis and its reduction under high glucose conditions may be a novel contributor to the glucotoxicity of β-cells. In addition, the GLP-1 analogue liraglutide seems to interfere in this process and may exert its beneficial effects in diabetes by affecting insulin production via restoration of ERp46 expression.