H2S, Endoplasmic Reticulum Stress, and Apoptosis of Insulin-secreting Beta Cells

Guangdong Yang,Wei Yang,Lingyun Wu,Rui Wang

doi:10.1074/jbc.m700605200

Guangdong Yang, Wei Yang + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m700605200

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Abstract

Cystathionine gamma-lyase (CSE) is a key enzyme in the trans-sulfuration pathway, which uses L-cysteine to produce hydrogen sulfide (H2S). Functional changes of pancreatic beta cells induced by endogenous H2S have been reported, but the effect of the CSE/H2S system on pancreatic beta cell survival has not been known. In this study, we demonstrate that H2Sat physiologically relevant concentrations induced apoptosis of INS-1E cells, an insulin-secreting beta cell line. Transfection of INS-1E cells with a recombinant defective adenovirus containing the CSE gene (Ad-CSE) resulted in a significant increase in CSE expression and H2S production. Ad-CSE transfection also stimulated apoptosis. The other two end products of CSE-catalyzed enzymatic reaction, ammonium and pyruvate, had no effects on INS-1E cell apoptosis, indicating that overexpression of CSE may stimulate INS-1E cell apoptosis via increased endogenous production of H2S. Both exogenous H2S (100 microM) and Ad-CSE transfection inhibited ERK1/2 but activated p38 MAPK. Interestingly, BiP and CHOP, two indicators of endoplasmic reticulum (ER) stress, were up-regulated in H2S-and CSE-mediated apoptosis in INS-1E cells. After suppressing CHOP mRNA expression, H2S-induced apoptosis of INS-1E cells was significantly decreased. Inhibition of p38 MAPK, but not of ERK1/2, inhibited the expression of BiP and CHOP and decreased H2S-stimulated apoptosis, suggesting that p38 MAPK activation functions upstream of ER stress to initiate H2S-induced apoptosis. It is concluded that H2S induces apoptosis of insulin-secreting beta cells by enhancing ER stress via p38 MAPK activation. Our findings may help unmask a novel role of CSE/H2S system in regulating pancreatic functions under physiological condition and in diabetes.

Highlights

Cystathionine ␥-lyase (CSE,5 EC 4.4.1.1) is a key pyridoxal 5Ј-phosphate-dependent enzyme in the trans-sulfuration pathway, which uses L-cysteine to produce hydrogen sulfide (H2S), a novel and important gasotransmitter [1,2,3]
Significant increase in CSE proteins was detected after transfection with adenovirus containing the CSE gene (Ad-CSE) (Fig. 1B)
These results indicate that p38 mitogen-activated protein kinase (MAPK) kinase activation functions upstream of endoplasmic reticulum (ER) stress induced by H2S

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—INS1-E cells derived from a rat insulinoma Cells were first incubated in the serum-free medium for 12 h and 10% serum added together with different treatments. To measure H2S concentration, 200 ␮l of culture media from each treatment were collected and added to microcentrifuge tubes containing zinc acetate (1% w/v, 600 ␮l) to trap H2S. H2S in the sampled culture media interacts with N,N-dimethyl-p-phenylenediamine sulfate to form methylene blue, and the absorbance of the resulting solution was determined at 670 nm [17]. After different treatments the cells were collected and homogenized in 20 mM potassium phosphate buffer (pH 7.8), and the homogenate was centrifuged at 12,000 ϫ g for 20 min at 4 °C. Neg-siRNA at different concentrations and transfection reagent complex were added to the cells in serum-free medium for 4 h. NegsiRNA was used to transfect INS-1E cells

Determination of mRNA Level by

RESULTS

DISCUSSION