Hydroxynonenal causes Langerhans cell degeneration in the pancreas of Japanese macaque monkeys

Abstract

AbstractBackgroundFor their functions of insulin biosynthesis and glucose- and fatty acid- induced insulin secretion, the Langerhans β-cells require an intracellular milieu rich in oxygen. This requirement makes β-cells, with their constitutively low antioxidative defense, susceptible to the oxidative stress. Although much progress has been made in identifying its molecular basis in the experimental systems, whether the oxidative stress due to excessive fatty acids plays a crucial role in the Langerhans degeneration in primates is still debated.MethodsFocusing on Hsp70.1, which has dual functions as a molecular chaperone and lysosomal stabilizer, the mechanism of lipotoxicity to the Langerhans islet cells was studied using Japanese macaque monkeys (Macaca fuscata) after the consecutive injections of the lipid peroxidation product hydroxynonenal. Based on the ‘calpain-cathepsin hypothesis’ of ischemic neuronal death formulated in 1998, calpain activation, Hsp70.1 cleavage, and lysosomal integrity were studied by immunofluorescence histochemistry, electron microscopy and Western blotting.ResultsLight microscopy showed higher vacuole formation in the treated islet cells than in the control cells. Electron microscopy showed that vacuolar changes that were identified as enlarged rough endoplasmic reticula occurred mainly in β-cells followed by δ-cells. Intriguingly, both cell types showed a marked decrease in insulin and somatostatin granules. Furthermore, they exhibited marked increases in peroxisomes, autophagosomes/autolysosomes, lysosomal and peroxisomal membrane rupture/permeabilization, and mitochondrial degeneration. Disrupted peroxisomes were often localized in the close vicinity of degenerating mitochondria or autolysosomes. Immunofluorescence histochemical analysis showed an increased colocalization of activated μ-calpain and Hsp70.1 with the extralysosomal release of cathepsin B. Western blotting showed increases in μ-calpain activation, Hsp70.1 cleavage, and hydroxynonenal receptor GPR109A expression.ConclusionsTaken together, these data implicate hydroxynonenal in both the carbonylation of Hsp70.1 and the activation of μ-calpain. The calpain-mediated cleavage of the carbonyl group on Hsp70.1 after the hydroxynonenal-mediated carbonylation of Hsp70.1, may cause lysosomal membrane rupture/permeabilization. The low defense of primate Langerhans cells against exogenous hydroxynonenal and peroxisomally-generated hydrogen peroxide, was presumably overwhelmed to facilitate cell degeneration.