The role of GRP94 chaperone in proinsulin processing

MARZEC, MT; PEDERSEN, S; CHETA, O; PERONE, MJ; GHIASI, SM; MANDRUP-POULSEN, T

Munich

Congreso; 52nd European Association for the Study of Diabetes (EASD); 2016

Background and aims: Insulin availability is a determinant of glucose homeostasis and insufficient insulin production is the underlying cause of diabetes. Insulin, like any other secreted protein, needs folding in the endoplasmic reticulum (ER) to attain its proper three-dimensional structure. Folding failure leads to insulin degradation and beta cell stress. While insulin biosynthesis and secretion have been extensively studied, surprisingly little is known about the specific folding requirements of proinsulin and the necessary ER chaperones. Glucose Regulated Protein 94 (GRP94), a resident chaperone of the ER, is essential in folding of insulin-like growth factors 1 and 2 (IGF). Insulin and IGFs are evolutionarily linked and share structural homology. Recent publications indicate that GRP94 is part of a complex required for preproinsulin transport to the ER and subsequent insulin secretion. Thus, we hypothesize that proinsulin folding is crucially dependent on the function of GRP94. Aim 1: To define functional impact of GRP94 on insulin production and secretion. Aim 2: To outline structural characteristics of the GRP94/proinsulin complex. Materials and methods: Cellular models of beta cells, freshly isolated rat and human islets will be subjected to shRNA and pharmacological treatment to diminish expression and activity of GRP94. Results are presented as mean ± s.e.m. with statistical differences determined using two-tailed Student?s t test or ANOVA. Results: 1. >95% GRP94 knock-down (KD) in cell lines and dispersed human islets was achieved by shRNA targeting GRP94. These models were tested for glucose induced insulin secretion. Control cells increased (in response to 20mM glucose concentration) 3-10 fold their insulin secretion while GRP94 KD cells failed to do so. 2. Similar results were obtained when cells, rat and human islets were pre-treated with GRP94 specific inhibitor. 3. We analyzed the ability of GRP94 KD cells to restore insulin content after a period of forced secretion (high to low glucose concentration shift). Insulin content was inversely correlated to the degree of KD of GRP94 with control cells able to build up its insulin content 4 fold within 1h while GRP94 KD cells showed no such an increase. 4. Analysis of GRINCH cells by confocal microscopy revealed: a) In 3mM glucose conditions insulin secretory granules aligned close to the cellular membrane in control cells, while GRP94 KD cells failed to produce similar pattern exhibiting distorted cellular morphology with expanded ER and accumulation of proinsulin. b) In 20mM glucose, control cells secreted membrane localized granules while GRP94 KD cells failed in that respect. In addition, control cells showed strong proinsulin signal resembling Golgi apparatus while GRP94 KD cells showed much more dispersed localization. 5. Analysis by SDS-PAGE for the formation of configurational isomers of proinsulin under reducing and non-reducing conditions discovered in GRP94 KD a) increased presence of disulfide isomers of proinsulin; b) lower amount of pro- and mature insulin (Insulin gene transcription was only minimally lower in GRP94 KD). 6. Co-immunoprecipitation experiment shows GFP-tagged proinsulin precipitating GRP94. Conclusion: Our preliminary results indicate that GRP94 is necessary for proinsulin processing into mature insulin and that it directly interacts with it or at least is a part of proinsulin folding complex in ER.