Islet cell stress induced by insulin-degrading enzyme deficiency promotes regeneration and protection from autoimmune diabetes.

Tuning of protein homeostasis through mobilization of the unfolded protein response (UPR) is key to the capacity of pancreatic beta cells to cope with variable demand for insulin. Here, we asked how insulin-degrading enzyme (IDE) affects beta cell adaptation to metabolic and immune stress. C57BL/6 and autoimmune non-obese diabetic (NOD) mice lacking IDE were exposed to proteotoxic, metabolic, and immune stress. IDE deficiency induced a low-level UPR with islet hypertrophy at the steady state, rapamycin-sensitive beta cell proliferation enhanced by proteotoxic stress, and beta cell decompensation upon high-fat feeding. IDE deficiency also enhanced the UPR triggered by proteotoxic stress in human EndoC-ßH1 cells. In Ide-/- NOD mice, islet inflammation specifically induced regenerating islet-derived protein 2, a protein attenuating autoimmune inflammation. These findings establish a role of IDE in islet cell protein homeostasis, demonstrate how its absence induces metabolic decompensation despite beta cell proliferation, and UPR-independent islet regeneration in the presence of inflammation.

Pancreatic islet cell stress induced by insulin-degrading enzyme deficiency promotes islet regeneration and protection from autoimmune diabetes.

Appropriate tuning of protein homeostasis through mobilization of the unfolded protein response (UPR) is key to the capacity of pancreatic beta cells to cope with highly variable demand for insulin synthesis. An efficient UPR ensures a sufficient beta cell mass and secretory output but can also affect beta cell resilience to autoimmune aggression. The factors regulating protein homeostasis in the face of metabolic and immune challenges are insufficiently understood. We examined beta cell adaptation to stress in mice deficient for insulin-degrading enzyme (IDE), a ubiquitous protease with high affinity for insulin and genetic association with type 2 diabetes. IDE deficiency induced a low-level UPR in both C57BL/6 and autoimmune non-obese diabetic (NOD) mice, associated with rapamycin-sensitive beta cell proliferation strongly enhanced by proteotoxic stress. Moreover, in NOD mice, IDE deficiency protected from spontaneous diabetes and triggered an additional independent pathway, conditional on the presence of islet inflammation but inhibited by proteotoxic stress, highlighted by strong upregulation of regenerating islet-derived protein 2, a protein attenuating autoimmune inflammation. Our findings establish a key role of IDE in islet cell protein homeostasis, identify a link between low-level UPR and proliferation, and reveal an UPR-independent anti-inflammatory islet cell response uncovered in the absence of IDE of potential interest in autoimmune diabetes.

Sensitivity of mass spectrometry analysis depends on the shape of the filtration unit used for filter aided sample preparation (FASP).

Efficient protein solubilization using detergents is required for in-depth proteome analysis, but successful LC-MS/MS analysis greatly depends on proper detergents removal. A commonly used sample processing method is the filter-aided sample preparation (FASP), which allows protein digestion and detergent removal on the same filtration device. Many optimizations of the FASP protocol have been published, but there is no information on the influence of the filtration unit typology on the detergents removal. The aim of this study was to compare the performance of conic and flat bottom filtration units in terms of number of proteins identified by LC-MS/MS. We have analyzed 1, 10 and 100 µg of total cell lysate prepared using lysis buffer with different SDS concentrations. We compared the FASP protocol using conic and flat bottom filtration units to ethanol precipitation method. Subsequently, we applied our most performant protocol to single murine pancreatic islet, and identified up to 2463 protein using FASP versus 1169 proteins using ethanol precipitation. We conclude that FASP performance depends strongly on the filter shape: flat bottom devices are better suited for low-protein samples, as they allow better SDS removal leading to the identification of greater number of proteins.

Establishment and characterization of a panel of human uveal melanoma xenografts derived from primary and/or metastatic tumors.

PURPOSE: Uveal melanoma is the most common primary intraocular malignant tumor in adults and is defined by a poor natural outcome, as 50% of patients die from metastases. The aim of this study was to develop and characterize a panel of human uveal melanoma xenografts transplanted into immunodeficient mice. EXPERIMENTAL DESIGN: Ninety tumor specimens were grafted into severe combined immunodeficient mice, and 25 transplantable xenografts were then established (28%). Relationship between tumor graft and clinical, biological, and therapeutic features of the patients included were investigated. Characterization of 16 xenografts included histology, molecular analyses by immunohistochemistry, genetic alteration analysis (single-nucleotide polymorphism), and specific tumor antigen expression by quantitative reverse transcription-PCR. Pharmacologic characterization (chemosensitivity) was also done in four models using two drugs, temozolomide and fotemustine, currently used in the clinical management of uveal melanoma. RESULTS: Take rate of human uveal melanoma was 28% (25 of 90). Tumor take was independent of size, histologic parameters, or chromosome 3 monosomy but was significantly higher in metastatic tumors. Interestingly, in vivo tumor growth was prognostic for a lower metastasis-free survival in patients with primary tumors. A high concordance between the patients' tumors and their corresponding xenografts was found for all parameters tested (histology, genetic profile, and tumor antigen expression). Finally, the four xenografts studied displayed different response profiles to chemotherapeutic agents. CONCLUSIONS: Based on these results, this panel of 16 uveal melanoma xenografts represents a useful preclinical tool for both pharmacologic and biological assessments.