Autophagy is a major regulator of beta cell insulin homeostasis.

AIMS/HYPOTHESIS: We studied the role of protein degradation pathways in the regulation of insulin production and secretion and hypothesised that autophagy regulates proinsulin degradation, thereby modulating beta cell function. METHODS: Proinsulin localisation in autophagosomes was demonstrated by confocal and electron microscopy. Autophagy was inhibited by knockdown of autophagy-related (ATG) proteins and using the H(+)-ATPase inhibitor bafilomycin-A1. Proinsulin and insulin content and secretion were assessed in static incubations by ELISA and RIA. RESULTS: Confocal and electron microscopy showed proinsulin localised in autophagosomes and lysosomes. Beta-Atg7 (-/-) mice had proinsulin-containing sequestosome 1 (p62 [also known as SQSTM1])(+) aggregates in beta cells, indicating proinsulin is regulated by autophagy in vivo. Short-term bafilomycin-A1 treatment and ATG5/7 knockdown increased steady-state proinsulin and hormone precursor chromogranin A content. ATG5/7 knockdown also increased glucose- and non-fuel-stimulated insulin secretion. Finally, mutated forms of proinsulin that are irreparably misfolded and trapped in the endoplasmic reticulum are more resistant to degradation by autophagy. CONCLUSIONS/INTERPRETATION: In the beta cell, transport-competent secretory peptide precursors, including proinsulin, are regulated by autophagy, whereas efficient clearance of transport-incompetent mutated forms of proinsulin by alternative degradative pathways may be necessary to avoid beta cell proteotoxicity. Reduction of autophagic degradation of proinsulin increases its residency in the secretory pathway, followed by enhanced secretion in response to stimuli.

LGR5 and Nanog identify stem cell signature of pancreas beta cells which initiate pancreatic cancer.

Pancreas cancer, is the fourth leading cause of cancer death but its cell of origin is controversial. We compared the localization of stem cells in normal and cancerous pancreas using antibodies to the stem cell markers Nanog and LGR5. Here we show, for the first time, that LGR5 is expressed in normal pancreas, exclusively in the islets of Langerhans and it is co-localized, surprisingly, with Nanog and insulin in clusters of beta cells. In cancerous pancreas Nanog and LGR5 are expressed in the remaining islets and in all ductal cancer cells. We observed insulin staining among the ductal cancer cells, but not in metastases. This indicates that the islet's beta cells, expressing LGR5 and Nanog markers are the initiating cells of pancreas cancer, which migrated from the islets to form the ductal cancerous tissue, probably after mutation and de-differentiation. This discovery may facilitate treatment of this devastating cancer.

Inhibition of mTORC1 by ER stress impairs neonatal ß-cell expansion and predisposes to diabetes in the Akita mouse.

Unresolved ER stress followed by cell death is recognized as the main cause of a multitude of pathologies including neonatal diabetes. A systematic analysis of the mechanisms of ß-cell loss and dysfunction in Akita mice, in which a mutation in the proinsulin gene causes a severe form of permanent neonatal diabetes, showed no increase in ß-cell apoptosis throughout life. Surprisingly, we found that the main mechanism leading to ß-cell dysfunction is marked impairment of ß-cell growth during the early postnatal life due to transient inhibition of mTORC1, which governs postnatal ß-cell growth and differentiation. Importantly, restoration of mTORC1 activity in neonate ß-cells was sufficient to rescue postnatal ß-cell growth, and to improve diabetes. We propose a scenario for the development of permanent neonatal diabetes, possibly also common forms of diabetes, where early-life events inducing ER stress affect ß-cell mass expansion due to mTOR inhibition.

Modulation of c-kit expression in pancreatic adenocarcinoma: a novel stem cell marker responsible for the progression of the disease.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers because of late symptoms and resistance to chemotherapy and radiation therapy. We have investigated the appearance of c-kit, a stem cell marker, in both normal adult pancreatic tissue and in cancerous tissue. Apart from some very pale staining of islets of Langerhans, normal pancreas was devoid of staining with antibodies to c-kit. In contrast, in cancerous tissue that still preserves the overall integrity of the pancreatic tissue, there was a clear labeling in islets of Langerhans, which seemed to be co-localized with insulin containing ß cells. In other cases, where the pancreatic tissue was completely deteriorated, intensive labeling was clearly evident in remnants of both the exocrine and the endocrine tissues. The duct cells of the adenocarcinoma were moderately but clearly labeled with antibodies to c-kit. In contrast, in metastasis of PDAC, very intensive labeling of c-kit was evident. The location of KRAS, which is strongly associated with PDAC, was also analyzed at the initial stages of the disease, when islets of Langerhans still preserve their integrity to a large extent. KRAS was found exclusively in islets of Langerhans and overlapped in its location with insulin and c-kit expressing cells. It is suggested that the modulation of the expression of c-kit, visualized by antibodies to the oncogene molecule, may play an important role in the formation and progression of PDAC. The absence of c-kit in normal pancreas and its appearance in PDAC is probably due to a mutational event, which probably allows conversion of the ß cells into cancer stem cells (CSC). Co-expression of both c-kit and KRAS, typical markers for CSC with overlapping with insulin in islets of Langerhans, strongly support the notion that ß-cells play a central role in the development of PDAC. The use of specific drugs that can attenuate the kinase activity of c-kit or target KRAS expressing cancer cells should be tested in order to attenuate the progression of this lethal disease.