Collection protocol for human pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation. Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing β-cells followed by lower proportions of glucagon-containing α-cells and somatostatin-containing δ-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes. Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.     

The ductal origin of structural and functional heterogeneity between pancreatic islets

Islets form in the pancreas after the first endocrine cells have arisen as either single cells or small cell clusters in the epithelial cords. These cords constitute the developing pancreas in one of its earliest recognizable stages. Islet formation begins at the time the cords transform into a branching ductal system, continues while the ductal system expands, and finally stops before the exocrine tissue of ducts and acini reaches its final expansion. Thus, islets continuously arise from founder cells located in the branching and ramifying ducts. Islets arising from proximal duct cells locate between the exocrine lobules, develop strong autonomic and sensory innervations, and pass their blood to efferent veins (insulo-venous efferent system). Islets arising from cells of more distal ducts locate within the exocrine lobules, respond to nerve impulses ending at neighbouring blood vessels, and pass their blood to the surrounding acini (insulo-acinar portal system). Consequently, the section of the ductal system from which an islet arises determines to a large extent its future neighbouring tissue, architecture, properties, and functions. We note that islets interlobular in position are frequently found in rodents (rats and mice), whereas intralobularly-located, peripheral duct islets prevail in humans and cattle. Also, we expound on bovine foetal Laguesse islets as a prominent foetal type of type 1 interlobular neuro-insular complexes, similar to neuro-insular associations frequently found in rodents. Finally, we consider the probable physiological and pathophysiological implications of the different islet positions within and between species.     

Human-IAPP disrupts the autophagy/lysosomal pathway in pancreatic β-cells: protective role of p62-positive cytoplasmic inclusions

In type II diabetes (T2DM), there is a deficit in β-cells, increased β-cell apoptosis and formation of intracellular membrane-permeant oligomers of islet amyloid polypeptide (IAPP). Human-IAPP (h-IAPP) is an amyloidogenic protein co-expressed with insulin by β-cells. IAPP expression is increased with obesity, the major risk factor for T2DM. In this study we report that increased expression of human-IAPP led to impaired autophagy, due at least in part to the disruption of lysosome-dependant degradation. This action of IAPP to alter lysosomal clearance in vivo depends on its propensity to form toxic oligomers and is independent of the confounding effect of hyperglycemia. We report that the scaffold protein p62 that delivers polyubiquitinated proteins to autophagy may have a protective role against human-IAPP-induced apoptosis, apparently by sequestrating protein targets for degradation. Finally, we found that inhibition of lysosomal degradation increases vulnerability of β-cells to h-IAPP-induced toxicity and, conversely, stimulation of autophagy protects β-cells from h-IAPP-induced apoptosis. Collectively, these data imply an important role for the p62/autophagy/lysosomal degradation system in protection against toxic oligomer-induced apoptosis.