Beta cell identity changes with mild hyperglycemia: Implications for function, growth, and vulnerability.

OBJECTIVE: As diabetes develops, marked reductions of insulin secretion are associated with very modest elevations of glucose. We wondered if these glucose changes disrupt beta cell differentiation enough to account for the altered function. METHODS: Rats were subjected to 90% partial pancreatectomies and those with only mild glucose elevations 4 weeks or 10 weeks after surgery had major alterations of gene expression in their islets as determined by RNAseq. RESULTS: Changes associated with glucose toxicity demonstrated that many of the critical genes responsible for insulin secretion were downregulated while the expression of normally suppressed genes increased. Also, there were marked changes in genes associated with replication, aging, senescence, stress, inflammation, and increased expression of genes controlling both class I and II MHC antigens. CONCLUSIONS: These findings suggest that mild glucose elevations in the early stages of diabetes lead to phenotypic changes that adversely affect beta cell function, growth, and vulnerability.

Generation of Pancreatic Ductal Organoids and Whole-Mount Immunostaining of Intact Organoids.

Traditionally, studies of cells and tissues have been performed on isolated primary cells or immortalized cell lines by culturing them in 2D culture dishes or flasks. However, a caveat regarding 2D culture is that the cells poorly recapitulate their in vivo counterparts, mainly due to a lack of 3D cell-cell and cell-extracellular matrix interactions. In recent years, the development of in vitro organoids as 3D culture has gained substantial attention as a model to study different tissues. In adults, pancreatic ductal cells are considered as a source of stem or progenitor cells, so developing new methods to study ductal cells would be useful. Here, we provide a protocol to isolate mouse pancreatic ductal cells and a cost-effective protocol to generate 3D organoid structures from such ductal cells. Additionally, we have devised a protocol for immunostaining of intact whole organoids without sectioning. © 2018 by John Wiley & Sons, Inc.

Heterogeneity of SOX9 and HNF1ß in Pancreatic Ducts Is Dynamic.

Pancreatic duct epithelial cells have been suggested as a source of progenitors for pancreatic growth and regeneration. However, genetic lineage-tracing experiments with pancreatic duct-specific Cre expression have given conflicting results. Using immunofluorescence and flow cytometry, we show heterogeneous expression of both HNF1ß and SOX9 in adult human and murine ductal epithelium. Their expression was dynamic and diminished significantly after induced replication. Purified pancreatic duct cells formed organoid structures in 3D culture, and heterogeneity of expression of Hnf1ß and Sox9 was maintained even after passaging. Using antibodies against a second cell surface molecule CD51 (human) or CD24 (mouse), we could isolate living subpopulations of duct cells enriched for high or low expression of HNF1ß and SOX9. Only the CD24high (Hnfßhigh/Sox9high) subpopulation was able to form organoids.

ß Cell Aging Markers Have Heterogeneous Distribution and Are Induced by Insulin Resistance.

We hypothesized that the known heterogeneity of pancreatic ß cells was due to subpopulations of ß cells at different stages of their life cycle with different functional capacities and that further changes occur with metabolic stress and aging. We identified new markers of aging in ß cells, including IGF1R. In ß cells IGF1R expression correlated with age, dysfunction, and expression of known age markers p16ink4a, p53BP1, and senescence-associated ß-galactosidase. The new markers showed striking heterogeneity both within and between islets in both mouse and human pancreas. Acute induction of insulin resistance with an insulin receptor antagonist or chronic ER stress resulted in increased expression of aging markers, providing insight into how metabolic stress might accelerate dysfunction and decline of ß cells. These novel findings about ß cell and islet heterogeneity, and how they change with age, open up an entirely new set of questions about the pathogenesis of type 2 diabetes.

Human Islet Morphology Revisited: Human and Rodent Islets Are Not So Different After All.

There has been great interest in understanding how human islets differ from rodent islets. Three major issues about human islet morphology have remained controversial over recent decades: 1) the proportion of the islet made up of ß-cells; 2) whether islet cell types have a non-random mantle-core pattern, as seen in rodents, or are randomly scattered throughout the islet; 3) the relation of the different cell types to the blood vessels within the islet, which has implications for intraislet function. We re-examined these issues on immunostained sections of non-diabetic adult human pancreas. The composition of the islets can vary by the analysis method (number vs volume) and by the sampling of islets by size. The majority of adult human islets have clear, non-random clustering of ß-cells and blood vessels that penetrate into the ß-cell cores. We conclude that although there is far more variability in islet composition both within each human pancreas and among different human pancreas than in rodent pancreas, the islet architecture is not so different between the species. The intrapancreatic variability raises important questions about how islets evolve and function throughout life and how this might relate to the pathogenesis of diabetes.

Reduced Ki67 Staining in the Postmortem State Calls Into Question Past Conclusions About the Lack of Turnover of Adult Human ß-Cells.

Some report that adult human ß-cells do not replicate, but we postulate this assumption is erroneous due a postmortem decline in replication markers such as Ki67. Our earlier report showed that Ki67-marked ß-cells were rarely found in human cadaveric pancreases but were in the range of 0.2-0.5% in human islets transplanted into mice. This study subjected 4-week-old mice to autopsy conditions that typically occur with humans. Mice were killed, left at room temperature for 3 h, and then placed at 4°C for 3, 9, or 21 h. There was a rapid marked fall in Ki67 staining of ß-cells compared with those fixed immediately. Values at death were 6.9 ± 0.9% (n = 6) after a 24-h fast, 4.1 ± 0.9% (n = 6) at 3 h room temperature, 2.7 ± 0.7% (n = 5) at 6 h, 1.6 ± 0.6% (n = 5) at 12 h, and 2.9 ± 0.8% (n = 5) at 24 h. Similar postmortem conditions in newborn pigs resulted in very similar declines in Ki67 staining of their ß-cells. These data support the hypothesis that conclusions on the lack of replication of adult human ß-cells are incorrect and suggest that adult human ß-cells replicate at a low but quantitatively meaningful rate.