Dynamic Changes in Pancreatic Endocrine Cell Abundance, Distribution, and Function in Antigen-Induced and Spontaneous Autoimmune Diabetes

OBJECTIVE

Insulin deficiency in type 1 diabetes and in rodent autoimmune diabetes models is caused by β-cell–specific killing by autoreactive T-cells. Less is known about β-cell numbers and phenotype remaining at diabetes onset and the fate of other pancreatic endocrine cellular constituents.

RESEARCH DESIGN AND METHODS

We applied multicolor flow cytometry, confocal microscopy, and immunohistochemistry, supported by quantitative RT-PCR, to simultaneously track pancreatic endocrine cell frequencies and phenotypes during a T-cell–mediated β-cell–destructive process using two independent autoimmune diabetes models, an inducible autoantigen-specific model and the spontaneously diabetic NOD mouse.

RESULTS

The proportion of pancreatic insulin-positive β-cells to glucagon-positive α-cells was about 4:1 in nondiabetic mice. Islets isolated from newly diabetic mice exhibited the expected severe β-cell depletion accompanied by phenotypic β-cell changes (i.e., hypertrophy and degranulation), but they also revealed a substantial loss of α-cells, which was further confirmed by quantitative immunohistochemisty. While maintaining normal randomly timed serum glucagon levels, newly diabetic mice displayed an impaired glucagon secretory response to non–insulin-induced hypoglycemia.

CONCLUSIONS

Systematically applying multicolor flow cytometry and immunohistochemistry to track declining β-cell numbers in recently diabetic mice revealed an altered endocrine cell composition that is consistent with a prominent and unexpected islet α-cell loss. These alterations were observed in induced and spontaneous autoimmune diabetes models, became apparent at diabetes onset, and differed markedly within islets compared with sub–islet-sized endocrine cell clusters and among pancreatic lobes. We propose that these changes are adaptive in nature, possibly fueled by worsening glycemia and regenerative processes.Although much has been learned about β-cell development and β-cell biology and function in vitro (using isolated pancreatic islets), studies designed to examine β-cell phenotype in vivo have suffered from technical limitations. For instance, currently available techniques to quantify pancreatic β-cell mass rely on laborious histomorphometric techniques (1) or on assumptions that β-cell mass correlates with β-cell function (stimulated C-peptide release) (2) or total pancreatic insulin content (3,4). Furthermore, quantifying the different endocrine islet cellular constituents by staining for the hormones produced (i.e., glucagon by α-cells, insulin by β-cells, somatostatin by δ-cells, and pancreatic polypeptide by PP-cells) has to date been challenging, relying again mostly on histomorphometry, and automated image processing setups typically allow only single-parameter analysis (5,6).Although preparative fluorescence-activated cell sorting of islet β-cells has been attempted (7–10), wider application has been limited by the lack of islet endocrine cell surface markers and insufficient resolution by autofluorescence, especially in species other than rats (11). Fluorescence reagents with islet granule affinity to identify β-cells in both mice (12) and humans (13) have limited utility, presumably because β-cells are degranulated by hyperglycemia. Other methods, such as quantitative (q)RT-PCR and analytical multicolor/multiparameter flow cytometry, capable of precise phenotypic and functional assessment, have been hampered by both the notorious difficulty to reliably prepare pancreatic RNA (14) and the fact that the pancreas is a heterogeneous organ comprised of irregularly intermixed exocrine and endocrine tissues. Even so-called “purified” isolated pancreatic islets from naïve mice (or other mammals) represent a multitude of specialized cell types (15), which is further complicated in animals with autoimmune diabetes when abundant inflammatory cells invade the islets (16).Recognizing these limitations, we adapted flow cytometry techniques for pancreatic studies and, together with qRT-PCR, confocal immunofluorescence microscopy, and quantitative immunohistochemistry, characterized the pancreatic endocrine islet cell components in naïve and recently diabetic mice. We now report that pancreatic islets isolated from mice developing T-cell–mediated β-cell–specific autoimmune diabetes demonstrate an unexpected glucagon-positive α-cell loss roughly commensurate with the expected β-cell loss.