Organ donor pancreases for the study of human islet cell histology and pathophysiology: a precious and valuable resource

Direct in vivo assessment of pancreatic islet-cells for the study of the pathophysiology of diabetes in humans is hampered by anatomical and technological hurdles. To date, most of the information that has been generated is derived from histological studies performed on pancreatic tissue from autopsy, surgery, in vivo biopsy or organ donation. Each approach has its advantages and disadvantages (as summarised in this commentary); however, in this edition of Diabetologia, Kusmartseva et al (https://doi.org/10.1007/s00125-017-4494-x) provide further evidence to support the use of organ donor pancreases for the study of human diabetes. They show that length of terminal hospitalisation of organ donors prior to death does not seem to influence the frequency of inflammatory cells infiltrating the pancreas and the replication of beta cells. These findings are reassuring, demonstrating the reliability of this precious and valuable resource for human islet cells research

Pancreatic Beta Cell Identity in Humans and the Role of Type 2 Diabetes

Pancreatic beta cells uniquely synthetize and release insulin. Specific molecular, functional as well as ultrastructural traits characterize their insulin secretion properties and survival phenotype. In this review we focus on human islet/beta cells, and describe the changes that occur in type 2 diabetes and could play roles in the disease as well as represent possible targets for therapeutical interventions. These include transcription factors, molecules involved in glucose metabolism and insulin granule handling. Quantitative and qualitative insulin release patterns and their changes in type 2 diabetes are also associated with ultrastructural features involving the insulin granules, the mitochondria, and the endoplasmic reticulum

Metabolic response of Insulinoma 1E cells to glucose stimulation studied by fluorescence lifetime imaging

A cascade of highly regulated biochemical processes connects glucose stimulation to insulin secretion in specialized cells of mammalian pancreas, the β-cells. Given the importance of this process for systemic glucose homeostasis, noninvasive and fast strategies capable to monitor the response to glucose in living cells are highly desirable. Here, we use the phasor-based approach to Fluorescence Lifetime IMaging (FLIM) microscopy to quantify the ratio between protein-bound and free Nicotinamide adenine dinucleotide (phosphate) species in their reduced form (NAD(P)H), and the Insulinoma cell line INS-1E as a β-like cellular model. Phasor-FLIM analysis shows that the bound/free ratio of NAD(P)H species increases upon pulsed glucose stimulation. Such response is impaired by 48-hours preincubation of cells under hyperglycemic conditions. Phasor-FLIM concomitantly monitors the appearance of long-lifetime species (LLS) as characteristic products of hyperglycemia-induced oxidative stress

MicroRNA expression analysis of in vitro dedifferentiated human pancreatic islet cells reveals the activation of the pluripotency-related microRNA cluster miR-302s

β-cell dedifferentiation has been recently suggested as an additional mechanism contributing to type-1 and to type-2 diabetes pathogenesis. Moreover, several studies demonstrated that in vitro culture of native human pancreatic islets derived from non-diabetic donors resulted in the generation of an undifferentiated cell population. Additional evidence from in vitro human β-cell lineage tracing experiments, demonstrated that dedifferentiated cells derive from β-cells, thus representing a potential in vitro model of β-cell dedifferentiation. Here, we report the microRNA expression profiles analysis of in vitro dedifferentiated islet cells in comparison to mature human native pancreatic islets. We identified 13 microRNAs upregulated and 110 downregulated in islet cells upon in vitro dedifferentiation. Interestingly, among upregulated microRNAs, we observed the activation of microRNA miR-302s cluster, previously defined as pluripotency-associated. Bioinformatic analysis indicated that miR-302s are predicted to target several genes involved in the control of β-cell/epithelial phenotype maintenance; accordingly, such genes were downregulated upon human islet in vitro dedifferentiation. Moreover, we uncovered that cell-cell contacts are needed to maintain low/null expression levels of miR-302. In conclusion, we showed that miR-302 microRNA cluster genes are involved in in vitro dedifferentiation of human pancreatic islet cells and inhibits the expression of multiple genes involved in the maintenance of β-cell mature phenotype

Response of young and adult birds to the same environmental variables and different spatial scales during post breeding period

Context: How do young birds achieve spatial knowledge about the environment during the initial stages of their life? They may follow adults, so gaining social information and learning; alternatively, young birds may acquire knowledge of the environment themselves by experiencing habitat and landscape features. If learning is at least partially independent of adults then young birds should respond to landscape composition at finer spatial scale than adults, who possess knowledge over a larger area. Objectives: We studied the responses of juvenile, immature and adult Caspian Gull Larus cachinnans to the same habitat and landscape variables, but at several spatial scales (ranging from 2.5 to 15\ua0km), during post-breeding period. Methods: We surveyed 61 fish ponds (foraging patches) in southern Poland and counted Caspian gulls. Results: Juvenile birds responded at finer spatial scales to the factors than did adults. Immature birds showed complicated, intermediate responses to spatial scale. The abundance of juvenile birds was mostly correlated with the landscape composition (positively with the cover of corridors and negatively with barriers). Adult abundance was positively related to foraging patch quality (fish stock), which clearly required previous spatial experience of the environment. The abundance of all age classes were moderately correlated with each other indicating that social behaviour may also contribute to the learning of the environment. Conclusions: This study shows that as birds mature, they respond differently to components of their environment at different spatial scales. This has considerable ecological consequences for their distribution across environments

Angiotensin I-converting enzyme type 2 expression is increased in pancreatic islets of type 2 diabetic donors

Aims: Angiotensin I-converting enzyme type 2 (ACE2), a pivotal SARS-CoV-2 receptor, has been shown to be expressed in multiple cells, including human pancreatic beta-cells. A putative bidirectional relationship between SARS-CoV-2 infection and diabetes has been suggested, confirming the hypothesis that viral infection in beta-cells may lead to new-onset diabetes or worse glycometabolic control in diabetic patients. However, whether ACE2 expression levels are altered in beta-cells of diabetic patients has not yet been investigated. Here, we aimed to elucidate the in situ expression pattern of ACE2 in Type 2 diabetes (T2D) with respect to non-diabetic donors which may account for a higher susceptibility to SARS-CoV-2 infection in beta-cells.Material and Methods: Angiotensin I-converting enzyme type 2 immunofluorescence analysis using two antibodies alongside insulin staining was performed on formalin-fixed paraffin embedded pancreatic sections obtained from n = 20 T2D and n = 20 non-diabetic (ND) multiorgan donors. Intensity and colocalisation analyses were performed on a total of 1082 pancreatic islets. Macrophage detection was performed using anti-CD68 immunohistochemistry on serial sections from the same donors.Results: Using two different antibodies, ACE2 expression was confirmed in beta-cells and in pancreas microvasculature. Angiotensin I-converting enzyme type 2 expression was increased in pancreatic islets of T2D donors in comparison to ND controls alongside with a higher colocalisation rate between ACE2 and insulin using both anti-ACE2 antibodies. CD68(+) cells tended to be increased in T2D pancreata, in line with higher ACE2 expression observed in serial sections.Conclusions: Higher ACE2 expression in T2D islets might increase their susceptibility to SARS-CoV-2 infection during COVID-19 in T2D patients, thus worsening glycometabolic outcomes and disease severity

IFN-α induces a preferential long-lasting expression of MHC class I in human pancreatic beta cells

Aims/hypothesis IFN-α, a cytokine expressed in human islets from individuals affected by type 1 diabetes, plays a key role in the pathogenesis of diabetes by upregulating inflammation, endoplasmic reticulum (ER) stress and MHC class I overexpression, three hallmarks of islet histology in early type 1 diabetes. We tested whether expression of these mediators of beta cell loss is reversible upon IFN-α withdrawal or IFN-α pathway inhibition. Methods IFN-α-induced MHC class I overexpression, ER stress and inflammation were evaluated by flow cytometry, immunofluorescence and real-time PCR in human EndoC-βH1 cells or human islets exposed to IFN-α with or without the presence of Janus kinase (JAK) inhibitors. Protein expression was evaluated by western blot. Results IFN-α-induced expression of inflammatory and ER stress markers returned to baseline after 24–48 h following cytokine removal. In contrast, MHC class I overexpression at the cell surface persisted for at least 7 days. Treatment with JAK inhibitors, when added with IFN-α, prevented MHC class I overexpression, but when added 24 h after IFN-α exposure these inhibitors failed to accelerate MHC class I return to baseline. Conclusions/interpretation IFN-α mediates a long-lasting and preferential MHC class I overexpression in human beta cells, which is not affected by the subsequent addition of JAK inhibitors. These observations suggest that IFN-α-stimulated long-lasting MHC class I expression may amplify beta cell antigen presentation during the early phase of type 1 diabetes and that IFN-α inhibitors might need to be used at very early stages of the disease to be effective

Co-localization of acinar markers and insulin in pancreatic cells of subjects with type 2 diabetes

To search for clues suggesting that beta cells may generate by transdifferentiation in humans, we assessed the presence of cells double positive for exocrine (amylase, carboxypeptidase A) and endocrine (insulin) markers in the pancreas of non-diabetic individuals (ND) and patients with type 2 diabetes (T2D). Samples from twelve ND and twelve matched T2D multiorgan donors were studied by electron microscopy, including amylase and insulin immunogold labeling; carboxypeptidase A immunofluorescence light microscopy assessment was also performed. In the pancreas from four T2D donors, cells containing both zymogen-like and insulin-like granules were observed, scattered in the exocrine compartment. Nature of granules was confirmed by immunogold labeling for amylase and insulin. Double positive cells ranged from 0.82 to 1.74 per mm2, corresponding to 0.26±0.045% of the counted exocrine cells. Intriguingly, cells of the innate immune systems (mast cells and/or macrophages) were adjacent to 33.3±13.6% of these hybrid cells. No cells showing co-localization of amylase and insulin were found in ND samples by electron microscopy. Similarly, cells containing both carboxypeptidase A and insulin were more frequently observed in the diabetic pancreata. These results demonstrate more abundant presence of cells containing both acinar markers and insulin in the pancreas of T2D subjects, which suggests possible conversion from one cellular type to the other and specific association with the diseased condition

Histopathology and ex vivo insulin secretion of pancreatic islets in gestational diabetes: A case report

Gestational diabetes (GD) results from insufficient endogenous insulin supply. No information is available on features of islet cells in human GD. Herein, we describe several properties of islets from a woman with GD. Immunohistochemical stainings and EM analyses were performed on pancreatic samples. Islet isolation was achieved by enzymatic dissociation and density gradient centrifugation. Ex vivo insulin secretion was studied in response to fuel secretagogues. Control islets were obtained from matched non-pregnant, non-diabetic women. Total insulin positive area was lower in GD, mainly due to the presence of smaller islets. β-cell apoptosis and the presence of Ki67 positive islet cells were similar in GD and controls, whereas the amount of insulin positive cells in or close to the ducts was decreased in GD. Ex vivo insulin secretion did not differ between GD and non-pregnant, non-diabetic islets. These findings suggest that in this case of human GD there might mainly be a defect of β-cell amount, not due to increased apoptosis, but possibly to insufficient regeneration