Pseudoislet Aggregation of Pancreatic ß-Cells Improves Glucose Stimulated Insulin Secretion by Altering Glucose Metabolism and Increasing ATP Production.

Appropriate glucose-stimulated insulin secretion (GSIS) by pancreatic ß-cells is an essential component of blood glucose homeostasis. Configuration of ß-cells as 3D pseudoislets (PI) improves the GSIS response compared to 2D monolayer (ML) culture. The aim of this study was to determine the underlying mechanisms. MIN6 ß-cells were grown as ML or PI for 5 days. Human islets were isolated from patients without diabetes. Function was assessed by GSIS and metabolic capacity using the Seahorse bioanalyser. Connexin 36 was downregulated using inducible shRNA. Culturing MIN6 as PI improved GSIS. MIN6 PI showed higher glucose-stimulated oxygen consumption (OCR) and extracellular acidification (ECAR) rates. Further analysis showed the higher ECAR was, at least in part, a consequence of increased glycolysis. Intact human islets also showed glucose-stimulated increases in both OCR and ECAR rates, although the latter was smaller in magnitude compared to MIN6 PI. The higher rates of glucose-stimulated ATP production in MIN6 PI were consistent with increased enzyme activity of key glycolytic and TCA cycle enzymes. There was no impact of connexin 36 knockdown on GSIS or ATP production. Configuration of ß-cells as PI improves GSIS by increasing the metabolic capacity of the cells, allowing higher ATP production in response to glucose.

Exendin-4 stimulates autophagy in pancreatic ß-cells via the RAPGEF/EPAC-Ca2+-PPP3/calcineurin-TFEB axis.

Macroautophagy/autophagy is critical for the regulation of pancreatic ß-cell mass and its deregulation has been implicated in the pathogenesis of type 2 diabetes (T2D). We have previously shown that treatment of pancreatic ß-cells with the GLP1R (glucagon like peptide 1 receptor) agonist exendin-4 stimulates autophagic flux in a setting of chronic nutrient excess. The aim of this study was to identify the underlying pathways contributing to enhanced autophagic flux.Pancreatic ß-cells (INS-1E),mouse and human islets were treated with glucolipotoxic stress (0.5 mM palmitate and 25 mM glucose) in the presence of exendin-4. Consistent with our previous work, exendin-4 stimulated autophagic flux. Using chemical inhibitors and siRNA knockdown, we identified RAPGEF4/EPAC2 (Rap guanine nucleotide exchange factor 4) and downstream calcium signaling to be essential for regulation of autophagic flux by exendin-4. This pathway was independent of AMPK and MTOR signaling. Further analysis identified PPP3/calcineurin and its downstream regulator TFEB (transcription factor EB) as key proteins mediating exendin-4 induced autophagy. Importantly, inhibition of this pathway prevented exendin-4-mediated cell survival and overexpression of TFEB mimicked the cell protective effects of exendin-4 in INS-1E and human islets. Moreover, treatment of db/db mice with exendin-4 for 21 days increased the expression of lysosomal markers within the pancreatic islets. Collectively our data identify the RAPGEF4/EPAC2-calcium-PPP3/calcineurin-TFEB axis as a key mediator of autophagic flux, lysosomal function and cell survival in pancreatic ß-cells. Pharmacological modulation of this axis may offer a novel therapeutic target for the treatment of T2D.Abbreviations: AKT1/protein kinase B: AKT serine/threonine kinase 1; AMPK: 5' AMP-activated protein kinase; CAMKK: calcium/calmodulin-dependent protein kinase kinase; cAMP: cyclic adenosine monophosphate; CASP3: caspase 3; CREB: cAMP response element-binding protein; CTSD: cathepsin D; Ex4: exendin-4(1-39); GLP-1: glucagon like peptide 1; GLP1R: glucagon like peptide 1 receptor; GLT: glucolipotoxicity; INS: insulin; MTOR: mechanistic target of rapamycin kinase; NFAT: nuclear factor of activated T-cells; PPP3/calcineurin: protein phosphatase 3; PRKA/PKA: protein kinase cAMP activated; RAPGEF3/EPAC1: Rap guanine nucleotide exchange factor 3; RAPGEF4/EPAC2: Rap guanine nucleotide exchange factor 4; SQSTM1/p62: sequestosome 1; T2D: type 2 diabetes; TFEB: transcription factor EB.

Development and Application of a Semi quantitative Scoring Method for Ultrastructural Assessment of Acute Stress in Pancreatic Islets.

BACKGROUND: Pancreas and islet transplantation outcomes are negatively impacted by injury to the endocrine cells from acute stress during donor death, organ procurement, processing, and transplant procedures. Here, we report a novel electron microscopy scoring system, the Newcastle Pancreas Endocrine Stress Score (NPESS). METHODS: NPESS was adapted and expanded from our previously validated method for scoring pancreatic exocrine acinar cells, yielding a 4-point scale (0-3) classifying ultrastructural pathology in endocrine cell nuclei, mitochondria, endoplasmic reticulum, cytoplasmic vacuolization, and secretory granule depletion, with a maximum additive score of 15. We applied NPESS in a cohort of deceased organ donors after brainstem (DBD) and circulatory (DCD) death with a wide range of cold ischemic times (3.6-35.9 h) including 3 donors with type 1 and 3 with type 2 diabetes to assess islets in situ (n = 30) in addition to pancreata (n = 3) pre- and postislet isolation. RESULTS: In DBD pancreata, NPESS correlated with cold ischemic time (head: r = 0.55; P = 0.02) and mirrored exocrine score (r = 0.48; P = 0.01). When stratified by endocrine phenotype, cells with granules of heterogeneous morphology had higher scores than α, ß, and δ cells (P < 0.0001). Cells of mixed endocrine-exocrine morphology were observed in association with increased NPESS (P = 0.02). Islet isolation was associated with improved NPESS (in situ: 8.39 ± 0.77 [Mean ± SD]; postisolation: 5.44 ± 0.31; P = 0.04). CONCLUSIONS: NPESS provides a robust method for semiquantitative scoring of subcellular ultrastructural changes in human pancreatic endocrine cells in situ and following islet isolation with utility for unbiased evaluation of acute stress in organ transplantation research.

Development and validation of a quantitative electron microscopy score to assess acute cellular stress in the human exocrine pancreas.

The pancreas is particularly sensitive to acute cellular stress, but this has been difficult to evaluate using light microscopy. Pancreatic ischaemia associated with deceased organ donation negatively impacts whole-organ and isolated-islet transplantation outcomes. Post-mortem changes have also hampered accurate interpretation of ante-mortem pancreatic pathology. A rigorous histological scoring system accurately quantifying ischaemia is required to experimentally evaluate innovations in organ preservation and to increase rigour in clinical/research evaluation of underlying pancreatic pathology. We developed and validated an unbiased electron microscopy (EM) score of acute pancreatic exocrine cellular stress in deceased organ donor cohorts (development [n = 28] and validation [n = 16]). Standardised assessment led to clearly described numerical scores (0-3) for nuclear, mitochondrial and endoplasmic reticulum (ER) morphology and intracellular vacuolisation; with a maximum (worst) aggregate total score of 12. In the Validation cohort, a trend towards higher scores was observed for tail versus head regions (nucleus score following donation after brainstem death [DBD]: head 0.67 ± 0.19; tail 0.86 ± 0.11; p = 0.027) and donation after circulatory death (DCD) versus DBD (mitochondrial score: DCD (head + tail) 2.59 ± 0.16; DBD (head + tail) 2.38 ± 0.21; p = 0.004). Significant mitochondrial changes were seen ubiquitously even with short cold ischaemia, whereas nuclear and vacuolisation changes remained mild even after prolonged ischaemia. ER score correlated with cold ischaemia time (CIT) following DBD (pancreatic tail region: r = 0.796; p = 0.018). No relationships between CIT and EM scores were observed following DCD. In conclusion, we have developed and validated a novel EM score providing standardised quantitative assessment of subcellular ultrastructural morphology in pancreatic acinar cells. This provides a robust novel tool for gold standard measurement of acute cellular stress in studies evaluating surrogate measures of peri-transplant ischaemia, organ preservation technologies and in samples obtained for detailed pathological examination of underlying pancreatic pathology.

Glucagon-Like Peptide 1 Protects Pancreatic ß-Cells From Death by Increasing Autophagic Flux and Restoring Lysosomal Function.

Studies in animal models of type 2 diabetes have shown that glucagon-like peptide 1 (GLP-1) receptor agonists prevent ß-cell loss. Whether GLP-1 mediates ß-cell survival via the key lysosomal-mediated process of autophagy is unknown. In this study, we report that treatment of INS-1E ß-cells and primary islets with glucolipotoxicity (0.5 mmol/L palmitate and 25 mmol/L glucose) increases LC3 II, a marker of autophagy. Further analysis indicates a blockage in autophagic flux associated with lysosomal dysfunction. Accumulation of defective lysosomes leads to lysosomal membrane permeabilization and release of cathepsin D, which contributes to cell death. Our data further demonstrated defects in autophagic flux and lysosomal staining in human samples of type 2 diabetes. Cotreatment with the GLP-1 receptor agonist exendin-4 reversed the lysosomal dysfunction, relieving the impairment in autophagic flux and further stimulated autophagy. Small interfering RNA knockdown showed the restoration of autophagic flux is also essential for the protective effects of exendin-4. Collectively, our data highlight lysosomal dysfunction as a critical mediator of ß-cell loss and shows that exendin-4 improves cell survival via restoration of lysosomal function and autophagic flux. Modulation of autophagy/lysosomal homeostasis may thus define a novel therapeutic strategy for type 2 diabetes, with the GLP-1 signaling pathway as a potential focus.

Downregulation of TGF-ßRII in T effector cells leads to increased resistance to TGF-ß-mediated suppression of autoimmune responses in type I diabetes.

Tregs play an important role in controlling immune responses, particularly autoimmunity. In NOD mouse model, an excellent model for autoimmune diabetes, transfer of Tregs was shown to prevent diabetes, whereas depletion of Tregs in vivo enhanced disease progression, suggesting that Treg dysfunction contributes to the pathogenesis of diabetes. However, the mechanisms leading to Treg dysfunction and their role in diabetes progression has remained unclear. In this study we assessed quantitative and qualitative changes in Tregs during the development of autoimmune diabetes in NOD. We compared female NOD with males that have similar predisposition to but a lower incidence of diabetes and found that Treg numbers remained unchanged between 6 to 16 weeks of age in both groups. Although female Tregs produced lower TGF-ß compared to male, regulatory function of female Tregs was only marginally inferior to male upon GAD65 autoantigen stimulation. GAD65-reactive female Teffectors were more responsive and progressively became refractory to regulation compared to male effectors, in part due to lower expression of TGF-ß RII, accounting for reduced sensitivity to Tregs. Moreover, we unexpectedly found that TGF-ß suppressed IFN-γ production to GAD65 antigen in male, not in female responders. These data suggest that TGF-ß plays a major role in Teff resistance to regulation and Treg dysfunction, and may account for autoimmune diabetes. Our study implies that development of a successful supplemental Treg therapy for halting autoimmunity may require further understanding of Teff responses to regulation in order to generate highly effective Tregs.

Nucleolar staining cannot be used as a screening test for the scleroderma marker anti-RNA polymerase I/III antibodies.

OBJECTIVE: Anti-RNA polymerase I/III (anti-RNAP I/III) antibodies are clinically useful markers of scleroderma, and their presence is associated with diffuse skin disease and an increased risk of cardiac and kidney involvement. Although RNAP I antibodies localize to the nucleolus, nucleolar staining by many anti-RNAP antibody-positive sera is not always observed. Nucleolar staining by anti-RNAP antibody-positive sera was examined by double staining with antifibrillarin antibodies to evaluate whether nucleolar staining can be used as a screening test for anti-RNAP I/III antibodies. In addition, the relationships between nucleolar staining and levels of anti-RNAP III antibodies were examined by enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IP) assay. METHODS: Sera were tested using immunofluorescent antinuclear antibodies on HEp-2 cell slides, by anti-RNAP III ELISA, and by IP assay using (35)S-labeled K562 cell extract. Nucleolar staining by anti-RNAP antibody IP-positive sera was confirmed by double staining using antifibrillarin monoclonal antibodies. The levels of anti-RNAP III antibodies were quantitated by ELISA and by IP assay using a serially diluted reference serum as a standard, and their relationship was analyzed. RESULTS: All 18 anti-RNAP I/III antibody-positive sera showed nuclear speckled patterns, but nucleolar staining was readily noticeable in only 44% of the sera. A positive correlation was found between ELISA and IP units for anti-RNAP III antibodies. The levels of anti-RNAP III antibodies and anti-RNAP I antibodies correlated well, with the exception of a few sera. Levels of anti-RNAP III antibodies were low in sera with nucleolar staining, whereas several sera with high levels of anti-RNAP I antibodies clearly showed nucleolar staining. CONCLUSION: Although some sera positive for anti-RNAP I/III antibodies clearly stain nucleoli, nucleolar staining is inconsistent and cannot be used to screen for anti-RNAP I/III antibodies.