AIMS/OBJECTIVE:p16INK4A is a cyclin-dependent kinase inhibitor that plays important roles in pancreatic islet beta-cell maturation, function, and senescence. As staining pattern consistency and reproducibility have been challenging in the field, our objective was to validate a monoclonal antibody for the detection of p16INK4A in mouse and human fixed pancreas sections. METHODS:Four commercial monoclonal antibodies reported to react against mouse p16INK4A were tested by fluorescent immunohistochemistry on positive and negative control formalin-fixed paraffin-embedded pancreas sections from two different mouse strains. Lot-to-lot consistency was also evaluated. Fixed human donor pancreas sections were evaluated for reactivity with the monoclonal p16INK4A antibody that performed best on mouse tissues. RESULTS:One commercial monoclonal antibody robustly detected a nuclear antigen in both mouse and human pancreas sections, consistent with the previously reported p16INK4A staining pattern, and its role as a cyclin-dependent kinase inhibitor in beta cells. This antibody also showed lot-to-lot consistency in staining pattern. CONCLUSION:Our findings highlight the importance of antibody validation for p16INK4A detection in mouse and human pancreas sections and suggest caution in using untested commercially available antibodies to this protein for immunohistochemistry of the pancreas.
Accurate quantification of islet mass is critical for the preclinical evaluation and therapeutic application of stem cell-derived pancreatic islet organoids. Traditional methods, including Ricordi's islet equivalent (IEQ) approach and its equivalent circle diameter adaptations, often overestimate islet volume due to reliance on maximal diameters and discrete size bins. To address these limitations, we developed an automated image segmentation and three-dimensional modeling framework to quantify individual islet clusters from brightfield images. Clusters were fitted with ellipses and modeled as ellipsoids using rotation about either the minor or major axis, allowing IEQs to be calculated continuously relative to a reference 150 μm spherical islet. Major-axis (prolate) rotation provided the most conservative and physically plausible volume estimates, whereas minor-axis (oblate) rotation and diameter-based approaches systematically overestimated IEQs. Functional assessment with glucose-stimulated insulin secretion assays across multiple size categories demonstrated consistent insulin output for clusters below 250 μm, supporting the reproducibility of our 3D differentiation system. In vivo, streptozotocin-induced diabetic mice transplanted with islet doses based on major-axis modeling exhibited faster and more stable restoration of glycemia compared with groups receiving doses derived from overestimated approaches. These findings establish that major-axis ellipsoid modeling offers a mathematically consistent, conservative, and biologically relevant method for estimating IEQs, providing a practical framework to guide dosing in preclinical studies and supporting the translational development of stem cell-derived islet therapies.
Diabetes mellitus and end-stage renal disease (ESRD) are major causes of morbidity and mortality worldwide.Pancreatic islet transplantation offers potential insulin independence. Different sites and their suitability for islet transplantation have been investigated, including renal subcapsular and intraportal sites, among others. Various limitations and complications have been reported, such as the number of donor islet species, islet isolation, and site of transplantation. Combining islets and kidney transplantation in diabetic patients suffering from ESRD has several advantages.This review compares the experimental experience of the renal subcapsular and intraportal sites for islet allo- and xeno-transplantation. The subcapsular site appears preferable in rodent models. The intraportal site has been more successful in large animals. Combined islet-kidneys have been suggested as a treatment targeting both ESRD and type 1 diabetes. With our increasing ability to genetically-engineer pigs, and the availability of novel immunosuppressive agents, xenotransplantation is an emerging therapeutic option.
BACKGROUND:Activation of islet stellate cells (ISCs) contributes to islet fibrosis and diabetes progression through excessive extracellular matrix secretion and lipid loss. Annexin A1 (ANXA1) has been reported to modulate lipid metabolism in other tissues, but its role in ISCs remains unclear. METHODS:ISCs were isolated from 9-and 28-week-old db/m and db/db mice. Lipid content analysis, qRT‒PCR, and Western blotting were used to assess lipid metabolism-related molecules. ANXA1 expression was analyzed by immunohistochemistry and Western blotting. Recombinant ANXA1 was co-cultured with db/db ISCs to evaluate lipid synthesis and lipolysis. The interaction between ANXA1 and peroxisome proliferator-activated receptor alpha (PPARα) was examined by immunoprecipitation. RESULTS:Activation of ISCs markedly reduced intracellular triglycerides, with decreased Diacylglycerol Acyltransferase 1/2 (DGAT1/2) and increased adipose triglyceride lipase (ATGL) and hormone-sensitive triglyceride lipase (HSL) expression. ANXA1 was detected in islets, MIN6 cells, and their culture supernatants. Recombinant ANXA1 treatment lowered triglyceride levels and upregulated PPARα and its downstream genes, acyl-CoA oxidase 1 (ACOX1) and cytochrome P450 4 A (CYP4A); these effects were enhanced by a PPARα agonist but reversed by inhibition. Immunofluorescence and coimmunoprecipitation confirmed that PPARα acts as a key mediator of ANXA1-regulated triglyceride metabolism in ISCs. CONCLUSION:ANXA1 promotes ISCs activation by enhancing triglyceride catabolism through the PPARα signaling pathway, suggesting a novel therapeutic target for islet fibrosis.
OBJECTIVE:Mitochondria are central to energy production and cellular homeostasis. Beyond importing diverse RNAs, they also encode hundreds of their own non-coding RNAs, contributing to a complex and dynamic RNA landscape. Early-life nutritional insults, such as fetal and postnatal protein deficiency, can impair mitochondrial function and increase the long-term diabetes risk. However, the mitochondrial non-coding transcriptome of pancreatic islets, particularly its responsiveness to nutritional cues, remains largely unexplored. METHODS:We performed RNA sequencing to profile small non-coding RNAs in mitochondrial fractions of islet cells from offspring of rats exposed to low-protein (LP) or control diets during gestation and lactation and employed mRNA-miRNA network analysis to explore the potential regulatory roles of differentially expressed mitomiRs in LP-exposed pups. RESULTS:Protein deficiency during gestation and lactation led to a profound remodeling of the small non-coding RNA landscape in whole islets, with microRNAs and piRNAs showing the most pronounced changes. In mitochondrial fractions, LP exposure resulted in a striking shift in microRNA composition, with 33 mitomiRs detected in control islets versus 23 in LP-exposed rats, and only 5 shared between groups. Notably, ten mitomiRs were selectively depleted from the cytosol and enriched in mitochondria of LP-exposed islets. Amongst these, miR-10a-5p and miR-126a-5p, are predicted to target genes involved in mitochondrial metabolism and structural organization. CONCLUSION:Early-life protein restriction triggers a highly selective reorganization of the mitomiR landscape in pancreatic islets. The identified mitomiRs may serve as regulators of mitochondrial function and intracellular signaling, potentially influencing β-cell metabolic coupling and contributing to diabetes susceptibility.
