The nuclear localization sequence and C-terminus of parathyroid hormone-related protein regulate normal pancreatic islet development and function.

Parathyroid hormone-related protein (PTHrP) is a pleiotropic hormone essential for morphogenesis, tissue differentiation, as well as cell regulation and function. PTHrP is expressed by pancreatic beta cells which are responsible for insulin secretion. Previous studies have reported that N-terminal PTHrP stimulated proliferation in beta cells in rodents. We have developed a knockin mouse model (PTHrP Δ/Δ) lacking the C-terminal and nuclear localization sequence (NLS) of PTHrP. These mice die at ∼day 5, are severely stunted in growth, weigh 54% less than control mice at day 1-2 and eventually fail to grow. PTHrP Δ/Δ mice are also hypoinsulinemic and hypoglycemic yet have nutrient intake proportional to size. To characterize the pancreatic islets in these mice, islets (∼10-20) were isolated from 2 to 5 day-old-mice using collagenase digestion. Islets from PTHrP Δ/Δ mice were smaller in size but secreted more insulin than littermate controls. PTHrP Δ/Δ and control mice islets were exposed to various glucose concentrations and intracellular calcium, the trigger for insulin release, was elevated for glucose concentrations of 8-20 mM. Immunofluorescence staining showed less glucagon-stained area in islets from PTHrP Δ/Δ mice (∼250 µm2) compared to islets from control mice (∼900 µm2), and ELISA confirmed there was reduced glucagon content. These data collectively demonstrate increased insulin secretion and reduced glucagon at the islet level, which may contribute to the observed hypoglycemia and early death in PTHrP Δ/Δ mice. Thus, the C-terminus and NLS of PTHrP are crucial to life, including regulation of glucose homeostasis and islet function.

G protein-coupled receptors as regulators of pancreatic islet functionality.

Glucose homeostasis is maintained by hormones secreted from different types of pancreatic islets and its dysregulation can result in diseases including diabetes mellitus. The secretion of hormones from pancreatic islets is highly complex and tightly controlled by G protein-coupled receptors (GPCRs). Moreover, GPCR signaling may play a role in enhancing islet cell replication and proliferation. Thus, targeting GPCRs offers a promising strategy for regulating the functionality of pancreatic islets. Here, available RNAseq datasets from human and mouse islets were used to identify the GPCR expression profile and the impact of GPCR signaling for normal islet functionality is discussed.

Islet vascularization is regulated by primary endothelial cilia via VEGF-A-dependent signaling.

Islet vascularization is essential for intact islet function and glucose homeostasis. We have previously shown that primary cilia directly regulate insulin secretion. However, it remains unclear whether they are also implicated in islet vascularization. At eight weeks, murine Bbs4-/-islets show significantly lower intra-islet capillary density with enlarged diameters. Transplanted Bbs4-/- islets exhibit delayed re-vascularization and reduced vascular fenestration after engraftment, partially impairing vascular permeability and glucose delivery to ß-cells. We identified primary cilia on endothelial cells as the underlying cause of this regulation, via the vascular endothelial growth factor-A (VEGF-A)/VEGF receptor 2 (VEGFR2) pathway. In vitro silencing of ciliary genes in endothelial cells disrupts VEGF-A/VEGFR2 internalization and downstream signaling. Consequently, key features of angiogenesis including proliferation and migration are attenuated in human BBS4 silenced endothelial cells. We conclude that endothelial cell primary cilia regulate islet vascularization and vascular barrier function via the VEGF-A/VEGFR2 signaling pathway.

Transcriptomic and Quantitative Proteomic Profiling Reveals Signaling Pathways Critical for Pancreatic Islet Maturation.

Pancreatic ß-cell dysfunction and reduced insulin secretion play a key role in the pathogenesis of diabetes. Fetal and neonatal islets are functionally immature and have blunted glucose responsiveness and decreased insulin secretion in response to stimuli and are far more proliferative. However, the mechanisms underlying functional immaturity are not well understood. Pancreatic islets are composed of a mixture of different cell types, and the microenvironment of islets and interactions between these cell types are critical for ß-cell development and maturation. RNA sequencing and quantitative proteomic data from intact islets isolated from fetal (embryonic day 19) and 2-week-old Sprague-Dawley rats were integrated to compare their gene and protein expression profiles. Ingenuity Pathway Analysis (IPA) was also applied to elucidate pathways and upstream regulators modulating functional maturation of islets. By integrating transcriptome and proteomic data, 917 differentially expressed genes/proteins were identified with a false discovery rate of less than 0.05. A total of 411 and 506 of them were upregulated and downregulated in the 2-week-old islets, respectively. IPA revealed novel critical pathways associated with functional maturation of islets, such as AMPK (adenosine monophosphate-activated protein kinase) and aryl hydrocarbon receptor signaling, as well as the importance of lipid homeostasis/signaling and neuronal function. Furthermore, we also identified many proteins enriched either in fetal or 2-week-old islets related to extracellular matrix and cell communication, suggesting that these pathways play critical roles in islet maturation. Our present study identified novel pathways for mature islet function in addition to confirming previously reported mechanisms, and provided new mechanistic insights for future research on diabetes prevention and treatment.

Long-Term Expansion of Pancreatic Islet Organoids from Resident Procr+ Progenitors.

It has generally proven challenging to produce functional ß cells in vitro. Here, we describe a previously unidentified protein C receptor positive (Procr+) cell population in adult mouse pancreas through single-cell RNA sequencing (scRNA-seq). The cells reside in islets, do not express differentiation markers, and feature epithelial-to-mesenchymal transition characteristics. By genetic lineage tracing, Procr+ islet cells undergo clonal expansion and generate all four endocrine cell types during adult homeostasis. Sorted Procr+ cells, representing ∼1% of islet cells, can robustly form islet-like organoids when cultured at clonal density. Exponential expansion can be maintained over long periods by serial passaging, while differentiation can be induced at any time point in culture. ß cells dominate in differentiated islet organoids, while α, δ, and PP cells occur at lower frequencies. The organoids are glucose-responsive and insulin-secreting. Upon transplantation in diabetic mice, these organoids reverse disease. These findings demonstrate that the adult mouse pancreatic islet contains a population of Procr+ endocrine progenitors.

Kindlin-2 modulates MafA and ß-catenin expression to regulate ß-cell function and mass in mice.

ß-Cell dysfunction and reduction in ß-cell mass are hallmark events of diabetes mellitus. Here we show that ß-cells express abundant Kindlin-2 and deleting its expression causes severe diabetes-like phenotypes without markedly causing peripheral insulin resistance. Kindlin-2, through its C-terminal region, binds to and stabilizes MafA, which activates insulin expression. Kindlin-2 loss impairs insulin secretion in primary human and mouse islets in vitro and in mice by reducing, at least in part, Ca2+ release in ß-cells. Kindlin-2 loss activates GSK-3ß and downregulates ß-catenin, leading to reduced ß-cell proliferation and mass. Kindlin-2 loss reduces the percentage of ß-cells and concomitantly increases that of α-cells during early pancreatic development. Genetic activation of ß-catenin in ß-cells restores the diabetes-like phenotypes induced by Kindlin-2 loss. Finally, the inducible deletion of ß-cell Kindlin-2 causes diabetic phenotypes in adult mice. Collectively, our results establish an important function of Kindlin-2 and provide a potential therapeutic target for diabetes.

Mouse Pancreas Stem/Progenitor Cells Get Augmented by Streptozotocin and Regenerate Diabetic Pancreas After Partial Pancreatectomy.

Existence of stem cells in adult pancreas remains contentious. Single cells suspensions obtained by collagenase and trypsin digestion separately from adult mouse pancreas and pancreatic islets were spun at 1000 rpm (250 g) to collect the cells. At this speed the stem/ progenitor cells remained buoyant and were further enriched by spinning the supernatant at 3000 rpm (1000 g). Two distinct populations of stem cells were detected including pluripotent, very small (2-6 µm) embryonic-like stem cells (VSELs) that expressed nuclear OCT-4A and pluripotent transcripts (Oct-4A, Sox2, Nanog, Stella) and slightly bigger progenitors, pancreatic stem cells (PSCs) that expressed cytoplasmic OCT-4B and PDX-1. Streptozotocin treated diabetic pancreas showed an increase in numbers of VSELs (2-6 µm, 7AAD-, LIN-CD45-SCA1+ cells) and up-regulation of transcripts specific for stem/ progenitor cells. Diabetic mice were further subjected to partial pancreatectomy to study involvement of VSELs/ PSCs during regeneration. VSELs/ PSCs were mobilized in large numbers, were observed in the lumen of blood vessels and PCNA expression suggested their proliferation. Initially, new acini assembled to regenerate the exocrine pancreas and later by Day 30, neogenesis of islets was observed in the vicinity of the blood vessels and pancreatic ducts by the differentiation of endogenous VSELs/ PSCs which may be targeted to regenerate diabetic pancreas in clinical settings.

Atorvastatin Targets the Islet Mevalonate Pathway to Dysregulate mTOR Signaling and Reduce ß-Cell Functional Mass.

Statins are cholesterol-lowering agents that increase the incidence of diabetes and impair glucose tolerance via their detrimental effects on nonhepatic tissues, such as pancreatic islets, but the underlying mechanism has not been determined. In atorvastatin (ator)-treated high-fat diet-fed mice, we found reduced pancreatic ß-cell size and ß-cell mass, fewer mature insulin granules, and reduced insulin secretion and glucose tolerance. Transcriptome profiling of primary pancreatic islets showed that ator inhibited the expression of pancreatic transcription factor, mechanistic target of rapamycin (mTOR) signaling, and small G protein (sGP) genes. Supplementation of the mevalonate pathway intermediate geranylgeranyl pyrophosphate (GGPP), which is produced by 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, significantly restored the attenuated mTOR activity, v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) expression, and ß-cell function after ator, lovastatin, rosuvastatin, and fluvastatin treatment; this effect was potentially mediated by sGP prenylation. Rab5a, the sGP in pancreatic islets most affected by ator treatment, was found to positively regulate mTOR signaling and ß-cell function. Rab5a knockdown mimicked the effect of ator treatment on ß-cells. Thus, ator impairs ß-cell function by regulating sGPs, for example, Rab5a, which subsequently attenuates islet mTOR signaling and reduces functional ß-cell mass. GGPP supplementation could constitute a new approach for preventing statin-induced hyperglycemia.

Characterization of Transplantable Insulinoma Cells.

This chapter describes the propagation and characterization of transplantable insulinoma cells as model of insulin-producing pancreatic islet cells in the rat. Here, the cells are propagated by transplantation into rats followed by harvesting after growth for approximately 1 month. The cells are then purified by Percoll density gradient centrifugation and characterized by pulse-chase radiolabelling and immunoprecipitation of the insulin-related peptides. The results show that the transplantable insulinoma cells produce insulin in a manner similar to that found in normal pancreatic islets.

An important step towards a prevascularized islet microencapsulation device: in vivo prevascularization by combination of mesenchymal stem cells on micropatterned membranes.

Extrahepatic transplantation of islets of Langerhans could aid in better survival of islets after transplantation. When islets are transfused into the liver 60-70% of them are lost immediately after transplantation. An important factor for a successful extrahepatic transplantation is a well-vascularized tissue surrounding the implant. There are many strategies known for enhancing vessel formation such as adding cells with endothelial potential, the combination with angiogenic factors and / or applying surface topography at the exposed surface of the device. Previously we developed porous, micropatterned membranes which can be applied as a lid for an islet encapsulation device and we showed that the surface topography induces human umbilical vein endothelial cell (HUVEC) alignment and interconnection. This was achieved without the addition of hydrogels, often used in angiogenesis assays. In this work, we went one step further towards clinical implementation of the device by combining this micropatterned lid with Mesenchymal Stem Cells (MSCs) to facilitate prevascularization in vivo. As for HUVECs, the micropatterned membranes induced MSC alignment and organization in vitro, an important contributor to vessel formation, whereas in vivo (subcutaneous rat model) they contributed to improved implant prevascularization. In fact, the combination of MSCs seeded on the micropatterned membrane induced the highest vessel formation score in 80% of the sections.

Accurate, strong, and stable reporting of choroid plexus epithelial cells in transgenic mice using a human transthyretin BAC.

BACKGROUND: Choroid plexus epithelial cells express high levels of transthyretin, produce cerebrospinal fluid and many of its proteins, and make up the blood-cerebrospinal fluid barrier. Choroid plexus epithelial cells are vital to brain health and may be involved in neurological diseases. Transgenic mice containing fluorescent and luminescent reporters of these cells would facilitate their study in health and disease, but prior transgenic reporters lost expression over the early postnatal period. METHODS: Human bacterial artificial chromosomes in which the transthyretin coding sequence was replaced with DNA for tdTomato or luciferase 2 were used in pronuclear injections to produce transgenic mice. These mice were characterized by visualizing red fluorescence, immunostaining, real-time reverse transcription polymerase chain reaction, and luciferase enzyme assay. RESULTS: Reporters were faithfully expressed in cells that express transthyretin constitutively, including choroid plexus epithelial cells, retinal pigment epithelium, pancreatic islets, and liver. Expression of tdTomato in choroid plexus began at the appropriate embryonic age, being detectable by E11.5. Relative levels of tdTomato transcript in the liver and choroid plexus paralleled relative levels of transcripts for transthyretin. Expression remained robust over the first postnatal year, although choroid plexus transcripts of tdTomato declined slightly with age whereas transthyretin remained constant. TdTomato expression patterns were consistent across three founder lines, displayed no sex differences, and were stable across several generations. Two of the tdTomato lines were bred to homozygosity, and homozygous mice are healthy and fertile. The usefulness of tdTomato reporters in visualizing and analyzing live Transwell cultures was demonstrated. Luciferase activity was very high in homogenates of choroid plexus and continued to be expressed through adulthood. Luciferase also was detectable in eye and pancreas. CONCLUSIONS: Transgenic mice bearing fluorescent and luminescent reporters of transthyretin should prove useful for tracking transplanted choroid plexus epithelial cells, for purifying the cells, and for reporting their derivation from stem cells. They also should prove useful for studying transthyretin synthesis by other cell types, as transthyretin has been implicated in many functions and conditions, including clearance of ß-amyloid peptides associated with Alzheimer's disease, heat shock in neurons, processing of neuropeptides, nerve regeneration, astrocyte metabolism, and transthyretin amyloidosis.

Pancreas regeneration.

The pancreas is made from two distinct components: the exocrine pancreas, a reservoir of digestive enzymes, and the endocrine islets, the source of the vital metabolic hormone insulin. Human islets possess limited regenerative ability; loss of islet ß-cells in diseases such as type 1 diabetes requires therapeutic intervention. The leading strategy for restoration of ß-cell mass is through the generation and transplantation of new ß-cells derived from human pluripotent stem cells. Other approaches include stimulating endogenous ß-cell proliferation, reprogramming non-ß-cells to ß-like cells, and harvesting islets from genetically engineered animals. Together these approaches form a rich pipeline of therapeutic development for pancreatic regeneration.

In vivo imaging of emerging endocrine cells reveals a requirement for PI3K-regulated motility in pancreatic islet morphogenesis.

The three-dimensional architecture of the pancreatic islet is integral to beta cell function, but the process of islet formation remains poorly understood due to the difficulties of imaging internal organs with cellular resolution. Within transparent zebrafish larvae, the developing pancreas is relatively superficial and thus amenable to live imaging approaches. We performed in vivo time-lapse and longitudinal imaging studies to follow islet development, visualizing both naturally occurring islet cells and cells arising with an accelerated timecourse following an induction approach. These studies revealed previously unappreciated fine dynamic protrusions projecting between neighboring and distant endocrine cells. Using pharmacological compound and toxin interference approaches, and single-cell analysis of morphology and cell dynamics, we determined that endocrine cell motility is regulated by phosphoinositide 3-kinase (PI3K) and G-protein-coupled receptor (GPCR) signaling. Linking cell dynamics to islet formation, perturbation of protrusion formation disrupted endocrine cell coalescence, and correlated with decreased islet cell differentiation. These studies identified novel cell behaviors contributing to islet morphogenesis, and suggest a model in which dynamic exploratory filopodia establish cell-cell contacts that subsequently promote cell clustering.

Targeted Mutation of NGN3 Gene Disrupts Pancreatic Endocrine Cell Development in Pigs.

The domestic pig is an attractive model for biomedical research because of similarities in anatomy and physiology to humans. However, key gaps remain in our understanding of the role of developmental genes in pig, limiting its full potential. In this publication, the role of NEUROGENIN 3 (NGN3), a transcription factor involved in endocrine pancreas development has been investigated by CRISPR/Cas9 gene ablation. Precomplexed Cas9 ribonucleoproteins targeting NGN3 were injected into in vivo derived porcine embryos, and transferred into surrogate females. On day 60 of pregnancy, nine fetuses were collected for genotypic and phenotypic analysis. One of the piglets was identified as an in-frame biallelic knockout (Δ2/Δ2), which showed a loss of putative NGN3-downstream target genes: NEUROD1 and PAX4, as well as insulin, glucagon, somatostatin and pancreatic polypeptide-Y. Fibroblasts from this fetus were used in somatic cell nuclear transfer to generate clonal animals to qualify the effect of mutation on embryonic lethality. Three live piglets were born, received colostrum and suckled normally, but experienced extreme weight loss over a 24 to 36-hour period requiring humane euthanasia. Expression of pancreatic endocrine hormones: insulin, glucagon, and somatostatin were lost. The data support a critical role of NGN3 in porcine endocrine pancreas development.

Immaturity of insulin secretion by pancreatic islets isolated from one human neonate.

Human ß-cells are functionally mature by the age of 1 year. The timeline and mechanisms of this maturation are unknown owing to the exceptional availability of testable tissue. Here, we report the first in vitro study of insulin secretion by islets from a 5-day-old newborn. Glucose was inefficient alone, but induced insulin secretion, which was concentration-dependent, showed a biphasic time-course and was of similar magnitude as in infant islets when ß-cell cyclic adenosine monophosphate was raised by forskolin. Tolbutamide alone was effective in low glucose, but its effect was not augmented by high glucose. Metabolic amplification by glucose was thus inoperative, in contrast to amplification by cyclic adenosine monophosphate. Newborn islets showed high basal insulin secretion that could be inhibited by diazoxide or omission of CaCl2 . Postnatal acquisition of functional maturity by human ß-cells implicates control of basal secretion and production of metabolic signals able to activate both triggering and amplifying pathways of insulin secretion.

Changes in expression of C2cd4c in pancreatic endocrine cells during pancreatic development.

C2cd4c, encoded by a gene belonging to the C2cd4 family, contains a C2 domain conserved across species and is localized to the cytoplasm. To examine the role of C2cd4c in the pancreas, we studied its localization and generated C2cd4c knockout (KO) mice. C2cd4c was expressed in pancreatic endocrine progenitors at early embryonic stages. When endocrine cells arise from their precursors, C2cd4c is gradually confined to the insulin- and pancreatic polypeptide-expressing cells of the endocrine. In the adult pancreas, C2cd4c is restricted to the beta cells. C2cd4c KO mice showed normal embryonic pancreatic development and adult pancreatic function. Thus, our results suggest that C2cd4c is dispensable for pancreatic development.

Lin28 and let-7 in the Metabolic Physiology of Aging.

The Lin28/let-7 molecular switch has emerged as a central regulator of growth signaling pathways and metabolic enzymes. Initially discovered to regulate developmental timing in the nematode, the Lin28/let-7 pathway of RNA regulation has gained prominence for its role in mammalian stem cells, cancer cells, tissue development, and aging. By regulating RNAs, the pathway coordinates cellular growth and cellular metabolism to influence metabolic physiology. Here, we review this regulatory mechanism and its impact on cancers, which reactivate Lin28, cardiovascular diseases, which implicate let-7, human genome-wide association studies (GWAS) of growth, and metabolic diseases, which implicate the Lin28/let-7 pathway. We also highlight questions relating to Barker's Hypothesis and the potential actions of the Lin28/let-7 pathway on programming long-lasting epigenetic effects.

Expanded Hematopoietic Progenitor Cells Reselected for High Aldehyde Dehydrogenase Activity Demonstrate Islet Regenerative Functions.

Human umbilical cord blood (UCB) hematopoietic progenitor cells (HPC) purified for high aldehyde dehydrogenase activity (ALDH(hi) ) stimulate islet regeneration after transplantation into mice with streptozotocin-induced ß cell deletion. However, ALDH(hi) cells represent a rare progenitor subset and widespread use of UCB ALDH(hi) cells to stimulate islet regeneration will require progenitor cell expansion without loss of islet regenerative functions. Here we demonstrate that prospectively purified UCB ALDH(hi) cells expand efficiently under serum-free, xeno-free conditions with minimal growth factor supplementation. Consistent with the concept that ALDH-activity is decreased as progenitor cells differentiate, kinetic analyses over 9 days revealed the frequency of ALDH(hi) cells diminished as culture time progressed such that total ALDH(hi) cell number was maximal (increased 3-fold) at day 6. Subsequently, day 6 expanded cells (bulk cells) were sorted after culture to reselect differentiated progeny with low ALDH-activity (ALDH(lo) subset) from less differentiated progeny with high ALDH-activity (ALDH(hi) subset). The ALDH(hi) subset retained primitive cell surface marker coexpression (32.0% ± 7.0% CD34(+) /CD38(-) cells, 37.0% ± 6.9% CD34(+) /CD133(+) cells), and demonstrated increased hematopoietic colony forming cell function compared with the ALDH(lo) subset. Notably, bulk cells or ALDH(lo) cells did not possess the functional capacity to lower hyperglycemia after transplantation into streptozotocin-treated NOD/SCID mice. However, transplantation of the repurified ALDH(hi) subset significantly reduced hyperglycemia, improved glucose tolerance, and increased islet-associated cell proliferation and capillary formation. Thus, expansion and delivery of reselected UCB cells that retain high ALDH-activity after short-term culture represents an improved strategy for the development of cellular therapies to enhance islet regeneration in situ.

Pharmacological characterization and antidiabetic activity of a long-acting glucagon-like peptide-1 analogue conjugated to an antithrombin III-binding pentasaccharide.

AIMS: To examine the biological characteristics of a novel glucagon-like peptide-1 (GLP-1) conjugate, in which an antithrombin III (ATIII)-binding pentasaccharide is conjugated to d-Ala(8) GLP-1 using a tetraethylene glycol linker. METHODS: We assessed GLP-1 receptor binding, cAMP generation and insulin secretory activity of the GLP-1 conjugate in vitro. Circulating half-life, glucose homeostatic and subchronic therapeutic effectiveness were then examined in vivo. RESULTS: The half-life of the GLP-1 conjugate in mice was ∼11 h. In vitro insulin secretion from clonal ß cells and islets was increased (p < 0.001) by the conjugate. The conjugate had half maximum effective concentration values of 1.3 × 10(-7) and 9.9 × 10(-8) M for displacement of (125) I-GLP-1 in competitive GLP-1 receptor binding and cAMP generation, respectively. Glucose tolerance in normal mice, immediately and 4 h after conjugate injection, resulted in significant (p < 0.001) improvements in blood glucose. These effects persisted for >48 h after administration. Daily treatment (21 days) of high-fat-fed and ob/ob mice with 25 nmol/kg conjugate resulted in significant improvement in glucose tolerance (p < 0.001) and reductions in glycated haemoglobin (HbA1c; p < 0.01) equivalent to or better than with exenatide or liraglutide. Treatment of C57BL/KsJ db/db mice for 15 days with 100 nmol/kg conjugate significantly (p < 0.001) reduced glucose and raised plasma insulin. Oral glucose tolerance was significantly (p < 0.001) improved and both 24-h glucose profile (p < 0.001) and HbA1c levels (p < 0.001) were reduced. Islet size (p < 0.001) and pancreatic insulin content were increased without change of islet cell proliferation or apoptosis. CONCLUSION: These data show that d-Ala(8) GLP-1(Lys(37) ) pentasaccharide exerts significant antidiabetic actions and has a projected pharmacokinetic/pharmacodynamic profile that merits further evaluation in humans for a possible once-weekly dosing regimen.

Pretreatment of donor pigs with a diet rich in soybean oil increases the yield of isolated islets.

INTRODUCTION: The pig is considered the donor species of choice for islet xenotransplantation. However, isolation of porcine islets is difficult, particularly from young pigs. Early life exposure to a high-fat diet (HFD) reportedly encourages islet ß-cell expansion in neonatal rodents and improves islet viability in culture from pretreated weanling pigs. In this study, we examined the influence of young donor pretreatment with a soybean oil-enriched HFD on porcine islet mass and yield after islet isolation. MATERIALS AND METHODS: Postweaning and between days 70 and 250, pigs were fed either a standard diet (control group; n = 5) or an HFD (experimental group; n = 6). Biochemical blood parameters and acute C-peptide response to intravenous glucose were monitored before pancreas procurement. The study was blinded to objectively evaluate the influence of treated diet. After procurement, pancreas biopsy samples were taken from control and pretreated donor pigs to assess islet number by using a dithizone scoring method and histologic islet area fraction determination. Control and HFD donor pig islets were isolated by using our standard isolation protocol to determine islet yield. Islet isolation characteristics and islet quality were assessed in both groups, and the results were compared. RESULTS: There were no significant differences in the donor characteristics (age, body weight, glucose disposal rate, acute C-peptide response to intravenous glucose, cholesterol, and aspartate aminotransferase) except fasting blood glucose level between the control and treatment groups (84 ± 6 vs 99 ± 12 mg/dL; P = .0317). The stimulated insulin and C-peptide levels between groups were similar. However, the dithizone score was slightly higher in the treatment group compared with the control group (95.4 ± 38.5 vs 62.6 ± 23.9; P = .1208). Digestion time, digested pancreas weight, pellet volume, and the fragility index were similar in both groups. However, the average islet count (islet equivalent number/g pancreas) at the digest level was significantly higher in the HFD group than in the control group (1578 ± 994 vs 738 ± 202; P = .0344). The functional viability of 2- and 7 day-cultured islets, as assessed by using oxygen consumption rate corrected for DNA, was similar in both groups. CONCLUSIONS: Pretreatment of pigs with HFD enriched with soybean oil could potentially be used to improve the islet mass in donor pigs. Further studies are needed to confirm and optimize the use of HFD for the purpose of increasing islet yield from young donor pigs.