Maternal Under- and Over-Nutrition during Gestation Causes Islet Hypertrophy and Sex-Specific Changes to Pancreas DNA Methylation in Fetal Sheep

The mechanisms by which fetal programming predisposes offspring to reduced β-cell function later in life are poorly understood. We hypothesized that maternal under- and over-nutrition during gestation would negatively affect offspring pancreas development and alter DNA methylation patterns. Pregnant ewes (n = 78) were fed 100, 60, or 140% of NRC requirements beginning at d 30.2 ± 0.2 of gestation. The fetuses are referred to as CON, RES, and OVER, respectively. Fetal pancreas tissue was collected at d 90 or 135 of gestation or within 24 h of birth. Tissue was preserved for histological (n = 8 to 9 offspring per treatment per time point) and DNA methylation analyses (n = 3 to 4 fetuses per treatment per sex). At d 135, OVER exhibited an increased islet size, reduced islet number, and greater insulin positive area compared with CON (p ≤ 0.03). An increased islet size was also observed at d 135 in RES (p ≤ 0.03) compared with CON. Cellular proliferation was reduced at birth in OVER vs. CON (p = 0.01). In the RES vs. CON females, 62% of the differentially methylated regions (DMRs) were hypomethylated (p ≤ 0.001). In the RES vs. CON males, 93% of the DMRs were hypermethylated (p ≤ 0.001). In OVER, 66 and 80% of the DMRs were hypermethylated in the female and male offspring compared with CON (p ≤ 0.001). In conclusion, changes to maternal diet during pregnancy affects the islet hypertrophy and cellular proliferation of the offspring at early post-natal time points. Additionally, changes in DNA methylation patterns appear to be in a diet-specific and sex-dependent manner.

Gut Microbiome Prolongs an Inhibitory Effect of Korean Red Ginseng on High-Fat-Diet-Induced Mouse Obesity

Although the anti-obesity effect of Korean red ginseng (Panax ginseng Meyer) has been revealed, its underlying mechanisms are not clearly understood. Here, we demonstrate an involvement of gut microbiome in the inhibitory effect of Korean red ginseng on high-fat-diet (HFD)-induced mouse obesity, and further provides information on the effects of saponin-containing red ginseng extract (SGE) and saponin-depleted red ginseng extract (GE). Mice were fed with either SGE or GE every third day for one month, and their food intakes, fat weights, plasma glucose, and insulin and leptin levels were measured. Immunofluorescence assays were conducted to measure pancreatic islet size. Stools from the mice were subjected to metagenomic analysis. Both SGE and GE attenuated HFD-induced gain of body weight, reducing HFD-induced increase of food intakes and fat weights. They also reduced HFD-increased plasma glucose, insulin, and leptin levels, decreased both fasting and postprandial glucose concentrations, and improved both insulin resistance and glucose intolerance. Immunofluorescence assays revealed that they blocked HFD-induced increase of pancreatic islet size. Our pyrosequencing of the 16S rRNA gene V3 region from stools revealed that both SGE and GE modulated HFD-altered composition of gut microbiota. Therefore, we conclude that Korean red ginseng inhibits HFD-induced obesity and diabetes by altering gut microbiome.

Human islet expression levels of Prostaglandin E2 synthetic enzymes, but not prostaglandin EP3 receptor, are positively correlated with markers of β-cell function and mass in non-diabetic obesity

We and others previously reported increased signaling through the Prostaglandin E3 Receptor (EP3), a G protein-coupled receptor (GPCR) for the arachidonic acid metabolite, prostaglandin E2 (PGE2), is associated with β-cell dysfunction of type 2 diabetes (T2D). Yet, the relationship between PGE2 production and signaling and β-cell function during the progression to T2D remains unclear. In this work, we assessed gene expression from a panel of cadaveric human islets from 40 non-diabetic donors with BMI values spanning the spectrum from lean to high-risk obesity. Interleukin-6 (gene symbol: IL6) and cyclooxygenase-2 (COX-2) (gene symbol: PTGS2) mRNA levels were positively correlated with donor body mass index (BMI), while EP3 (gene symbol: PTGER3) was not. IL6 was itself strongly correlated with PTGS2 and all but one of the other PGE2 synthetic pathway genes tested. About half of the islet preparations were used in glucose-stimulated- and incretin-potentiated insulin secretion assays using an EP3-specific antagonist, confirming functionally-relevant up-regulation of PGE2 production. Islets from obese donors showed no inherent β-cell dysfunction and were at least equally as glucose- and incretin-responsive as islets from non-obese donors. Furthermore, insulin content, a marker of islet size known to be associated with donor BMI, was also significantly and positively correlated with islet PTGS2 expression. We conclude up-regulated islet PGE2 production and signaling may be a necessary part of the β-cell adaption response, compensating for obesity and insulin resistance. Analysis of plasma PGE2 metabolite levels from a clinical cohort reveal these findings are not in conflict with the concept of further elevations in PGE2 production contributing to T2D-related β-cell dysfunction where islet EP3 expression has also been up-regulated.Graphical Abstract

Chronic Fetal Leucine Infusion Does Not Potentiate Glucose-Stimulated Insulin Secretion or Affect Pancreatic Islet Development in Late-Gestation Growth-Restricted Fetal Sheep

ABSTRACT Background Growth-restricted fetuses have attenuated glucose-stimulated insulin secretion (GSIS), smaller pancreatic islets, less pancreatic β-cells, and less pancreatic vascularization compared with normally growing fetuses. Infusion of leucine into normal late-gestation fetal sheep potentiates GSIS, as well as increases pancreatic islet size, the proportion of the pancreas and islet comprising β-cells, and pancreatic and islet vascularity. In addition, leucine stimulates hepatocyte growth factor (HGF ) mRNA expression in islet endothelial cells isolated from normal fetal sheep. Objective We hypothesized that a 9-d leucine infusion would potentiate GSIS and increase pancreatic islet size, β-cells, and vascularity in intrauterine fetal growth restriction (IUGR) fetal sheep. We also hypothesized that leucine would stimulate HGF mRNA in islet endothelial cells isolated from IUGR fetal sheep. Methods Late-gestation Columbia-Rambouillet IUGR fetal sheep (singleton or twin) underwent surgeries to place vascular sampling and infusion catheters. Fetuses were randomly allocated to receive a 9-d leucine infusion to achieve a 50–100% increase in leucine concentrations or a control saline infusion. GSIS was measured and pancreas tissue was processed for histologic analysis. Pancreatic islet endothelial cells were isolated from IUGR fetal sheep and incubated with supplemental leucine. Data were analyzed by mixed-models ANOVA; Student, Mann-Whitney, or a paired t test; or a test of equality of proportions. Results Chronic leucine infusion in IUGR fetuses did not affect GSIS, islet size, the proportion of the pancreas comprising β-cells, or pancreatic or pancreatic islet vascularity. In isolated islet endothelial cells from IUGR fetuses, HGF mRNA expression was not affected by supplemental leucine. Conclusions IUGR fetal sheep islets are not responsive to a 9-d leucine infusion with respect to insulin secretion or any histologic features measured. This is in contrast to the response in normally growing fetuses. These results are important when considering nutritional strategies to prevent the adverse islet and β-cell consequences in IUGR fetuses.

Morphometric Study of Changes in GAD 65 Expressing Beta Cell Mass In The Islets Of Langerhans In Pregnant Mice

INTRODUCTION: This study explored the adaptive changes in pancreatic islets by assessing the maternal pancreatic β-cell mass (PβCM), islets size, number, shape, distribution and vascularity using the anti-GAD65 Ab as a marker in pregnant mice. MATERIALS AND METHODS: Fourty female Swiss-Webster mice were randomly divided into two age matched groups of 20 animals each, pregnant and control (non-pregnant) groups. The pregnant mice were sacrificed at Day 19 of gestation and the control group was sacrificed 19 days after observation in the same environmental conditions. Specimens of pancreata were fixed in 10% formal-saline, and processed for light microscopy. Paraffin sections were stained with chromogen DAB. Image scope (APERIO) with Image J software was used for morphometric study. RESULTS: Islets cells showed specific reaction with GAD65 Ab. There was an increase in the cellularity and vascularity of the PβCM. A significant increase in the islets area of the pregnant group ranging between 32-723 µm2 in comparison with the control group that ranged between 5–210 µm2 , the islet size and numbers also increased; there were numerous newly formed islets and coalescence of adjacent islets, positive reactivity of β-cells toward antiGAD65 during pregnancy, the mean of positive granulation in PβCM in pregnant group was 8273.5±SE 831.35 while in control group was 34±SE 0.2. CONCLUSION: The changes for alteration in PβCM such as increase in cells number, size, positive anti-GAD65 reactivity and their vascularity during pregnancy is related to the adaptation for hormonal and nutritional demands of both foetus and mother.

Comparison of Islet Characterization from Use of Standard Crude Collagenase to GMP-Grade Collagenase Enzyme Blends in Preweaned Porcine Islet Isolations

For the advancement of porcine xenotransplantation for clinical use in type 1 diabetes mellitus, the concerns of a sustainable and safe digestion enzyme blend must be overcome. Incorporating good manufacturing practices (GMP) can facilitate this through utilizing GMP-grade enzymes. In conjunction, still taking into account the cost-effectiveness, a wide concern. We evaluated how GMP-grade enzyme blends impact our piglet islets and their long-term effects. Preweaned porcine islets (PPIs) were isolated from 8- to 10-day-old pigs. Digestion enzyme blends, collagenase type V (Type V), collagenase AF-1 GMP-grade with collagenase NB 6 GMP-grade (AF-1 and NB 6), and collagenase AF-1 GMP-grade with collagenase neutral protease AF GMP-grade (AF-1 and NP AF) were compared. Islet quality control assessments, islet yield, viability, and function, were performed on days 3 and 7, and cell content was performed on day 7. GMP-grade AF-1 and NB 6 (17,209 ± 2,730 islet equivalent per gram of pancreatic tissue [IE/g] on day 3, 9,001 ± 1,034 IE/g on day 7) and AF-1 and NP AF (17,214 ± 3,901 IE/g on day 3, 8,833 ± 2,398 IE/g on day 7) showed a significant increase in islet yield compared to Type V (4,618 ± 1,240 IE/g on day 3, 1,923 ± 704 IE/g on day 7). Islet size, viability, and function showed comparable results in all enzyme blends. There was no significant difference in islet cellular content between enzyme blends. This study demonstrated a comparison of GMP-grade collagenase enzyme blends and a standard crude collagenase enzyme in preweaned-aged porcine, a novel topic in this age. GMP-grade enzyme blends of AF-1 and NB 6 and AF-1 and NP AF resulted in substantially higher yields and as effective PPIs compared to Type V. In the long run, considering costs, integrity, and sustainability, GMP-grade enzyme blends are more favorable for clinical application due to high reproducibility in comparison to undefined manufacturing processes of standard enzymes.

Histopathological picture of pancreatic islets in dogs with congenital portosystemic shunt

Hypoglycemia is frequently found in dogs suffering from portosystemic shunt (PSS). However, the mechanisms leading to abnormal blood glucose concentration in such dogs have not been studied. Therefore, investigations were undertaken to study the structure of pancreatic islets in 25 patients with congenital PSS (cPSS). Material for morphometry and histopathology was taken during the surgical closing of the abnormal blood vessel. A total of 75 islets (3 randomly chosen from each patient) were analyzed, and their average size was compared to reference values for dogs that were considered as 50-325 μm. The average size of 47 (63%) islets was below 50 μm, whereas the mean dimension of the largest islet did not exceeded 80 μm. The average islet size in all patients was 46 (SD 13.3) μm and in only 2 dogs (8%) all 3 analyzed islets were bigger than the lower reference value. Histopathological examination revealed cytoplasmic vesicles in pancreatic cells, as well as extracellular, homogenous, acidophilic deposits in pancreatic islets. These results indicate that in dogs suffering from cPSS the pancreatic islets are smaller than the reference values and their cells may contain abnormal structures.

Pancreas-specific miR-216a regulates proliferation and endocrine and exocrine cell function in vivo

Pancreas is a vital organ composed of exocrine and endocrine cells that aid digestion of food and regulate blood glucose levels. Perturbations in the function of pancreatic cells leads to the development of life-burdening and/or threatening diseases such as diabetes and pancreatic cancer. Thus, it is critical to understand the molecular check-points that maintain normal pancreas physiology. MicroRNAs (miRNAs) are small non-coding RNAs involved in regulating gene expression in normal and diseased tissues. Several miRNAs have tissue-specific patterns consistent with crucial functions in many biological processes. Yet, there is limited knowledge about the role of pancreas-specific miRNAs in pancreatic pathologies. Here, we report that miR-216a is a conserved, pancreas-specific miRNA that is expressed in both endocrine and exocrine cells. Deletion of miR-216a in mice leads to reduced β-cell mass and a reduction in islet size under both chow and high-fat diet feeding conditions. We show that inhibition of miR-216a increases apoptosis and decreases cell proliferation in β- and exocrine cells. Smad7 is upregulated in miR-216a deficient islets and cell cycle and proliferation are among the most significantly regulated biological processes in miR-216 knockout pancreata. Re-introduction of miR-216a in the pancreatic cancer line, PANC-1, increases cell migration more than 2-fold. In vivo, deletion of miR-216a in the pancreatic cancer prone mouse line KrasG12D;Ptf1aCreER inhibits the propensity of pancreatic cancer precursor lesions. Our study identifies miR-216a as an important pancreas-specific miRNA which may have implications for both diabetes and pancreatic cancer.

Impaired glucose tolerance, glucagon, and insulin responses in mice lacking the loop diuretic-sensitive Nkcc2a transporter

The Na+K+2Cl− cotransporter-2 ( Nkcc2, Slc12a1) is abundantly expressed in the kidney and its inhibition with the loop-diuretics bumetanide and furosemide has been linked to transient or permanent hyperglycemia in mice and humans. Notably, Slc12a1 is expressed at low levels in hypothalamic neurons and in insulin-secreting β-cells of the endocrine pancreas. The present study was designed to determine if global elimination of one of the Slc12a1 products, i.e., Nkcc2 variant a ( Nkcc2a), the main splice version of Nkcc2 found in insulin-secreting β-cells, has an impact on the insulin and glucagon secretory responses and fuel homeostasis in vivo. We have used dynamic tests of glucose homeostasis in wild-type mice and mice lacking both alleles of Nkcc2a ( Nkcc2aKO) and assessed their islet secretory responses in vitro. Under basal conditions, Nkcc2aKO mice have impaired glucose homeostasis characterized by increased blood glucose, intolerance to the sugar, delayed/blunted in vivo insulin and glucagon responses to glucose, and increased glycemic responses to the gluconeogenic substrate alanine. Further, we provide evidence of conserved quantitative secretory responses of Nkcc2aKO islets within a context of increased islet size related to hyperplastic/hypertrophic glucagon- and insulin-positive cells (α-cells and β-cells, respectively), normal total islet Cl− content, and reduced β-cell expression of the Cl− extruder Kcc2.