Pleiotrophin Expression and Actions in Pancreatic ß-Cells.

Pleiotrophin (PTN) is a heparin-binding cytokine that is widely expressed during early development and increases in maternal circulation during pregnancy.Aged PTN-deficient mice exhibit insulin resistance, suggesting a role in metabolic control. The objectives of this study were to determine if PTN is expressed in mouse pancreatic ß-cells in young vs. adult animals, and its effects on DNA synthesis, ß-cell gene expression and glucose-stimulated insulin secretion (GSIS). The Ptn gene was expressed in isolated fractions of young mouse ß-cells, especially within immature ß-cells with low glucose transporter 2 expression. Expression was retained in the adult pancreas but did not significantly change during pregnancy. PTN and its receptor, phosphotyrosine phosphatase-ß/ζ, were also expressed in the proliferative INS1E ß-cell line. Fluorescence immunohistochemistry showed that PTN peptide was present in islets of Langerhans in adult mice, associated predominantly with ß-cells. The percentage of ß-cells staining for PTN did not alter during mouse pregnancy, but intense staining was seen during ß-cell regeneration in young mice following depletion of ß-cells with streptozotocin. Incubation of INS1E cells with PTN resulted in an increased DNA synthesis as measured by Ki67 localization and increased expression of Pdx1 and insulin. However, both DNA synthesis and GSIS were not altered by PTN in isolated adult mouse islets. The findings show that Ptn is expressed in mouse ß-cells in young and adult life and could potentially contribute to adaptive increases in ß-cell mass during early life or pregnancy.

Acetone Ingestion Mimics a Fasting State to Improve Glucose Tolerance in a Mouse Model of Gestational Hyperglycemia.

Gestational diabetes mellitus results, in part, from a sub-optimal ß-cell mass (BCM) during pregnancy. Artemisinins were reported to increase BCM in models of diabetes by α- to ß-cell conversion leading to enhanced glucose tolerance. We used a mouse model of gestational glucose intolerance to compare the effects of an artemisinin (artesunate) on glycemia of pregnant mice with vehicle treatment (acetone) or no treatment. Animals were treated daily from gestational days (GD) 0.5 to 6.5. An intraperitoneal glucose tolerance test was performed prior to euthanasia at GD18.5 or post-partum. Glucose tolerance was significantly improved in both pregnant and non-pregnant mice with both artesunate and vehicle-alone treatment, suggesting the outcome was primarily due to the acetone vehicle. In non-pregnant, acetone-treated animals, improved glucose tolerance was associated with a higher BCM and a significant increase in bihormonal insulin and glucagon-containing pancreatic islet cells, suggesting α- to ß-cell conversion. BCM did not differ with treatment during pregnancy or post-partum. However, placental weight was higher in acetone-treated animals and was associated with an upregulation of apelinergic genes. Acetone-treated animals had reduced weight gain during treatment despite comparable food consumption to non-treated mice, suggesting transient effects on nutrient uptake. The mean duodenal and ileum villus height was reduced following exposure to acetone. We conclude that acetone treatment may mimic transient fasting, resulting in a subsequent improvement in glucose tolerance during pregnancy.

Ontology of the apelinergic system in mouse pancreas during pregnancy and relationship with ß-cell mass.

The apelin receptor (Aplnr) and its ligands, Apelin and Apela, contribute to metabolic control. The insulin resistance associated with pregnancy is accommodated by an expansion of pancreatic ß-cell mass (BCM) and increased insulin secretion, involving the proliferation of insulin-expressing, glucose transporter 2-low (Ins+Glut2LO) progenitor cells. We examined changes in the apelinergic system during normal mouse pregnancy and in pregnancies complicated by glucose intolerance with reduced BCM. Expression of Aplnr, Apelin and Apela was quantified in Ins+Glut2LO cells isolated from mouse pancreata and found to be significantly higher than in mature ß-cells by DNA microarray and qPCR. Apelin was localized to most ß-cells by immunohistochemistry although Aplnr was predominantly associated with Ins+Glut2LO cells. Aplnr-staining cells increased three- to four-fold during pregnancy being maximal at gestational days (GD) 9-12 but were significantly reduced in glucose intolerant mice. Apelin-13 increased ß-cell proliferation in isolated mouse islets and INS1E cells, but not glucose-stimulated insulin secretion. Glucose intolerant pregnant mice had significantly elevated serum Apelin levels at GD 9 associated with an increased presence of placental IL-6. Placental expression of the apelinergic axis remained unaltered, however. Results show that the apelinergic system is highly expressed in pancreatic ß-cell progenitors and may contribute to ß-cell proliferation in pregnancy.

Increased alpha and beta cell mass during mouse pregnancy is not dependent on transdifferentiation.

Maternal pancreatic beta-cell mass (BCM) increases during pregnancy to compensate for relative insulin resistance. If BCM expansion is suboptimal, gestational diabetes mellitus can develop. Alpha-cell mass (ACM) also changes during pregnancy, but there is a lack of information about α-cell plasticity in pregnancy and whether α- to ß-cell transdifferentiation can occur. To investigate this, we used a mouse model of gestational glucose intolerance induced by feeding low-protein (LP) diet from conception until weaning and compared pregnant female offspring to control diet-fed animals. Control and LP pancreata were collected for immunohistochemical analysis and serum glucagon levels were measured. In order to lineage trace α- to ß-cell conversion, we utilized transgenic mice expressing yellow fluorescent protein behind the proglucagon gene promoter (Gcg-Cre/YFP) and collected pancreata for histology at various gestational timepoints. Alpha-cell proliferation increased significantly at gestational day (GD) 9.5 in control pregnancies resulting in an increased ACM at GD18.5, and this was significantly reduced in LP animals. Despite these changes, serum glucagon was higher in LP mice at GD18.5. Pregnant Gcg-Cre/YFP mice showed no increase in the abundance of insulin+YFP+glucagon- cells (phenotypic ß-cells). A second population of insulin+YFP+glucagon+ cells was identified which also did not alter during pregnancy. However, there was an altered anatomical distribution within islets with fewer insulin+YFP+glucagon- cells but more insulin+YFP+glucagon+ cells being present in the islet mantle at GD18.5. These findings demonstrate that dynamic changes in ACM occur during normal pregnancy and were altered in glucose-intolerant pregnancies.

Altered pancreas remodeling following glucose intolerance in pregnancy in mice.

Gestational diabetes mellitus increases the risk of dysglycemia postpartum, in part, due to pancreatic ß-cell dysfunction. However, no histological evidence exists comparing endocrine pancreas after healthy and glucose-intolerant pregnancies. This study sought to address this knowledge gap, in addition to exploring the contribution of an inflammatory environment to changes in endocrine pancreas after parturition. We used a previously established mouse model of gestational glucose intolerance induced by dietary low protein insult from conception until weaning. Pancreas and adipose samples were collected at 7, 30 and 90 days postpartum for histomorphometric and cytokine analyses, respectively. Glucose tolerance tests were performed prior to euthanasia and blood was collected via cardiac puncture. Pregnant female mice born to dams fed a low protein diet previously shown to develop glucose intolerance at late gestation relative to controls continued to be glucose intolerant until 1 month postpartum. However, glucose tolerance normalized by 3 months postpartum. Glucose intolerance at 7 days postpartum was associated with lower beta- and alpha-cell fractional areas and higher adipose levels of pro-inflammatory cytokine, interleukin-6. By 3 months postpartum, a compensatory increase in the number of small islets and a higher insulin to glucagon ratio likely enabled euglycemia to be attained in the previously glucose-intolerant mice. The results show that impairments in endocrine pancreas compensation in hyperglycemic pregnancy persist after parturition and contribute to prolonged glucose intolerance. These impairments may increase the susceptibility to development of future type 2 diabetes.

A mouse model of gestational glucose intolerance through exposure to a low protein diet during fetal and neonatal development.

KEY POINTS: Pancreatic ß-cell dysfunction is hypothesized to be the significant determinant of gestational diabetes pathogenesis, however pancreatic samples from patients are scarce. This study reports a novel mouse model of gestational glucose intolerance in pregnancy, originating from previous nutrition restriction in utero, in which glucose intolerance was restricted to late gestation as is seen in human gestational diabetes. Glucose intolerance was attributed to reduced ß-cell proliferation, leading to impaired gestational ß-cell mass expansion in maternal endocrine pancreas, in addition to reduced glucose-stimulated insulin secretion. This model reproduces some of the features of gestational diabetes and is suitable for testing safe therapeutic interventions that increase ß-cell mass during pregnancy and prevent or reverse gestational glucose intolerance. ABSTRACT: Gestational diabetes mellitus (GDM) is an increasingly prevalent form of diabetes that appears during pregnancy. Pathological studies link a failure to adaptively increase maternal pancreatic ß-cell mass (BCM) in pregnancy to GDM. Due to the scarcity of pancreatic samples from GDM patients, we sought to develop a novel mouse model for impaired gestational glucose tolerance. Mature female C57Bl/6 mouse offspring (F1) born to dams fed either a control (C) or low-protein (LP) diet during gestation and lactation were randomly allocated into two subsequent study groups: pregnant (CP, LPP) or non-pregnant (CNP, LPNP). Glucose tolerance tests were performed at gestational day (GD) 9, 12 and 18. Subsequently, pancreata were removed for fluorescence immunohistochemistry to assess α-cell mass (ACM), BCM and ß-cell proliferation. An additional group of animals was used to measure insulin secretion from isolated islets at GD18. LPP females displayed glucose intolerance compared to CP females at GD18 (P < 0.001). BCM increased threefold at GD18 in CP females. However, LPP females had reduced BCM expansion (P < 0.01) concurrent with reduced ß-cell proliferation at GD12 (P < 0.05). LPP females also had reduced ACM expansion at GD18 (P < 0.01). LPP islets had impaired glucose-stimulated insulin secretion in vitro compared to CP islets (P < 0.01). Therefore, impaired glucose tolerance during pregnancy is associated with a failure to adequately adapt BCM, as a result of reduced ß-cell proliferation, in addition to lower glucose-stimulated insulin secretion. This model could be used to evaluate novel interventions during pregnancy to increase BCM or function as a strategy to prevent/reverse GDM.

Offspring of Mice Exposed to a Low-Protein Diet in Utero Demonstrate Changes in mTOR Signaling in Pancreatic Islets of Langerhans, Associated with Altered Glucagon and Insulin Expression and a Lower ß-Cell Mass.

Low birth weight is a risk factor for gestational and type 2 diabetes (T2D). Since mammalian target of rapamycin (mTOR) controls pancreatic ß-cell mass and hormone release, we hypothesized that nutritional insult in utero might permanently alter mTOR signaling. Mice were fed a low-protein (LP, 8%) or control (C, 20%) diet throughout pregnancy, and offspring examined until 130 days age. Mice receiving LP were born 12% smaller and ß-cell mass was significantly reduced throughout life. Islet mTOR levels were lower in LP-exposed mice and localized predominantly to α-rather than ß-cells. Incubation of isolated mouse islets with rapamycin significantly reduced cell proliferation while increasing apoptosis. mRNA levels for mTORC complex genes mTOR, Rictor and Raptor were elevated at 7 days in LP mice, as were the mTOR and Raptor proteins. Proglucagon gene expression was similarly increased, but not insulin or the immune/metabolic defense protein STING. In human and mouse pancreas STING was strongly associated with islet ß-cells. Results support long-term changes in islet mTOR signaling in response to nutritional insult in utero, with altered expression of glucagon and insulin and a reduced ß-cell mass. This may contribute to an increased risk of gestational or type 2 diabetes.

Direct comparison of the abilities of bone marrow mesenchymal versus hematopoietic stem cells to reverse hyperglycemia in diabetic NOD.SCID mice.

Both bone marrow-derived hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC) improve glycemic control in diabetic mice, but their kinetics and associated changes in pancreatic morphology have not been directly compared. Our goal was to examine the time course of improvements in glucose tolerance and associated changes in ß-cell mass and proliferation following transplantation of equivalent numbers of HSC or MSC from the same bone marrow into diabetic non-obese diabetic severe combined immune deficiency (NOD.SCID) mice. We used transgenic mice with a targeted expression of yellow fluorescent protein (YFP) driven by the Vav1 gene promoter to genetically tag HSC and progeny. HSC were separated from bone marrow by fluorescence-activated cell sorting and MSC following cell culture. Equivalent numbers of isolated HSC or MSC were transplanted directly into the pancreas of NOD.SCID mice previously made diabetic with streptozotocin. Glucose tolerance, serum insulin, ß-cell mass and ß-cell proliferation were examined up to 28 days following transplant. Transplantation with MSC improved glucose tolerance within 7 days and serum insulin levels increased, but with no increase in ß-cell mass. Mice transplanted with HSC showed improved glucose tolerance only after 3 weeks associated with increased ß-cell proliferation and mass. We conclude that single injections of either MSC or HSC transiently improved glycemic control in diabetic NOD.SCID mice, but with different time courses. However, only HSC infiltrated the islets and were associated with an expanded ß-cell mass. This suggests that MSC and HSC have differing mechanisms of action.

An increase in immature ß-cells lacking Glut2 precedes the expansion of ß-cell mass in the pregnant mouse.

A compensatory increase in ß-cell mass occurs during pregnancy to counter the associated insulin resistance, and a failure in adaptation is thought to contribute to gestational diabetes. Insulin-expressing but glucose-transporter-2-low (Ins+Glut2LO) progenitor cells are present in mouse and human pancreas, being predominantly located in extra-islet ß-cell clusters, and contribute to the regeneration of the endocrine pancreas following induced ablation. We therefore sought to investigate the contribution of Ins+Glut2LO cells to ß-cell mass expansion during pregnancy. Female C57Bl/6 mice were time mated and pancreata were collected at gestational days (GD) 6, 9, 12, 15, and 18, and postpartum D7 (n = 4/time-point) and compared to control (non-pregnant) animals. Beta cell mass, location, proliferation (Ki67+), and proportion of Ins+Glut2LO cells were measured using immunohistochemistry and bright field or confocal microscopy. Beta cell mass tripled by GD18 and ß-cell proliferation peaked at GD12 in islets (≥6 ß-cells) and small ß-cell clusters (1-5 ß-cells). The proportion and fraction of Ins+Glut2LO cells undergoing proliferation increased significantly at GD9 in both islets and clusters, preceding the increase in ß-cell mass and proliferation, and their proliferation within clusters persisted until GD15. The overall number of clusters increased significantly at GD9. Quantitative PCR showed a significant increase in Pdx1 presence at GD9 vs. GD18 or control pancreas, and Pdx1 was visualized by immunohistochemistry within both Ins+Glut2LO and Ins+Glut2HI cells within clusters. These results indicate that Ins+Glut2LO cells are likely to contribute to ß-cell mass expansion during pregnancy.

Decrease in Ins+Glut2LO ß-cells with advancing age in mouse and human pancreas.

The presence and location of resident pancreatic ß-cell progenitors is controversial. A subpopulation of insulin-expressing but glucose transporter-2-low (Ins+Glut2LO) cells may represent multipotent pancreatic progenitors in adult mouse and in human islets, and they are enriched in small, extra-islet ß-cell clusters (<5 ß cells) in mice. Here, we sought to identify and compare the ontogeny of these cells in mouse and human pancreata throughout life. Mouse pancreata were collected at postnatal days 7, 14, 21, 28, and at 3, 6, 12, and 18 months of age, and in the first 28 days after ß-cell mass depletion following streptozotocin (STZ) administration. Samples of human pancreas were examined during fetal life (22-30 weeks gestation), infancy (0-1 year), childhood (2-9), adolescence (10-17), and adulthood (18-80). Tissues were analyzed by immunohistochemistry for the expression and location of insulin, GLUT2 and Ki67. The proportion of ß cells within clusters relative to that in islets was higher in pancreas of human than of mouse at all ages examined, and decreased significantly at adolescence. In mice, the total number of Ins+Glut2LO cells decreased after 7 days concurrent with the proportion of clusters. These cells were more abundant in clusters than in islets in both species. A positive association existed between the appearance of new ß cells after the STZ treatment of young mice, particularly in clusters and smaller islets, and an increased proportional presence of Ins+Glut2LO cells during early ß-cell regeneration. These data suggest that Ins+Glut2LO cells are preferentially located within ß-cell clusters throughout life in pancreas of mouse and human, and may represent a source of ß-cell plasticity.

Insulin-positive, Glut2-low cells present within mouse pancreas exhibit lineage plasticity and are enriched within extra-islet endocrine cell clusters.

Regeneration of insulin-producing ß-cells from resident pancreas progenitors requires an understanding of both progenitor identity and lineage plasticity. One model suggested that a rare ß-cell sub-population within islets demonstrated multi-lineage plasticity. We hypothesized that ß-cells from young mice (postnatal day 7, P7) exhibit such plasticity and used a model of islet dedifferentiation toward a ductal epithelial-cell phenotype to test this theory. RIPCre;Z/AP(+/+) mice were used to lineage trace the fate of ß-cells during dedifferentiation culture by a human placental alkaline phosphatase (HPAP) reporter. There was a significant loss of HPAP-expressing ß-cells in culture, but remaining HPAP(+) cells lost insulin expression while gaining expression of the epithelial duct cell marker cytokeratin-19 (Ck19). Flow cytometry and recovery of ß-cell subpopulations from whole pancreas vs. islets suggest that the HPAP(+)Ck19(+) cells had derived from insulin-positive, glucose-transporter-2-low (Ins(+)Glut2(LO)) cells, representing 3.5% of all insulin-expressing cells. The majority of these cells were found outside of islets within clusters of <5 ß-cells. These insulin(+)Glut2(LO) cells demonstrated a greater proliferation rate in vivo and in vitro as compared to insulin(+)Glut2(+) cells at P7, were retained into adulthood, and a subset differentiated into endocrine, ductal, and neural lineages, illustrating substantial plasticity. Results were confirmed using RIPCre;ROSA- eYFP mice. Quantitative PCR data indicated these cells possess an immature ß-cell phenotype. These Ins(+)Glut2(LO) cells may represent a resident population of cells capable of forming new, functional ß-cells, and which may be potentially exploited for regenerative therapies in the future.

Featured Article: Beta cell specific pyruvate dehydrogenase alpha gene deletion results in a reduced islet number and ß-cell mass postnatally.

The ability of pancreatic ß-cells to undertake glucose-stimulated insulin secretion (GSIS) depends on the generation of adenosine triphosphate (ATP) within the mitochondria from pyruvate, a major rate-limiting enzyme being pyruvate dehydrogenase (PDH) complex (PDC). However, glucose metabolism also controls ß-cell mass. To examine the role of PDC in the regulation of pancreatic ß-cell development and maturation, we generated ß-cell-targeted PDHα subunit knock-out male mice (ß-PDHKO) and compared these with control males (ß-PDHCT) from birth until 6-8 weeks age. Pancreas morphology, transcription factor expression, pancreatic insulin content, and circulating glucose and insulin values were compared. Compared to ß-PDHCT male mice, ß-PDHKO animals had significantly reduced pancreatic insulin content from birth, a lower serum insulin content from day 15, and relative hyperglycemia from day 30. Isolated islets from ß-PDHKO mice demonstrated a reduced GSIS. The number of islets per pancreatic area, mean islet area, and the proportion of islet cells that were ß-cells were all reduced in ß-PDHKO animals. Similarly the number of insulin-immunopositive, extra-islet small endocrine cell clusters, a possible source of ß-cell progenitors, was lower in ß-PDHKO mice. Analysis of pancreatic expression of transcription factors responsible for ß-cell lineage commitment, proliferation, and maturation, Pdx1, Neurogenin3, and NeuroD1 showed that mRNA abundance was reduced in the ß-PDHKO. This demonstrates that PDC is not only required for insulin expression and glucose-stimulated secretion, but also directly influences ß-cell growth and maturity, and positions glucose metabolism as a direct regulator of ß-cell mass and plasticity.

NOX2 deficiency protects against streptozotocin-induced beta-cell destruction and development of diabetes in mice.

OBJECTIVE: The role of NOX2-containing NADPH oxidase in the development of diabetes is not fully understood. We hypothesized that NOX2 deficiency decreases reactive oxygen species (ROS) production and immune response and protects against streptozotocin (STZ)-induced ß-cell destruction and development of diabetes in mice. RESEARCH DESIGN AND METHODS: Five groups of mice--wild-type (WT), NOX2(-/-), WT treated with apocynin, and WT adoptively transferred with NOX2(-/-) or WT splenocytes--were treated with multiple-low-dose STZ. Blood glucose and insulin levels were monitored, and an intraperitoneal glucose tolerance test was performed. Isolated WT and NOX2(-/-) pancreatic islets were treated with cytokines for 48 h. RESULTS: Significantly lower blood glucose levels, higher insulin levels, and better glucose tolerance was observed in NOX2(-/-) mice and in WT mice adoptively transferred with NOX2(-/-) splenocytes compared with the respective control groups after STZ treatment. Compared with WT, ß-cell apoptosis, as determined by TUNEL staining, and insulitis were significantly decreased, whereas ß-cell mass was significantly increased in NOX2(-/-) mice. In response to cytokine stimulation, ROS production was significantly decreased, and insulin secretion was preserved in NOX2(-/-) compared with WT islets. Furthermore, proinflammatory cytokine release induced by concanavalin A was significantly decreased in NOX2(-/-) compared with WT splenocytes. CONCLUSIONS: NOX2 deficiency decreases ß-cell destruction and preserves islet function in STZ-induced diabetes by reducing ROS production, immune response, and ß-cell apoptosis.

Maternal hyperinsulinemia predisposes rat fetuses for hyperinsulinemia, and adult-onset obesity and maternal mild food restriction reverses this phenotype.

We have previously shown that artificial rearing of newborn female rat pups on a high-carbohydrate (HC) milk formula resulted in chronic hyperinsulinemia and adult-onset obesity (HC phenotype) and that the maternal HC phenotype was transmitted to their progeny (2-HC rats) because of fetal development in the HC female rat. The aims of this study were to investigate 1) the fetal adaptations that predisposed the progeny for the expression of the HC phenotype in adulthood and 2) whether the transfer of the HC phenotype to the progeny could be reversed by maternal food restriction. Fetal parameters such as plasma insulin and glucose levels, mRNA level of preproinsulin gene, pancreatic insulin content, and islet insulin secretory response in vitro were determined. On gestational day 21, 2-HC fetuses were hyperinsulinemic, had increased insulin content and mRNA level of the preproinsulin gene in their pancreata and demonstrated an altered glucose-stimulated insulin secretory response by isolated islets. Modification of the intrauterine environment in HC female rats was achieved by pair feeding them to the amount of diet consumed by age-matched control rats from the time of their weaning. This mild dietary restriction reversed their HC phenotype and also prevented the development of the HC phenotype in their progeny. These findings show that mal-programming of the progeny of the hyperinsulinemic-obese HC female for the expression of the HC phenotype is initiated in utero and that normalization of the maternal environment in HC female rats by mild food restriction resulted in the normal phenotype in their progeny.

Bone marrow-derived stem cells initiate pancreatic regeneration.

We show that transplantation of adult bone marrow-derived cells expressing c-kit reduces hyperglycemia in mice with streptozotocin-induced pancreatic damage. Although quantitative analysis of the pancreas revealed a low frequency of donor insulin-positive cells, these cells were not present at the onset of blood glucose reduction. Instead, the majority of transplanted cells were localized to ductal and islet structures, and their presence was accompanied by a proliferation of recipient pancreatic cells that resulted in insulin production. The capacity of transplanted bone marrow-derived stem cells to initiate endogenous pancreatic tissue regeneration represents a previously unrecognized means by which these cells can contribute to the restoration of organ function.