Mechanisms of beta-cell deficiency in gestational diabetes and strategies to reverse hyperglycemia

Gestational diabetes mellitus (GDM) is an increasingly prevalent form of diabetes that first appears during pregnancy, and reverses after parturition in most cases. Nonetheless, GDM is associated with adverse maternal and fetal health outcomes. There is currently no reliable method of intervention for GDM and a limited understanding of the mechanisms of impaired endocrine adaptability in GDM. In this thesis, I aimed to address these knowledge gaps by establishing a mouse model for the study of suboptimal endocrine adaptations during pregnancy. This was accomplished using a dietary low protein (LP) insult during fetal and neonatal development, which programs for suboptimal pancreas development in the offspring, and performing histomorphometric analyses on fixed pancreas tissues. Female offspring displayed glucose intolerance during their own pregnancy that was apparent by gestational day (GD) 18.5 and characterized by reduced β-cell mass (BCM) and α-cell mass (ACM) relative to control-fed animals. Using this model, I provided evidence that pancreatic maladaptations at GD18.5 persisted at postpartum day 7.5, contributing to glucose intolerance until 1 month after parturition. To provide mechanistic insights of reduced BCM expansion in GDM, I investigated the contribution of α- to β-cell transdifferentiation via immunofluorescence cell counting analysis of fixed pancreas tissues. I identified maladaptations of α-cell plasticity in glucose-intolerant mice, as demonstrated by reduced α-cell proliferation, leading to reduced ACM expansion relative to controls. Additionally, these animals presented with hyperglucagonemia. These findings demonstrated that, in addition to β-cells, insufficient pancreatic α-cell adaptations can also contribute to GDM pathogenesis. Although there were differences in the percentages of bihormonal (Insulin+Glucagon+) cells in LP vs. control pregnancy, genetic lineage tracing in control pregnancy using Glucagon-Cre/Rosa26-eYFP mice revealed a negligible amount of α- to β-cell transdifferentiation contributing to BCM expansion. Finally, I used the animal model to test a therapeutic intervention for GDM through the attempted manipulation of BCM using the artemisinin, artesunate. Artesunate-treated animals had improved glucose tolerance, although the glucose-lowering effect was attributed to the acetone vehicle. Collectively, this thesis has identified mechanisms of impaired endocrine pancreas adaptability in GDM and has established a mouse model that can be used to explore novel therapeutics

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

A compensatory increase in β-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 β-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 β-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 β-cell proliferation peaked at GD12 in islets (6 β-cells) and small β-cell clusters (1–5 β-cells). The proportion and fraction of Ins+Glut2LO cells undergoing proliferation increased significantly at GD9 in both islets and clusters, preceding the increase in β-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 β-cell mass expansion during pregnancy

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

Key points: Pancreatic β-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 β-cell proliferation, leading to impaired gestational β-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 β-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 β-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 β-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 \u3c 0.001). BCM increased threefold at GD18 in CP females. However, LPP females had reduced BCM expansion (P \u3c 0.01) concurrent with reduced β-cell proliferation at GD12 (P \u3c 0.05). LPP females also had reduced ACM expansion at GD18 (P \u3c 0.01). LPP islets had impaired glucose-stimulated insulin secretion in vitro compared to CP islets (P \u3c 0.01). Therefore, impaired glucose tolerance during pregnancy is associated with a failure to adequately adapt BCM, as a result of reduced β-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

Altered pancreas remodeling following glucose intolerance in pregnancy in mice

Gestational diabetes mellitus increases the risk of dysglycemia postpartum, in part, due to pancreatic β-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

An increase in immature β-cells lacking Glut2 precedes the expansion of β-cell mass in the pregnant mouse - Fig 3

<p><b>Presence of β-cell clusters (A -D) and islets (E & F) in mouse pancreas at gestational day (GD) 9.</b> Immunohistochemistry was used to localize insulin (red), Glut2 (green), and Ki67 (white), whilst nuclei were counter-stained with DAPI (blue). Panels A-C show the same cluster to demonstrate insulin (A), Glut2 (B) and the merged image (C). Ins⁺Glut2^LO cells within clusters or islets without nuclear staining for Ki67 are indicated by the solid arrows in A-C and E. An Ins⁺Glut2^LO cell within a cluster showing nuclear staining for Ki67 is shown by the dashed arrow in D. Ins⁺Glut2^HI cells immunopositive for both Ki67 and Glut 2 can be seen in E and F (dashed arrows).</p

Co-localization of Pdx1 (white) with insulin (red) or Glut2 (green) in mouse pancreas at gestational day (GD) 9.

<p>Nuclei were counter-stained with DAPI (blue). An islet of Langerhans is shown in A, and small β-cell clusters in B and C. Panel B shows a cluster containing Ins⁺Glut2^HI cells demonstrating the presence of nuclear Pdx1together with cytoplasmic insulin and Glut2 associated with the plasma membranes. Panel C shows an Ins⁺Glut2^LO cell (solid arrow) within a small cluster containing nuclear Pdx1as well as cytoplasmic insulin, but lacking detectable Glut2. To the right is shown an Ins⁺ cell with detectable Glut2 and nuclear-localized Pdx1(dashed arrow).</p

Fold changes in mRNA expression for transcription factors, at gestational days (GD) 9 and 18 relative to pancreas from non-pregnant female mice.

<p>Fold changes in mRNA expression for transcription factors, at gestational days (GD) 9 and 18 relative to pancreas from non-pregnant female mice.</p