Decrease in Ins⁺Glut2^LO β-cells with advancing age in mouse and human pancreas

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   Figure 1

   Lifespan of the mouse and human. The entire lifespan was examined in human (A) and mouse (B), with the ages of pancreatic tissue donor selected to illustrate major developmental stages, including early development, infancy, childhood/weaning, puberty/sexual maturation, and adulthood, as represented by each red star. When possible, developmental points were matched. A significant proportion of mouse islet development occurs postnatally, such that 20-week gestation in humans is analogous to postnatal day 8 in mice (Pan & Wright 2011); n ≥ 4 mice, and ≥3 humans/star.
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   Figure 2

   Beta cell cluster proportion decreases with age in mouse (A) and human (B) pancreas. Similar developmental points were grouped (day 7 (mouse) and fetal (human) samples, open bars; pre-weaning and sexual maturation (14-28 days) (mouse) and infancy/childhood/adolescence (human) samples, hatched bars; all adult time points, black bars). Beta cells were counted and allocated to the cluster (<5 β cells together) or islet (>5 β cells) compartments. (C, D, E) Representative images from human pancreas immunostained with insulin (green) illustrate changes during the maturation of islet structures, from fetal life (C, 30-week gestation female), puberty (D, 14-year-old female), and late adulthood (E, 72-year female). Data represent mean ± s.e.m. proportion (%) of total β-cells counted; *P < 0.05, **P < 0.01, ***P < 0.001 vs day 7 in mouse, and fetal in human; n > 3. Size bar denotes 50 μm.
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   Figure 3

   Insulin and GLUT2 immunostaining in human and mouse pancreas. Representative images of a human islet (A) and β-cell cluster (B) stained for insulin (green) and GLUT2 (red). Human Ins⁺Glut2^LO cells are shown in B (arrows). Proto-typical membrane immunostaining for GLUT2 (red) and cytoplasmic insulin (green) in a mouse islet (C), as well as an Ins⁺Glut2^LO cell in a mouse cluster (C, arrow). The non-membrane immunostaining of human GLUT2 was previously demonstrated by others (McCulloch et al. 2011), an effect of the TSA kit protocol. Size bar represents 50 μm.
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   Figure 4

   The Ins⁺Glut2^LO cell proportion decreases with age. Percent Ins⁺Glut2^LO cells relative to proportion of total β cells (A, B), islets (C, D), and clusters (E, F) as found in mouse (A, C, E) and human (B, D, F) pancreas. Data represent mean ± s.e.m.; *P < 0.05, **P < 0.01; ***P < 0.001 vs day 7 (A, C) or vs fetal (B, D, F); t-test 3 months vs 21 days (E); n > 5.
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   Figure 5

   Frequency of β-cell proliferation decreases with age. Proliferation of insulin-expressing β cells by Ki67 presence is shown in total β cells (A, B), islets (C, D), and clusters (E, F) within mouse (A, C, E) and human (B, D, F) pancreas samples. Scatter graphs show the spread of data points between individual pancreata, and R² values were calculated using non-linear regression analysis.
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   Figure 6

   Ins⁺Glut2^LOKi67⁺ cells are enriched within β-cell clusters. A β-cell cluster within a human fetal pancreas section demonstrates an Ins⁺Glut2^LOKi67⁺ cell (arrow) immunostained for insulin (green, B), GLUT2 (red, A), and Ki67 (yellow, B). Size bar denotes 50 μm.
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   Figure 7

   Partial regeneration of the endocrine pancreas in the young mouse following STZ administration. Blood glucose (A) and β-cell mass (B) following treatment with STZ on d 7 (closed circles/bars) or citrate buffer (open circles/bars) for up to 28 days. Data represent mean ± s.e.m.; *P < 0.05 vs day 7, *P < 0.05 vs control for same day; n > 5.
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   Figure 8

   The distribution of total β-cells, proliferating β-cells and Ins⁺Glut2^LO cell abundance demonstrate ontological and anatomical differences during partial regeneration of β-cell mass in the young mouse. Animals were treated with STZ on d 7 (closed bars) or vehicle alone (open bars) and subsequently examined on days 9, 14, 21 or 28. The changes in the percentage of Ins⁺ cells in clusters (<5 β-cells, i), small islets (5–15 β-cells, ii) or larger islets (>15 β-cells, iii) are shown in A, the percentage of Ki67- labeled Ins⁺ cells in B, and the percentage of Ins⁺Glut2^LO cells relative to all Ins⁺ cells in C. Within β-cell clusters the relative number of Ins⁺ cells decreased with age in control, but not STZ-treated mice. In clusters from control mice the percent Ins⁺Ki67⁺ cells significantly decreased with age, but not so after STZ treatment, whilst a transient increase in Ins⁺Ki67⁺ cells occurred in small and large islets at day 14 after STZ. The proportional presence of Ins⁺Glut2^LO cells in clusters did not differ between control or STZ-treated mice, but were increased after STZ at day 14 in both small and large islets. Data represent mean ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001 vs day 9; n > 5.

• © 2017 Society for Endocrinology