Non-β-cell progenitors of β-cells in pregnant mice

Abstract

Pregnancy is a normal physiological condition in which the maternal β-cell mass increases rapidly about two-fold to adapt to new metabolic challenges. We have used a lineage tracing of β-cells to analyse the origin of new β-cells during this rapid expansion in pregnancy. Double transgenic mice bearing a tamoxifen-dependent Cre-recombinase construct under the control of a rat insulin promoter, together with a reporter Z/AP gene, were generated. Then, in response to a pulse of tamoxifen before pregnancy, β-cells in these animals were marked irreversibly and heritably with the human placental alkaline phosphatase (HP AP). First, we conclude that the lineage tracing system was highly specific for β-cells. Secondly, we scored the proportion of the β-cells marked with HP AP during a subsequent chase period in pregnant and non-pregnant females. We observed a dilution in this labeling index in pregnant animal pancreata, compared to nonpregnant controls, during a single pregnancy in the chase period. To extend these observations we also analysed the labeling index in pancreata of animals during the second of two pregnancies in the chase period. The combined data revealed statistically-significant dilution during pregnancy, indicating a contribution to new beta cells from a non-β-cell source. Thus for the first time in a normal physiological condition, we have demonstrated not only β-cell duplication, but also the activation of a non-β-cell progenitor population. Further, there was no transdifferentiation of β-cells to other cell types in a two and half month period following labeling, including the period of pregnancy.

Keywords: non-β-cell progenitor; pancreas; pregnancy; β-cell duplication; β-cells.

Figure 1

The Cre/loxP-based lineage tracing system. (A) Double transgenic mice, carry one transgene that consists of the rat insulin promoter driving expression of the tamoxifen-inducible Cre-recombinase (RIP-CreER^TAM). The second transgene is a Z/AP reporter gene in which Cre-mediated recombination between loxP sites leads to expression of HP AP from a generally-active promoter. In the absence of tamoxifen, β-cells (red, B) are not marked by the HP AP reporter (green, D), but upon administration of tamoxifen the Cre-recombinase becomes active and mediates recombination at the loxP sites, hence causing deletion of transcriptional and translational stop signals upstream of the HP AP reporter gene (C). As a result the β-cells are irreversibly and heritably labelled for HP AP (D). Pancreatic sections were immunostained using anti-HP AP (green, B and D) and anti-insulin (red, B and D) antibodies. The pancreatic nuclei were stained using DAPI (blue, B and D). Bar scale corresponds to 40 µm.

Figure 2

Analysing the origin of new β-cells during pregnancy. Double transgenic female mice (Z/AP; RIP-CreER^TAM) were pulsed with tamoxifen to label their β-cells. Then during the chase period, pregnancy was induced in only one group of mice while the other animals were assigned to the non-pregnant control group. After the chase period, if the percentage of β-cells positive for HP AP in pancreas of the pregnant animals is identical to the non-pregnant controls we conclude that new β-cells are the progeny of pre-existing β-cells (Hypothesis 1). However, if there is a dilution of the percentage of HP AP-labelled β-cells in the pregnant animals, we conclude that a non-β-cell source gives rise to new β-cells, possibly in combination with a contribution from pre-existing β-cells (Hypothesis 2). Finally, if the percentage of islets totally devoid of any label is higher in pregnant animals than in the controls, one can deduce that whole new islets arise from non-β-cell progenitors (Hypothesis 3).

Figure 3

Responses to pregnancy. During pregnancy, the mean body mass increased by 45% (A), and the mean pancreas mass by 51% (B), as compared with their age-matched non-pregnant control counterparts. Mean β-cell mass in pregnant animals increased by 91% (C).

Figure 4

Lineage tracing in pregnancy. (A) The HP AP labelling index in non-pregnant control and pregnant female animal pancreata was calculated by forming the ratio of total number of pixels of cross-sectional area immunostained for HP AP to total number of pixels cross-sectional area immunostained for insulin, using Image J. The mean labelling index in the pancreata of non-pregnant animals is higher (0.44 ± 0.05) than in the pregnant mice (0.33 ± 0.06). (B) The proportion of islet section completely negative for HP AP is calculated by forming the ratio of the HP AP-negative islet cross-sectional area to the total islet cross-sectional area in the whole of five sections for each pancreas. The mean proportion of islet sections HP AP negative in the pregnant group is (0.19 ± 0.06) is higher than in their age-matched, non-pregnant control counterparts (0.11 ± 0.04) as seen in the graph. # designates a p-value <0.05 by paired two-tailed student t-test.

Figure 5

Photomicrographs of pancreatic sections from pregnant animals stained for insulin (red), HP AP (green), and nuclei (blue, DAPI). Small islet sections are either partly or wholly HP AP-positive (A), or completely HP AP-negative (dashed circle, B). Small islet sections represent either genuinely small islets or glancing sections of larger islets (C). Bar scale corresponds to 40 µm.

Figure 6

Insulin-positive cells associated with the ductal epithelium. Pancreas sections from pregnant female animals, stained for insulin (red), HP AP (green) and nuclei (blue, DAPI), show examples (yellow arrows) of HP AP-positive β-cells (A), or HP AP-negative β-cells (B), associated with duct epithelium. Bar scales correspond to 20 µm (A) and 40 µm (B).

Figure 7

HP AP labeling remains restricted to β-cells after a chase period. Pancreatic sections from non-pregnant control females animals stained for insulin (red), HP AP (green) and nuclei (blue, DAPI), showing that the HP AP label is restricted to β-cells at 2.5 months following the pulse of tamoxifen. Bar scale corresponds to 40 µm.