Figure 1.
Timelines for the 4 cohorts of pregnant mice.
GTT (glucose tolerance testing) was performed on all female mice prior to pregnancy. After timed mating, GTT was repeated on day 16.5 of gestation in cohort 1, and glucose-stimulated insulin secretion (GSIS) in cohort 2. Newborn pups in these 2 cohorts were weighed at day 1.5. Female offspring from cohort 1 were weighed weekly from weaning and timed mated at 10–12 weeks of age with a GTT at day 16.5. GTT and GSIS were performed prior to pregnancy. In cohort 3, BrdU was administered 16 hours before sacrifice at day 15.5 of gestation. In cohort 4, fetuses and islets were collected at day 16.5 of gestation.
Figure 2.
Glucose metabolism in ß-ARNT and floxed control females.
(A) Pre-gravid fasting and peak blood glucose levels in ß-ARNT (n = 19) and floxed control (n = 19) females. *p<0.05, t-test. Bars represent mean±SEM. (B) Late gestational fasting and peak blood glucose levels in ß-ARNT (n = 19) and floxed control (n = 19) females. **p<0.01, t-test. Bars represent mean±SEM. (C) Glucose tolerance curves for ß-ARNT and floxed control pregnancies. Each curve represents an individual mouse. (D) Fasting triglycerides in pregnant ß-ARNT and floxed control (FC) females. Bars represent mean±SEM. (E) Decreased first phase insulin secretion in non pregnant ß-ARNT versus floxed control females. *p<0.05. Data points represent mean±SEM. (F) Decreased first and second phase insulin secretion in pregnant ß-ARNT (n = 12) versus floxed control (n = 14) females. Insulin expressed in absolute values. *p<0.05. Data points represent mean±SEM. (G) Decreased first and second phase insulin secretion in pregnant ß-ARNT versus floxed control females. Insulin expressed as percentage of baseline. *p<0.05. Data points represent mean±SEM.
Figure 3.
(A) Weight of floxed control (FC) and ß-ARNT fetuses from diabetic pregnancies (DP) (n = 27 ß-ARNT and 14 FC) and non diabetic pregnancies (NDP) (n = 28 ß-ARNT and 32 FC). **p<0.01, Ŧ = 0.07, t-test. Significant main effect of DP on offspring weight on 2-way ANOVA, p = 0.001. Bars represent mean±SEM. (B) Blood glucose levels in floxed control and ß-ARNT fetuses from diabetic pregnancies and non diabetic pregnancies. Δ = 0.12, t-test. Bars represent mean±SEM. (C) Plasma insulin levels in floxed control and ß-ARNT fetuses from diabetic pregnancies (n = 25 ß-ARNT and 13 FC) and non diabetic pregnancies (n = 14 ß-ARNT and 18 FC). Bars represent mean±SEM. (D) Weight of floxed control and ß-ARNT pups from diabetic pregnancies (for all pups: n = 74 from DP and 41 from NDP, for pups in which genotype was known: n = 19 FC pups from DP, 9 from NDP, n = 9 ß-ARNT pups from DP and 10 from DP). *p<0.05, t-test. Bars represent mean±SEM. For this figure, black bars represent diabetic pregnancies and grey bars represent non diabetic pregnancies.
Figure 4.
Islet histology in ß-ARNT and floxed control mothers.
(A–B) Representative images of insulin immunofluorescence on islets from a floxed control (A) and ß-ARNT (B) mouse in late pregnancy. (C) Beta-cell mass in floxed control (FC) (n = 6) and ß-ARNT (n = 6) females in late pregnancy. p = ns, t-test. (D–E) Representative BrdU (red), insulin (green) and DAPI (blue) immunofluorescence on islets from a (D) floxed control and (E) ß-ARNT mouse in late pregnancy. (F) The BrdU labelling index was significantly decreased in insulin-positive islet cells of ß-ARNT versus floxed control (FC) dams (median 1.86 (1.72–2.55) versus 3.96 (1.86–6.6)) *p<0.05, Mann Whitney test). Data points represent individual mice. (G) The caspase labelling index was similar in insulin-positive islet cells of ß-ARNT versus floxed control dams (median 1.69 (0.48–1.26) versus 0.84 (0.56–3.01) p = NS, Mann Whitney test). Data points represent individual mice.
Figure 5.
Gene and protein expression: ARNT and cyclinD2.
(A) Gene expression in β-ARNT islets (white bars) expressed as percentage of gene expression in floxed control islets (black bars) (n = 13 β-ARNT and 13 FC). ***p<0.001, **p<0.01, *p<0.05, Ŧ p = 0.06, Δ p = 0.08, t-test. Bars represent mean±SEM. (B) Western blot of ARNT in islets from non pregnant and pregnant wild type females (top) Western blot of cyclinD2 in islets from non pregnant and pregnant floxed control and β-ARNT females (bottom). (C) Quantitation of ARNT protein in wild type female islets, expressed as fold change of non pregnant islets (n = 6 vs. 6). *p = 0.048, t-test. Bars represent mean±SEM. (D) Quantitation of cyclinD2 protein in non pregnant and pregnant β-ARNT (white bars) and floxed control (black bars) islets, expressed as fold change of non pregnant floxed control islets (n = 4 vs. 4). *p = 0.02, **p = 0.002, t-test. Bars represent mean±SEM.