Figure 1. Loss of Adrb2 in neonatal β-cells results in glucose intolerance and impaired insulin secretion in female mice.

(A) Adult (2-month-old) female Adrb2 cKO mice have elevated fasting blood glucose and are glucose intolerant. Means ± SEM for n = 6 control and seven mutant female mice. *p<0.05, ***p<0.001, t-test. (B) Area under the curve (AUC) for glucose tolerance. *p<0.05, t-test. (C) Glucose tolerance is unaffected in male Adrb2 cKO mice at 2 months. Means ± SEM for n = 6 control and nine mutant mice for glucose tolerance. (D) Area under the curve (AUC) for glucose tolerance in males. (E, F) Glucose-stimulated insulin secretion (GSIS) in vivo is reduced in female but not male Adrb2 cKO mice. Means ± SEM for n = 6 control and eight mutant female mice; n = 5 control and six mutant male mice *p<0.05, t-test. (G) Decreased basal insulin secretion and GSIS in isolated adult female Adrb2 cKO islets. Means ± SEM from n = 4 control and six mutant mice. **p<0.01, ****p<0.0001, two-way ANOVA with Bonferroni’s post-test. (H) Islet Adrb2 expression declines postnatally and is significantly lower in adult males and females compared to neonatal stages. For female islets, p<0.01, t-test for P60 compared to P6. For male islets, p<0.0001 for P60 compared to P6 (one sample t-test since male P6 values were normalized to (1). Adrb2 levels are higher in female islets compared to males at all timepoints assessed. *p<0.05, **p<0.01, t-test. Adrb2 expression in P2, P6, and P60 islets was assessed by qRT-PCR analyses and data were normalized to 18S rRNA. Results are means ±SEM and expressed as fold-change relative to P6 male islets for n = 3–5 mice/sex/age. (I) Neonatal β-cell-specific Adrb2 deletion elicits glucose intolerance in mice. Neonatal Adrb2 i-cKO mice were injected with TMX or vehicle on the day of birth and 1 day later (P0–P1), and glucose tolerance was tested when mice were 2 months old. Means ± SEM for n = 7 vehicle and 5 TMX-injected Adrb2 i-cKO mice. *p<00.5, **p<0.01, ***p<0.001, t-test. (J) AUC for glucose tolerance. *p<0.05, t-test. (K) Neonatal β-cell-specific Adrb2 deletion results in impaired GSIS. Means ± SEM for n = 4 vehicle and 4 TMX-injected Adrb2 i-cKO mice. *p<0.05, **p<0.01, t-test.

Figure 1—figure supplement 1. Adrb2 expression, insulin sensitivity and islet morphology in adult Adrb2 cKO mice, and effects of adult β-cell-specific Adrb2 deletion on glucose tolerance and insulin secretion.

(A) Adrb2 transcript levels are significantly decreased in P6 Adrb2 cKO pancreas, while other adrenergic receptors are unaltered. Transcript levels were normalized to 18S RNA. Results are means ±SEM and expressed as fold-change relative to control Adrb2^f/f values. n = 6 control and 6–8 mutant mice. ****p<0.0001, one sample t-test. (B) Adrb2 mRNA is significantly reduced in both female and male Adrb2 cKO adult (2 month) islets compared to same-sex controls. Note that in controls, Adrb2 expression is higher in female islets compared to males. n = 3 control mice per sex, five female mutant and three male mutant mice. *p<0.05, **p<0.01, ****p<0.0001, one-sample t-test. (C, D) Normal insulin sensitivity in male (C) and female (D) Adrb2 cKO mice. Means ± SEM for n = 3 female control mice, four male control, six mutant females and six mutant male mice. **p<0.01, ***p<0.001, one-sample t-test. (E) Pancreatic Adrb2 loss does not affect adult islet organization, but results in increased insulin immunoreactivity. Scale bar, 50 µm. (F) Increased insulin content in adult (2 month) Adrb2 cKO islets. n = 5–7 mice/genotype. **p<0.01, t-test. (G) Normal endocrine cell numbers in adult Adrb2 cKO islets. Means ± SEM from n = 4 mice/genotype, one-way ANOVA with Tukey’s post-test. (H) Adrb2 cKO mice have smaller islets relative to controls. Means ± SEM from n = 3 control and four mutant mice, **p<0.01, t-tests. (I, J) Adrb2 loss does not affect β-cell proliferation in adult islets. Scale bar, 50 µm. Quantification of EdU/insulin-double positive cells from n = 3 mice/genotype. (K) Adrb2 is enriched in neonatal β-cells. β-cells were isolated by FACS purification from MIP-GFP mice at postnatal day 6 (P6). n = 9 β-cell samples and four non-β-cell samples, **p<0.01, one sample t-test. (L) Adult β-cell Adrb2 loss does not affect glucose tolerance. Mature Adrb2 i-cKO mice were injected with tamoxifen (TMX) or corn oil (vehicle) at 5–6 weeks of age and glucose tolerance was tested 4 weeks later. Means ± SEM for n = 5 vehicle and 7 TMX-injected mice. (M) Area under the curve (AUC) for glucose tolerance. (N) Normal GSIS with adult β-cell Adrb2 deletion. Means ± SEM for n = 6 control and 5 TMX-injected mice. (O) Adult β-cell Adrb2 loss does not affect GSIS in Adrb2 i-cKO islets. Means ± SEM for n = 3 mice per condition. **p<0.01, two-way ANOVA with Bonferroni’s post-test.