PAX6 mutation alters circadian rhythm and β cell function in mice without affecting glucose tolerance

Abstract

The transcription factor PAX6 is involved in the development of the eye and pancreatic islets, besides being associated with sleep-wake cycles. Here, we investigated a point mutation in the RED subdomain of PAX6, previously described in a human patient, to present a comprehensive study of a homozygous Pax6 mutation in the context of adult mammalian metabolism and circadian rhythm. Pax6^Leca2 mice lack appropriate retinal structures for light perception and do not display normal daily rhythmic changes in energy metabolism. Despite β cell dysfunction and decreased insulin secretion, mutant mice have normal glucose tolerance. This is associated with reduced hepatic glucose production possibly due to altered circadian variation in expression of clock and metabolic genes, thereby evading hyperglycemia. Hence, our findings show that while the RED subdomain is important for β cell functional maturity, the Leca2 mutation impacts peripheral metabolism via loss of circadian rhythm, thus revealing pleiotropic effects of PAX6.

Fig. 1. Structural disorganization of the retina in homozygous Pax6^Leca2 mice.

a–h′ Representative immunofluorescence images displaying a, b expression of NEUN, c, d BRN3A, e, f Melanopsin, and g–h′ merged images of eyes and eye-like structures of 20-week-old male mice. n = 4. Arrow and arrowheads depict cell types as specified in the text. Scale bar, 100 µm. i Relative mRNA expressions of circadian genes in hypothalamus at different ZTs (normalized to WT ZT0) as specified in 14–16-week-old male mice. n = 4 (WT ZT0 n = 5, Leca2 ZT6 n = 3). p values in parentheses described in the graphs were acquired by applying CircWave analysis.

Fig. 2. Loss of circadian rhythm in Pax6^Leca2 mice.

a–d Indirect calorimetry measurements taken over 72 h displaying (WT n = 19, Leca2 n = 13) a oxygen consumption, b average oxygen consumption (***p < 0.001 one-way Welch’s ANOVA followed by Dunnett’s post-hoc test), c locomotor activity, and d average locomotor activity (***p < 0.001 one-way Welch’s ANOVA followed by Dunnett’s post-hoc test). Gray shade and black bars depict lights off and white shade and bars depict lights on. Fourteen-week-old male mice were used for this study. Temporal measurements of plasma corticosterone in e and melatonin in f at different ZTs as specified (WT n = 8, Leca2 n = 7). (*p < 0.05, **p < 0.01, ***p < 0.001 two-way ANOVA followed by Bonferroni’s post-hoc test. Thirteen- to fifteen-week-old male mice were used for this study. Error bars display ±s.e.m.

Fig. 3. Pax6^Leca2 mice display normal glucose tolerance in spite of impaired insulin secretion.

a Temporal measurement of blood glucose levels. b Average ad libitum body weight. Fourteen-week-old mice were used for these experiments. n = 9, *p < 0.05, **p < 0.01, ***p < 0.001 Student’s t-test and two-way ANOVA followed by Bonferroni’s post-hoc test. c Oral glucose tolerance test (oGTT) and d insulin measurements during the oGTT. WT n = 21 (c), n = 19 (d), Leca2 n = 12, **p < 0.01, ***p < 0.001 two-way ANOVA followed by Bonferroni’s post-hoc test. Total islet protein content of insulin (n = 9) in e and glucagon (n = 4) in f. *p < 0.05, **p < 0.01 Student’s t-test. g Glucose stimulated insulin secretion assay. WT n = 6, Leca2 n = 4, *p < 0.05, **p < 0.01 Student’s t-test. Ten- to twelve-week-old mice were used for these experiments. Error bars display ±s.e.m. in a, c, d, rest ±s.d.

Fig. 4. Leca2 mutation leads to changes in islet transcriptome.

a Heat map displaying differentially expressed genes (DEGs) in isolated islets. Genes were filtered for fold-change >1.5× and FDR < 10%. n = 4. b A comparative analysis of DEGs in Leca2 mutants and known PAX6 targets. c Relative islet mRNA expression of Pax6, Ins2, Pcx, Ffar1, Mafa, and Ucn3. n = 4 (WT Ucn3 n = 3, Leca2 Pcx n = 3), ns non-significant, *p < 0.05, **p < 0.01, ***p < 0.001 Welch’s and Student’s t-test. d Representative immunofluorescence images for GLUT2 in pancreatic islets. n = 3. Scale bar, 50 µm. e Representative immunofluorescence images displaying insulin and glucagon-positive cells in islets and quantifications of α and β mass, and their ratio. n = 5, >80 islets per mouse were analyzed. Scale bar, 100 µm. *p < 0.05, **p < 0.01 Welch’s t-test and Student’s t-test. Ten- to twelve-week-old mice were used for all experiments. Error bars display ±s.d.

Fig. 5. Decreased hepatic glucose production in Pax6^Leca2 mice.

a, b Linear regression model displaying a fat mass and b lean mass plotted against body mass of 14-week-old male mice. WT n = 23. Leca2 n = 25. c Intraperitoneal insulin tolerance test. WT n = 7, Leca2 n = 8, **p < 0.01, ***p < 0.001 Welch’s t-test and two-way ANOVA followed by Bonferroni’s post-hoc test. d–f Results from hyperinsulinemic-euglycemic clamp displaying d Glucose infusion rate (*p < 0.05, **p < 0.01, ***p < 0.001 two-way ANOVA followed by Bonferroni’s post-hoc test), e glucose uptake by peripheral tissues, and f hepatic glucose production (***p < 0.001 one-way ANOVA followed by Bonferroni’s post-hoc test). n = 7, 12–14-week-old male mice were used. Error bars display ±s.e.m. in c, d and ±s.d. in e–f.

Fig. 6. Lack of rhythmic changes in clock and metabolic genes in the liver of Pax6^Leca2 mice.

a, b Relative liver mRNA expressions of a circadian genes and b glucose metabolism at different ZTs (normalized to WT ZT0) as specified in 14–16-week-old male mice. n = 4 (WT ZT0 n = 5, Leca2 ZT6 n = 3). p values in parentheses described in the graphs were acquired by applying CircWave analysis.

Fig. 7. Graphical summary.

Primarily, the Leca2 mutation seems to directly affect pancreatic islets and eye development. However, decreased circulating insulin does not produce a hyperglycemic phenotype in Pax6^Leca2 mice. Instead, decreased hepatic glucose production as a consequence of loss of circadian rhythm results in normal glucose tolerance.