Prompted by the hypothesis that the circadian clock exerts effects on metabolism through cellular actions, we sought to dissect the impact of clock function within the pancreatic islet, a principal regulator of glucose homeostasis. We used real-time bioluminescence imaging in
isolated pancreatic islets from Per2Luc knock-in mice (mice expressing a period2–luciferase fusion protein) to determine whether the clock is expressed autonomously within pancreas14. Continuous monitoring of light emission from individual islets revealed a self-sustained high-
amplitude rhythm of PER2–LUC expression with a period length of 23.58 6 0.3 h (Fig. 1a, b and Supplementary Movies 1 and 2), which closely matched that of other peripheral tissues and the suprachiasmatic nucleus (Fig. 1b)14,15. The oscillation gradually dampened after 3 days, similar to pituitary and liver (Fig. 1b), and addition of 10 mM forskolin to islets led to immediate re-initiation of robust rhythms(Fig. 1c). Bioluminescence from individual islets from ClockD19/D19 mutant mice lacked a circadian rhythm, even after forskolin stimulation (Fig. 1c). Quantitative real-time polymerase chain reaction
(PCR) showed that Per2 RNA expression was reduced and rhythmicity abolished in islets from ClockD19/D19 mutant mice (Fig. 1d). Together, the PER2 protein and mRNA oscillation in wild-type islets, as well as the loss of rhythmicity of Per2 in ClockD19/D19 islets, provide evidence for a self-sustained clock in endocrine pancreas.
Because a major mechanism of circadian regulation involves the cycling of genes involved in cell metabolism and proliferation, we examined 24-h RNA rhythms of essential transcripts involved in insulin secretion and b-cell growth in isolated islets (Supplementary Fig. 1). ClockD19/D19 mutant islets showed decreased expression levels of genes downstream of CLOCK and BMAL1 that comprise the core circadian loop, as well as the D-box and ROR feedback loops (Fig. 1d). ClockD19/D19 mutant mice also showed decreased levels of expression and/or phase shifts of RNA oscillation of genes involved in insulin signalling (Insr, Irs2, PI3K (also called Pik3r1), Akt2), glucose sensing (Glut2 (Slc2a2), Gck), and islet growth and development (Ccnd1, Gsk3b, Hnf4a, Hnf1a, Pdx1, Neurod1) (Supplementary Fig. 1). The alterations in temporal patterns of gene expression in ClockD19/D19 mutant islets were distinct from those in ClockD19/D19 mutant liver, reflecting partitioning of metabolic functions within these two tissues at different times of day (Supplementary Figs 1 and 2 and Supplementary Description 1).
To determine whether molecular disruption of the clock in pancreas corresponds with abnormalities in the temporal control of glucose metabolism, we analysed 24-h glucose and insulin profiles in 8-month old ClockD19/D19 mutant mice and their wild-type littermates during ad libitum feeding. In ClockD19/D19 mutant mice, glucose levels were elevated across the entire light/dark cycle without a rise in insulin levels, whereas insulin rises in wild-type mice during the beginning of the feeding period (Fig. 2a, b). ClockD19/D19 mutant mice also displayed significantly elevated fasting glucose levels at both ZT2 and ZT14
(Zeitgeber time) (Supplementary Fig. 3e, f). Glucose tolerance tests further revealed a 50% reduction in insulin release, corresponding with elevated glucose levels in ClockD19/D19 mutant mice, particularly at the beginning of the dark period (Fig. 2c, d and Supplementary Description 2). The likelihood that impaired glucose tolerance in
ClockD19/D19 mutant mice involves a primary defect in pancreatic function was further supported by the finding that these animals have normal insultin tolerance.
For a better understanding of the impact of the circadian gene mutation on pancreatic function, we examined glucose-stimulated insulin secretion (GSIS) in isolated size-matched pancreatic islets from 8-month-old mice. Islets from ClockD19/D19 mice displayed a ,50% reduction in GSIS (Fig. 3a) and failed to respond to KCl (Fig. 3b), indicating a defect in insulin exocytosis. Consistent with a predominant defect in insulin release rather than glucose metabolism, we observed normal calcium flux in response to 12 mM glucose in ClockD19/D19 mutant compared to wild-type islets (Supplementary Fig. 5d, e). Also consistent with defects in exocytosis, islets from ClockD19/D19 mutant mice displayed diminished insulin secretory responses to the cyclase activators forskolin and exendin 4, as well as to 8-bromoadenosine 39,59-cyclic monophosphate (8-bromo-cAMP), localizing the impaired function of ClockD19/D19 mutant islets to a late stage in stimulus–secretion coupling16 (Fig. 3b). Finally, in agreement with an exocytic defect as the cause of decreased insulin release in circadian mutant mice, we did not observe a significant difference in either absolute insulin mRNA levels (Supplementary Fig. 1b) or in islet insulin content (wild type, 38.2 ng insulin per islet; ClockD19/D19, 32.9 ng insulin per islet, P 5 0.08).