Circadian clocks drive regeneration of pancreatic β-cells in mice

We aimed to understand the functional roles of islet cellular oscillators under diabetic conditions and during β-cell regeneration. We assessed diurnal regulation of β-cell proliferation and the transcriptional landscape in α- and residual β-cells following β-cell ablation in Insulin-rtTA/TET-DTA mice that simultaneously expressed α- and β-cell specific fluorescent reports. The mouse pancreatic islets were isolated over 24-h with 4-h interval, followed by separation of α- and β- cells using FACS sorting, RNA extraction and RNA sequencing. Acute hyperglycemia and loss of β-cell mass perturbed absolute expression levels and temporal transcriptome profiles in residual β-cells, whereas in neighboring α-cells only changes in temporal profiles were observed. Strikingly, compensatory regeneration of β-cells exhibited circadian rhythmicity. In arrhythmic Bmal1 deficient mice, massive β-cell ablation led to aggravated hyperglycemia, hyperglucagonemia and a fatal diabetes. No compensatory proliferation of β-cells was observed in arrhythmic mice, suggesting an essential role of circadian clocks in β-cell regeneration. Massive β-cells ablation was induced in ProGcg-Venus/RIP-Cherry/PER2::Luc/Insulin-rtTA/TET-DTA mice by administering 300 mg of doxycycline (DOX) in the drinking water supplemented with 2% sucrose. The control group received only water supplemented with 2 % sucrose. All the experiments were done in mice aged 6-8 weeks, under standard animal housing conditions with ad libitum access to food and water and under 12 h light/12 h dark cycles (LD). For the islet isolation experiments, male mice were subjected to night-restricted feeding 10 days prior to the experiments, and during the entire period of DOX treatment and sample collection. For the islet isolation across 24 h period, half of the animals were entrained by inverted LD and feeding cycles for 2 weeks preceding the experiments. Islets of Langerhans were isolated by standard procedure based on collagenase (Type XI, Sigma) digestion of pancreas followed by Ficoll purification (Petrenko et al., 2017a). Islet cells were gently dissociated by 0.05% trypsin (GIBCO) treatment, re-suspended in KRB solution (pH 7.4, supplemented with 0.3 % free fatty acid bovine serum albumin (BSA; Sigma), 1.4 mM glucose and 0.5 mM EDTA). α- and β-cell populations were separated by flow cytometry fluorescence activated cell sorting (FACS; Astrios sorter (Beckman Coulter)) based on fluorescence wavelength and intensity, cell singlet nature, size and viability as described previously (Petrenko et al., 2017b).