Fig 1.
Expression levels of main circadian genes and corresponding Glucose Stimulated Insulin Secretion (GSIS) at peaks of expression.
A: 24h expression profile of rev-erb α and β and Bmal1 in islets cultured for 24h and sampled every 6h (except first two sample times collected at 15:00 (ZT8) and 19:00 (ZT11)). B: Expression profiles at ZT8 and ZT20 of rev-erb α, rev-erb β, and Bmal1 in islets from normal or inversed cycle mice after 24h of culture. Islet isolation and sampling time are indicated in schematics. Data are from 4 animals for (A) and 3 animals for (B), with 2 technical replicates per animal and per ZT where applicable. White circles = Rev-erb α; White squares = Rev-erb β; Dark squares = Bmal1; *P<0.05 by ANOVA (A). C: Basal (2.8 mM glucose) and Glucose induced (16.7 mM glucose) insulin secretion from ZT8 and ZT20 islets. Grey bars = ZT8; Dark bars = ZT20; N = 20–60 experimental replicates across 6 animals per ZT. *P<0.05 using a t-test.
Fig 2.
No change in [Ca2+]i signaling, Na+, Ca2+ currents and exocytosis (measured via whole cell capacitance) between ZT8 and ZT20.
A: Glucose-induced [Ca2+]i transients in ZT8 or ZT20 islets. The glucose concentration was varied as indicated above the traces from a basal concentration of 2.8 mM glucose. Glucose-induced oscillations during the plateau phase were 0.085±0.0081 and 0.076±0.0054 in amplitude (Ratio of F340/F380), whilst oscillation period was 111±11 s and 92±7 s in ZT8 and ZT20 islets, respectively. n = 41 and n = 52 islets across a minimum of 6 animals for ZT8 and ZT20, respectively. B: Peak current (I)-voltage (V) relationship for voltage-gated Ca2+ currents recorded from single ZT8 or ZT20 β-cells using Ba2+ in the extracellular solution as a surrogate to Ca2+ during depolarizations from -70 mV to membrane potentials between -60 and +80 mV (n = 17 and 20 β-cells from 3 mice for ZT8 and ZT20, respectively). * p<0.05 using a t-test. The horizontal dotted line indicates zero current level. C: Na+ currents from single ZT8, n = 66 or ZT20, n = 73 β-cells across 3 mice per ZT. D: Increase in membrane capacitance (ΔC) during a train of ten 500 ms depolarizations from -70 mV to 0 mV (V) in single ZT8, n = 71 or ZT20, n = 72 β-cells across 3 mice per ZT. Traces are mean ± S.E.M. Grey lines = ZT8; Dark lines = ZT20 on all subpanels.
Fig 3.
Subtle effects on β-cells ultrastructure and hormone content during the 24h cycle.
A: Representative electron micrographs from β-cells and depiction of subgranule areas /granule types detected and analyzed. Scale bars: 0.5μm. B sizes and C numbers of region of interests (1, 2 and 3). Box plots groups of data according to their quartiles, of granule (1) and substructures areas (LDCV outer ring (2) or inner ring (3)) in ZT8 or ZT20 β-cells. The Mid line indicates the median whilst the small square within each box indicates the mean value. In panel C, data was averaged per cells. Panel D: mean insulin and (E:) glucagon content per islet ± S.E.M. for ZT8, n = 46 and 23 or ZT20, n = 45 and 22, respectively. Experimental replicates across a minimum of 6 mice per ZT. *P<0.05 using a t-test. F: mean DNA content per islet± S.E.M. for ZT8, n = 19 or ZT20, n = 23 replicates across a minimum of 3 mice per ZT. G: Proinsulin islet content in n = 57 for ZT8 islets (across 6 mice) and n = 57 for ZT20 islets (across 6 mice). Grey bars = ZT8; Dark bars = ZT20.
Fig 4.
Expression of genes of interest at ZT8 and ZT20.
A: Microarray data (GSE109882) unraveled a total of 301 transcripts significantly up or downregulated (see S1 Table for full details). 18 genes of interest (note that hemoglobin alpha and beta–Hba and Hbb- bore two probes on the microarray chip). Genes have been grouped by function. $ denotes genes identified in ref [14]. N = 5 experimental replicates for each group. B: relative expression of glucagon, Ins1, Ins2, Cav1.2 and Cav1.3 at ZT8 and ZT20 measured by qRT-PCR. Data are from 3 animals with 2 technical replicates per animal and per ZT.
Fig 5.
No change in exocytosis (measured via whole cell capacitance) between ZT8 and ZT12.
A: representative traces of 10x500ms depolarization evoked capacitance increases. B: Step increases in membrane capacitance (ΔCm) for each depolarization pulse. C: Total increase in capacitance over the whole train. Whole cell patch clamps performed on single ZT8, n = 19 or ZT12, n = 20 β-cells across 2 mice per ZT. Bars are mean ± S.E.M. light grey = ZT8; Dark grey = ZT12 on all subpanels.