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. 2010 Jul 29;466(7306):627-31.
doi: 10.1038/nature09253.

Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes

Biliana Marcheva  1 , Kathryn Moynihan Ramsey, Ethan D Buhr, Yumiko Kobayashi, Hong Su, Caroline H Ko, Ganka Ivanova, Chiaki Omura, Shelley Mo, Martha H Vitaterna, James P Lopez, Louis H Philipson, Christopher A Bradfield, Seth D Crosby, Lellean JeBailey, Xiaozhong Wang, Joseph S Takahashi, Joseph Bass
Affiliations

Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes

Biliana Marcheva et al. Nature. .

Abstract

The molecular clock maintains energy constancy by producing circadian oscillations of rate-limiting enzymes involved in tissue metabolism across the day and night. During periods of feeding, pancreatic islets secrete insulin to maintain glucose homeostasis, and although rhythmic control of insulin release is recognized to be dysregulated in humans with diabetes, it is not known how the circadian clock may affect this process. Here we show that pancreatic islets possess self-sustained circadian gene and protein oscillations of the transcription factors CLOCK and BMAL1. The phase of oscillation of islet genes involved in growth, glucose metabolism and insulin signalling is delayed in circadian mutant mice, and both Clock and Bmal1 (also called Arntl) mutants show impaired glucose tolerance, reduced insulin secretion and defects in size and proliferation of pancreatic islets that worsen with age. Clock disruption leads to transcriptome-wide alterations in the expression of islet genes involved in growth, survival and synaptic vesicle assembly. Notably, conditional ablation of the pancreatic clock causes diabetes mellitus due to defective beta-cell function at the very latest stage of stimulus-secretion coupling. These results demonstrate a role for the beta-cell clock in coordinating insulin secretion with the sleep-wake cycle, and reveal that ablation of the pancreatic clock can trigger the onset of diabetes mellitus.

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Figures

Figure 1
Figure 1. Cell autonomous oscillator in pancreas
(a) Islets from Per2Luc mice were imaged, and orange trace at right represents bioluminescence rhythm collected from the islet in the orange square (left). Traces from other islets are shown below. See also Supplemental Movies 1–2. (b) Periods of luminescence and damping rate in multiple tissues (mean ± S.E.M., n=6 mice/genotype). (c) Whole field and individual traces from WT and ClockΔ19/Δ19 islets. Red arrow indicates exposure to 10 µM forskolin for 1 hour. (d) Oscillation of clock genes in WT and ClockΔ19/Δ19 mutant islets across 24 hrs (mean ± S.E.M., n=4 mice/genotype/time point, 2-way ANOVA, *p<0.05).
Figure 2
Figure 2. Diabetic phenotypes in 8 month old circadian mutant mice
Ad lib fed (a) glucose and (b) insulin in ClockΔ19/Δ19 animals, shown as the average for time points during the light and dark periods (n=17). (c) Glucose tolerance (n=15–18) and (d) insulin secretion (n=8–10) in ClockΔ19/Δ19 mice at ZT14 following intraperitoneal glucose administration of 2 or 3g/kg body weight, respectively n=15–18). Data was analyzed by Student’s t-test (a–b) and 1-way ANOVA (c–d). p<0.05; **p<0.01; ***p<0.001. All values represent mean ± S.E.M.
Figure 3
Figure 3. Reduced islet size, proliferation, and insulin release in 8–10 month old circadian mutant mice
(a) Glucose-stimulated insulin release in isolated ClockΔ19/Δ19 islets compared to similar-sized WT islets (n=9–10 mice/genotype), normalized to % insulin content. (b) Insulin secretion from ClockΔ19/Δ19 islets in response to secretagogues (n=6–14). Insulin release was calculated as in (a), and ClockΔ19/Δ19 values are expressed as a percentage of WT. (c) Representative islet morphology in ClockΔ19/Δ19 and WT pancreata (body weight and pancreata weight were not different). (d) Size of islets isolated from ClockΔ19/Δ19 and Bmal1−/− mice compared to WT (n=6–9). (e) Ki67 staining of islet proliferation (ClockΔ19/Δ19 and WT, n=5–6). (f) Glucose-stimulated insulin secretion in Bmal1−/− islets compared to WT (n=5). (g) Insulin secretion from Bmal1−/− islets in response to secretagogues (n=6–10). Five islets per mouse were analyzed in triplicate for each test condition; data was analyzed by Student’s t-test. *p<0.05; **p<0.01, and values represent mean ± S.E.M.
Figure 4
Figure 4. Impaired glucose tolerance and islet insulin secretion in 2–4 mo old pancreas-specific circadian mutant mice
(a) Immunofluorescent staining of BMAL1 (red), insulin (blue), and glucagon (green) in PdxCre; Bmal1flx/flx and control islets (Scale bar, 25 m). (b) Immunofluorescent staining of BMAL1 (red) and DAPI (blue) in SCN of PdxCre; Bmal1flx/flx and control mice (Scale bar, 50 µm). (c) Oscillation of Bmal1, Rev-erbα, and s100a6 in islets and liver of PdxCre; Bmal1flx/flx mice at three sequential 8 hr time points (n=4 mice/genotype/time). (d) Blood glucose levels in ad lib fed PdxCre; Bmal1flx/flx mice, shown as the average for values in light and dark (n=9–10). (e) Glucose tolerance (n=6–8) and (f) insulin secretion (n=9–11) in PdxCre; Bmal1flx/flx mice at ZT2 following intraperitoneal glucose administration of 2 or 3g/kg body weight, respectively. (g) Insulin release in response to glucose in PdxCre; Bmal1flx/flx islets compared to size-matched control islets (n=5–7). (h) Insulin secretion from PdxCre; Bmal1flx/flx islets in response to secretagogues (n=5–7). For all studies, five islets per mouse were analyzed in triplicate for each concentration of glucose and secretagogue. Data was analyzed by 1-way (e,f) and 2-way (c) ANOVA, and Student’s t-test (d,g,h). *p<0.05; **p<0.01; ***p<0.001. For c,e,f, * denotes significance between Bmal1flx/flx and PdxCre;Bmal1flx/flx, and + denotes significance between PdxCre and PdxCre;Bmal1flx/flx. All values represent mean ± S.E.M.
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