Loss of Bmal1 leads to uncoupling and impaired glucose-stimulated insulin secretion in β-cells - PubMed (original) (raw)

Loss of Bmal1 leads to uncoupling and impaired glucose-stimulated insulin secretion in β-cells

Jeongkyung Lee et al. Islets. 2011 Nov-Dec.

Abstract

The circadian clock has been shown to regulate metabolic homeostasis. Mice with a deletion of Bmal1, a key component of the core molecular clock, develop hyperglycemia and hypoinsulinemia, suggesting β-cell dysfunction. However, the underlying mechanisms are not fully known. In this study, we investigated the mechanisms underlying the regulation of β-cell function by Bmal1. We studied β-cell function in global Bmal1-/- mice, in vivo and in isolated islets ex vivo, as well as in rat insulinoma cell lines with shRNA-mediated Bmal1 knockdown. Global Bmal1-/- mice develop diabetes secondary to a significant impairment in glucose-stimulated insulin secretion (GSIS). There is a blunting of GSIS in both isolated Bmal1-/- islets and in Bmal1 knockdown cells, as compared to controls, suggesting that this is secondary to a loss of cell-autonomous effect of Bmal1. In contrast to previous studies, in these Bmal1-/- mice on a C57Bl/6 background, the loss of stimulated insulin secretion, interestingly, is with glucose but not to other depolarizing secretagogues, suggesting that events downstream of membrane depolarization are largely normal in Bmal1-/- islets. This defect in GSIS occurs as a result increased mitochondrial uncoupling with consequent impairment of glucose-induced mitochondrial potential generation and ATP synthesis, due to an upregulation of Ucp2. Inhibition of Ucp2, in isolated islets, leads to a rescue of the glucose-induced ATP production and insulin secretion in Bmal1-/- islets. Thus, Bmal1 regulates mitochondrial energy metabolism to maintain normal GSIS and its disruption leads to diabetes due to a loss of GSIS.

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Figures

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Figure 1. Bmal1-/- mice develop diabetes with impaired GSIS. (A) Fasting plasma glucose at ZT-9. n = 11–12. (B) Fasting plasma insulin in 12 week old mice at ZT-9. n = 8. (C and D) Plasma glucose and insulin during glucose tolerance testing in 6 h fasted 14 week old mice at ZT-9. n = 4. All values are mean ± SEM *p ≤ 0.05.

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Figure 2. Bmal1-/- mice have normal insulin content. (A) Total pancreas insulin content expressed per mg protein. (B and C) Insulin content in isolated islets expressed per μg DNA or per equally sized islet. n = 3–5. All values are mean ± SEM; N.S., not significant.

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Figure 3. Bmal1-/- mice have impaired glucose-induced first-phase insulin secretion. (A and B) Acute insulin secretion after glucose (A) and L-Arginine (B) stimulation in 10 week old mice at ZT-9. n = 4–6. All values are mean ± SEM *p ≤ 0.05; N.S., not significant.

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Figure 4. Bmal1-/- islets have impaired insulin secretion with glucose but not depolarizing secretagogues. (A and B) Insulin secretion from (A) isolated islets (ten islets each from four individual mice of each genotype) and from (B) Bmal1 knockdown and scrambled control Ins-1 (832/13) cells (n = 6) on exposure to increasing glucose concentrations. Secretion is represented as a fold increase over basal insulin secretion at 2.8 mM glucose, assessed after normalization with total insulin content. (C) Insulin secretion from isolated islets (ten islets each from three individual mice of each genotype) on exposure to depolarizing secretagogues, 30 mM L-Arginine (L-Arg), 15 μM Glibenclamide (Glbn) and 30 mM KCl for 30 min in 2.8 mM glucose buffer. Y-axis represents secreted insulin in the presence of secretagogues as a fold increase over basal secretion in the absence of secretagogues displayed on a log-scale. (D) Insulin secretion during perifusion of isolated islets (n = 3–4 mice per genotype) with KRB containing glucose (2.8 G–2.8 mM glucose; 25 G–25 mM glucose) and KCl (30 mM in 2.8 mM glucose) from samples collected every minute. Results were normalized first to total insulin content of islets and then to the basal insulin secretion during the first 10 min under 2.8 mM glucose. **p ≤ 0.01 between the groups.

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Figure 5. Mitochondrial defects in OXPHOS coupling underlie GSIS defects in Bmal1-/- islets. (A) Red/green (590/530) fluorescence ratio of isolated islets (ten islets each from three individual mice for each genotype) after loading with JC-1 dye on exposure to 2.8 and 25 mM glucose concentrations. Values are represented as fold change over wild-type islets in 2.8 mM glucose. (B) Representative images of wild-type (WT) and Bmal1-/- mouse islets exposed to 2.8 mM glucose (top panels) or 25 mM glucose (bottom panels) after loading with JC-1 dye. Images taken are at emission 530 (green-indicating the cytosolic JC-1 dye) and at 590 nM (red-indicating the intra-mitochondrial dye). All values are mean ± SEM *p ≤ 0.05. (C) Representative image of immunohistochemistry of pancreas for Ucp2 showing intense brown staining only in Bmal1-/- islets but not in wild-type (WT) controls. Scale bar represents 20 μm. (D) Relative expression of Ucp2 transcript in isolated Bmal1-/- islets as compared with wild-type controls, after normalization to housekeeping genes. (E) Protein gel blot analysis of Upc2 and Bmal1 proteins from isolated islets (each lane represents 350–400 islets, pooled from two mice) in Bmal1-/- and wild-type control mice. (F) Relative expression of Ucp2 protein, as compared with wild-type control, after normalization to β-actin using ImageJ to quantitate the protein bands from (E).

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Figure 6. Inhibition of Ucp2 rescues GSIS in Bmal1-/- islets. (A) Fold change over basal ATP/ADP ratio measured in isolated islets after exposure to increasing glucose concentrations and a rescue after exposure to 10 μM genipin for 30 min. n = 3–5 (ten islets each from three to five individual mice of each genotype). (B) Insulin secretion from isolated islets on exposure to increasing glucose concentrations and a rescue of insulin secretion after exposure to 10 μM genipin for 60 min. n = 3–5 (ten islets each from three to five individual mice of each genotype). All values are mean ± SEM *p ≤ 0.05.

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