Abstract
Background and aims Mitochondria are highly dynamic organelles, fundamental to cellular energy homeostasis. Mitochondrial metabolism of glucose is essential for the initiation of insulin release from pancreatic beta cells. Whether mitochondrial ultra-structure, and the proteins controlling fission and fusion, are important for glucose recognition are unclear. Mitochondrial fusion is supported by proteins including mitofusin 1 (MFN1), mitofusin 2 (MFN2) and optic atrophy (OPA1), and fission by dynamin-related protein 1 (DRP1). Here, we generated mice with beta cell-selective, adult-restricted deletion of Mfn1 and Mfn2 (βMfn1/2-KO), and explored the impact on insulin secretion and glucose homeostasis in vivo and in vitro.
Materials and methods C57BL/6J mice bearing Mfn1 and Mfn2 alleles with loxP sites, were crossed to animals carrying an inducible Cre recombinase at the Pdx1 locus (PdxCreERT). Isolated islets were used for live beta cell fluorescence imaging of cytosolic (Cal-520) or mitochondrial (Pericam) free Ca2+ concentration and membrane potential (TMRE). Mitochondrial network characteristics were quantified using super resolution fluorescence and transmission electron microscopy. Beta cell-beta cell connectivity was assessed using the Pearson (R) analysis and Monte Carlo simulation in intact mouse islets. Intravital imaging was performed in mice injected with an adeno-associated virus to express the cytosolic Ca2+ sensor GCaMP6s selectively in beta cells and TMRM to visualise mitochondria using multiphoton microscopy.
Results βMfn1/2-KO mice displayed higher fasting glycaemia than control littermates at 14 weeks (8.6 vs 6.4 mmol/L, p>0.05) and a >five-fold decrease in plasma insulin post-intraperitoneal glucose injection (5-15 min, p<0.0001). Mitochondrial length, and glucose-induced Ca2+ accumulation, mitochondrial hyperpolarisation and beta cell connectivity were all significantly reduced in βMfn1/2-KO mouse islets. Examined by intravital imaging of the exteriorised pancreas, antiparallel changes in cytosolic Ca2+ and mitochondrial membrane potential, observed in control animals in vivo, were suppressed after Mfn1/2 deletion.
Conclusion Mitochondrial fusion and fission cycles are essential in the beta cell to maintain normal mitochondrial bioenergetics and glucose sensing both in vitro and in the living mouse. Such cycles may be disrupted in some forms of diabetes to impair mitochondrial function and, consequently, insulin secretion.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- [Ca2+]cyt
- Cytoplasmic Ca2+ concentration
- [Ca2+]mito
- Mitochondrial free Ca2+ concentration
- βMfn1/2-KO
- Beta cell-specific Mfn1/2-null (animal)
- Δψm
- Mitochondrial membrane potential
- FCCP
- Carbonyl cyanide-4-phenylhydrazone
- GSIS
- Glucose-stimulated insulin secretion
- KO
- Knockout
- MFN
- Mitofusin
- MOI
- Multiplicity of infection
- mtDNA
- mitochondrial DNA
- NA
- Numerical aperture
- qRT-PCR
- Quantitative RT-PCR
- R
- Pearson correlation coefficient
- TMRE
- Tetramethylrhodamine ethyl ester
- TMRM
- Tetramethylrhodamine methyl ester
- WT
- Wild-type