A Versatile, Portable Intravital Microscopy Platform for Studying Beta-cell Biology In Vivo

Reissaus, Christopher A.; Piñeros, Annie R.; Twigg, Ashley N.; Orr, Kara S.; Conteh, Abass M.; Martínez, Michelle; Kamocka, Malgorzata M.; Day, Richard N.; Tersey, Sarah A.; Mirmira, Raghavendra G.; Dunn, Kenneth W.; Linnemann, Amelia K.

doi:10.1038/s41598-019-44777-0

Cited by 41 publications

(51 citation statements)

References 38 publications

(49 reference statements)

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“…Isolated mouse islets were distended with Accutase for 1 min at 37° C as described previously (58). Islets were then transduced with 0.15 MOI of Ad.EV (expressing an empty vector) or Ad.hTFAM (expressing human-specific TFAM) for 48 hrs (viral particles purchased from Vector Biolabs).…”

Section: Adenoviral Transfectionmentioning

confidence: 99%

Mitofusin 1 and 2 regulation of mitochondrial DNA content is a critical determinant of glucose homeostasis

Sidarala

Zhu

Pearson

et al. 2021

Preprint

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Mitochondria are vital for β-cell function, yet the importance of mitochondrial fusion for glucose homeostasis is uncertain. Here, we report that the dynamin-like GTPases Mitofusin 1 and 2 (Mfn1 and Mfn2) are critical for glucose-stimulated insulin secretion (GSIS). Whereas Mfn1 and Mfn2 individually were dispensable for glucose control, combined Mfn1/2 deletion in β-cells induced mitochondrial fragmentation, reduced mtDNA content, and impaired respiratory function, ultimately resulting in severe glucose intolerance. Importantly, gene dosage studies revealed that Mfn1/2 control of glucose homeostasis is dependent on maintenance of mtDNA content, rather than mitochondrial structure. Mfn1/2 maintain mtDNA content by regulating the expression of the crucial mitochondrial transcription factor Tfam, as Tfam overexpression ameliorated the reduction in mtDNA content and GSIS in Mfn1/2-deficient β-cells. Further, mitofusin agonists rescued mtDNA content and GSIS in islets from db/db mice, a model of type 2 diabetes. Thus, Mfn1 and 2 act collectively to promote both optimal mitochondrial dynamics and mtDNA copy number in β-cells, and may be promising therapeutic targets to improve insulin release in diabetes.

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Section: Adenoviral Transfectionmentioning

confidence: 99%

Mitofusin 1 and 2 regulation of mitochondrial DNA content is a critical determinant of glucose homeostasis

Sidarala

Zhu

Pearson

et al. 2021

Preprint

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“…The in vivo imaging technique that we have been developing and its future enhancements should enable investigating this important phenomenon at the cellular level and to address its regulation in live animals. The recent development of abdominal imaging window (AIW) has made it possible to track internal organs by high resolution fluorescence microscopy for a prolonged period of time ( 26 , 27 ). Under favorable conditions, an animal could be imaged repeatedly up to several weeks.…”

Section: Discussionmentioning

confidence: 99%

In Vivo ZIMIR Imaging of Mouse Pancreatic Islet Cells Shows Oscillatory Insulin Secretion

et al. 2021

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Appropriate insulin secretion is essential for maintaining euglycemia, and impairment or loss of insulin release represents a causal event leading to diabetes. There have been extensive efforts of studying insulin secretion and its regulation using a variety of biological preparations, yet it remains challenging to monitor the dynamics of insulin secretion at the cellular level in the intact pancreas of living animals, where islet cells are supplied with physiological blood circulation and oxygenation, nerve innervation, and tissue support of surrounding exocrine cells. Herein we presented our pilot efforts of ZIMIR imaging in pancreatic islet cells in a living mouse. The imaging tracked insulin/Zn2+ release of individual islet β-cells in the intact pancreas with high spatiotemporal resolution, revealing a rhythmic secretion activity that appeared to be synchronized among islet β-cells. To facilitate probe delivery to islet cells, we also developed a chemogenetic approach by expressing the HaloTag protein on the cell surface. Finally, we demonstrated the application of a fluorescent granule zinc indicator, ZIGIR, as a selective and efficient islet cell marker in living animals through systemic delivery. We expect future optimization and integration of these approaches would enable longitudinal tracking of beta cell mass and function in vivo by optical imaging.

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“…vivo Two-photon imaging was adopted to allow visualisation of the intact pancreas, exposed through an abdominal incision on anaesthetised mice [49]. Islets previously infected in vivo with GCaMP6s and co-stained with TMRM immediately prior to data capture, were imaged for 18 min during which, cytosolic Ca 2+ oscillations and mitochondrial membrane depolarisation were recorded post IP injection of glucose in WT ( Fig.…”

Section: Glucose-induced Cytosolic Ca 2+ and δψM Changes Are Impairedmentioning

confidence: 99%

MitofusinsMfn1andMfn2are required to preserve glucose-but not incretin- stimulated beta cell connectivity and insulin secretion

Georgiadou

Muralidharan

Martínez

et al. 2020

Preprint

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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 ultrastructure, 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 (bMfn1/2-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 (Pdx CreERT ). Isolated islets were used for live beta cell fluorescence imaging of cytosolic (Cal-520) or mitochondrial (Pericam) free Ca 2+ concentration and membrane potential (TMRE). Mitochondrial network characteristics were quantified using super resolution fluorescence and transmission electron microscopy. Beta cellbeta 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 Ca 2+ sensor GCaMP6s selectively in beta cells and TMRM to visualise mitochondria using multiphoton microscopy. Results: bMfn1/2-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 Ca 2+ accumulation, mitochondrial hyperpolarisation and beta cell connectivity were all significantly reduced in bMfn1/2-KO mouse islets. Examined by intravital imaging of the exteriorised pancreas, antiparallel changes in cytosolic Ca 2+ and mitochondrial membrane potential, observed in control animals in vivo, were suppressed after Mfn1/2 deletion. 3 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. R Pearson correlation coefficient TMRE Tetramethylrhodamine ethyl ester TMRM Tetramethylrhodamine methyl ester WT Wild-type

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A Versatile, Portable Intravital Microscopy Platform for Studying Beta-cell Biology In Vivo

Cited by 41 publications

References 38 publications

Mitofusin 1 and 2 regulation of mitochondrial DNA content is a critical determinant of glucose homeostasis

Mitofusin 1 and 2 regulation of mitochondrial DNA content is a critical determinant of glucose homeostasis

In Vivo ZIMIR Imaging of Mouse Pancreatic Islet Cells Shows Oscillatory Insulin Secretion

MitofusinsMfn1andMfn2are required to preserve glucose-but not incretin- stimulated beta cell connectivity and insulin secretion

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