TY - JOUR T1 - Mitofusins <em>Mfn1</em> and <em>Mfn2</em> are required to preserve glucose-but not incretin- stimulated beta cell connectivity and insulin secretion JF - bioRxiv DO - 10.1101/2020.04.22.055384 SP - 2020.04.22.055384 AU - Eleni Georgiadou AU - Charanya Muralidharan AU - Michelle Martinez AU - Pauline Chabosseau AU - Alejandra Tomas AU - Fiona Yong Su Wern AU - Elina Akalestou AU - Theodoros Stylianides AU - Asger Wretlind AU - Cristina Legido-Quigley AU - Ben Jones AU - Livia Lopez Noriega AU - Yanwen Xu AU - Guoqiang Gu AU - Nour Alsabeeh AU - Céline Cruciani-Guglielmacci AU - Christophe Magnan AU - Mark Ibberson AU - Isabelle Leclerc AU - Yusuf Ali AU - Scott A. Soleimanpour AU - Amelia K. Linnemann AU - Tristan A. Rodriguez AU - Guy A. Rutter Y1 - 2021/01/01 UR - http://biorxiv.org/content/early/2021/08/13/2020.04.22.055384.abstract N2 - Aims/hypothesis Mitochondrial glucose metabolism is essential for stimulated insulin release from pancreatic beta cells. Whether mitochondrial networks may be important for glucose or incretin sensing has yet to be determined.Methods Here, we generated mice with beta cell-selective, adult-restricted deletion of the mitofusin genes Mfn1 and Mfn2 (βMfn1/2 dKO). Whole or dissociated pancreatic islets were used for live beta cell fluorescence imaging of cytosolic or mitochondrial Ca2+ concentration and ATP production or GSIS in response to increasing glucose concentrations or GLP-1 receptor agonists. Serum and blood samples were collected to examine oral and i.p. glucose tolerance.Results βMfn1/2 dKO mice displayed elevated fed and fasted glycaemia (p&lt;0.01, p&lt;0.001) and a &gt;five-fold decrease (p&lt;0.0001) in plasma insulin. Mitochondrial length, glucose-induced polarisation, ATP synthesis and cytosolic Ca2+ increases were all reduced (p&lt;0.05,p&lt;0.01,p&lt;0.0001) in dKO islets, and beta cell Ca2+ dynamics were suppressed in vivo (p&lt;0.001). In contrast, oral glucose tolerance was near normal in βMfn1/2 dKO mice (p&lt;0.05, p&lt;0.01) and GLP-1 or GIP receptor agonists largely corrected defective GSIS from isolated islets through an EPAC-dependent signalling activation.Conclusions/interpretation Mitochondrial fusion and fission cycles are thus essential in the beta cell to maintain normal glucose, but not incretin, sensing. Defects in these cycles in some forms of diabetes might therefore provide opportunities for novel incretin-based or other therapies.Impact of Mfn1/2 deletion on glucose and incretin stimulated-insulin secretion in beta cells. (A) In control animals, glucose is taken up by beta cells through GLUT2 and metabolised by mitochondria (elongated structure) through the citrate (TCA) cycle, leading to an increased mitochondrial proton motive force (hyperpolarised Δψm), accelerated ATP synthesis and O2 consumption rate (OCR). Consequently, the cytoplasmic ATP:ADP ratio rises, which causes closure of KATP channels, depolarisation of plasma membrane potential (ψm), opening of VDCCs and influx of cytosolic Ca2+. Elevated [Ca2+]cyt triggers a number of ATP-dependent processes including insulin secretion and improved beta-beta cell communication through connexin 36 (Cx36). (B) Following Mfn1/2 deletion (βMfn1/2 dKO), highly fragmented mitochondria were associated with reduced mitochondrial Ca2+ ([Ca2+]m) accumulation, leading to a less polarised Δψm, weaker OCR, lower mtDNA copy number and decreased ATP synthesis. This is expected to result in weaker ψm depolarisation, cytosolic Ca2+ influx and beta-beta cell connectivity due to lower expression of Cx36. Despite observing a higher number of docked insulin granules on the plasma membrane, insulin secretion was highly suppressed in these animals. This was also associated with increased beta cell death and reduced beta cell mass. (C) In response to incretins, insulin secretion is potentiated through the activation of GLP1-R and cAMP signalling involving PKA- and EPAC2-dependent pathways. Elevated [Ca2+]cyt triggers a number of ATP-dependent processes including insulin secretion and Ca2+ removal into the endoplasmic reticulum (ER).(D) In βMfn1/2 dKO cells, activation of the GLP1-R was shown to be linked with a potentiation of the EPAC2 pathway that is PKA independent, along with an increased ER Ca2+ uptake and improved beta-beta cell communication. How these ‘amplifying’ signals of glucose metabolism for insulin secretion are linked with fragmented mitochondria remains unknown. Red and bold arrows represent enhanced pathways; dashed arrows represent impaired pathways. This figure was produced using illustrations from Servier Medical Art, http://smart.servier.com/Research in context What is already known about this subject?Mitochondrial ultrastructural variations and number are altered in beta cells of human T2D patients [1].Mice lacking Opa1, which controls mitochondrial fusion and inner membrane cristae structure, in beta cells, develop hyperglycaemia and defects in GSIS [2].What is the key question?Is an interconnected mitochondrial network essential in primary mouse beta cells for normal insulin secretion and glucose homeostasis?What are the new findings?We generated mice with beta cell-selective, adult-restricted deletion of the mitofusin genes Mfn1 and Mfn2 and show that insulin secretion and glucose homeostasis are strongly reduced in vivo.Cytosolic and mitochondrial Ca2+ increases, Δψm, ATP production and beta cell connectivity are impaired in βMfn1/2 dKO animals.Incretins bypass the above defects through an exchange protein directly activated by cAMP (EPAC)-dependent signalling mechanism.How might this impact on clinical practice in the foreseeable future?The ability of incretins to bypass defects in mitochondrial function might be exploited by the design of new agonists which target this pathway.Competing Interest StatementGAR is a consultant for Sun Pharmaceuticals and has received grant support from Les Laboratories Servier[Ca2+]cytCytoplasmic Ca2+ concentration[Ca2+]mitoMitochondrial free Ca2+ concentrationAAAntimycin AAchAcetylcholineClec16aΔpancPancreatic islet specific Clec16a knock-outDiazDiazoxidedKOdouble knock-outEx4Exendin-4FCCPCarbonyl cyanide-4-phenylhydrazoneGIPGlucose-dependent insulinotropic peptideGLP-1Glucagon-like peptide-1GSISGlucose-stimulated insulin secretionIMMInner mitochondria membraneIPGTTIntraperitoneal glucose tolerance testOGTTOral gavage and glucose tolerance testOligoOligomycinOMMOuter mitochondrial membranerPearson correlation coefficientRotRotenoneTMRETetramethylrhodamine ethyl esterβMfn1/2 dKObeta cell specific Mitofusin 1 and 2 double knock-outΔψmMitochondrial membrane potential ER -