RESUMEN
Mechanisms coordinating pancreatic ß cell metabolism with insulin secretion are essential for glucose homeostasis. One key mechanism of ß cell nutrient sensing uses the mitochondrial GTP (mtGTP) cycle. In this cycle, mtGTP synthesized by succinyl-CoA synthetase (SCS) is hydrolyzed via mitochondrial PEPCK (PEPCK-M) to make phosphoenolpyruvate, a high-energy metabolite that integrates TCA cycling and anaplerosis with glucose-stimulated insulin secretion (GSIS). Several strategies, including xenotopic overexpression of yeast mitochondrial GTP/GDP exchanger (GGC1) and human ATP and GTP-specific SCS isoforms, demonstrated the importance of the mtGTP cycle. These studies confirmed that mtGTP triggers and amplifies normal GSIS and rescues defects in GSIS both in vitro and in vivo. Increased mtGTP synthesis enhanced calcium oscillations during GSIS. mtGTP also augmented mitochondrial mass, increased insulin granule number, and membrane proximity without triggering de-differentiation or metabolic fragility. These data highlight the importance of the mtGTP signal in nutrient sensing, insulin secretion, mitochondrial maintenance, and ß cell health.
Asunto(s)
Adenosina Trifosfato/metabolismo , Glucosa/metabolismo , Guanosina Trifosfato/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Mitocondrias/metabolismo , Succinato-CoA Ligasas/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular , Proliferación Celular/genética , Ciclo del Ácido Cítrico/genética , Homeostasis , Humanos , Secreción de Insulina/genética , Secreción de Insulina/fisiología , Células Secretoras de Insulina/enzimología , Células Secretoras de Insulina/ultraestructura , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Mitocondrias/enzimología , Mitocondrias/ultraestructura , Membranas Mitocondriales/metabolismo , Fosforilación Oxidativa , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Regulación hacia ArribaRESUMEN
Adeno-associated virus serotype 2 (AAV-2) can preferentially integrate its DNA into a 4-kb region of human chromosome 19, designated AAVS1. The nicking activity of AAV-2's Rep68 or Rep78 proteins is essential for preferential integration. These proteins nick at the viral origin of DNA replication and at a similar site within AAVS1. The current nicking model suggests that the strand containing the nicking site is separated from its complementary strand prior to nicking. In AAV serotypes 1 through 6, the nicking site is flanked by a sequence that is predicted to form a stem-loop with standard Watson-Crick base pairing. The region flanking the nicking site in AAVS1 (5'-GGCGGCGGT/TGGGGCTCG-3' [the slash indicates the nicking site]) lacks extensive potential for Watson-Crick base pairing. We therefore performed an empirical search for a stable secondary structure. By comparing the migration of radiolabeled oligonucleotides containing wild-type or mutated sequences from the AAVS1 nicking site to appropriate standards, on native and denaturing polyacrylamide gels, we have found evidence that this region forms a stable secondary structure. Further confirmation was provided by circular dichroism analyses. We identified six bases that appear to be important in forming this putative secondary structure. Mutation of five of these bases, within the context of a double-stranded nicking substrate, reduces the ability of the substrate to be nicked by Rep78 in vitro. Four of these five bases are outside the previously recognized GTTGG nicking site motif and include parts of the CTC motif that has been demonstrated to be important for integration targeting.
