RESUMEN
AIM: To determine the impact of a functional human islet clock on insulin secretion and gene transcription. METHODS: Efficient circadian clock disruption was achieved in human pancreatic islet cells by small interfering RNA-mediated knockdown of CLOCK. Human islet secretory function was assessed in the presence or absence of a functional circadian clock by stimulated insulin secretion assays, and by continuous around-the-clock monitoring of basal insulin secretion. Large-scale transcription analysis was accomplished by RNA sequencing, followed by quantitative RT-PCR analysis of selected targets. RESULTS: Circadian clock disruption resulted in a significant decrease in both acute and chronic glucose-stimulated insulin secretion. Moreover, basal insulin secretion by human islet cells synchronized in vitro exhibited a circadian pattern, which was perturbed upon clock disruption. RNA sequencing analysis suggested alterations in 352 transcript levels upon circadian clock disruption. Among them, key regulators of the insulin secretion pathway (GNAQ, ATP1A1, ATP5G2, KCNJ11) and transcripts required for granule maturation and release (VAMP3, STX6, SLC30A8) were affected. CONCLUSIONS: Using our newly developed experimental approach for efficient clock disruption in human pancreatic islet cells, we show for the first time that a functional ß-cell clock is required for proper basal and stimulated insulin secretion. Moreover, clock disruption has a profound impact on the human islet transcriptome, in particular, on the genes involved in insulin secretion.
Asunto(s)
Proteínas CLOCK/metabolismo , Relojes Circadianos , Hiperglucemia/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Proteínas CLOCK/antagonistas & inhibidores , Proteínas CLOCK/genética , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Células Cultivadas , Relojes Circadianos/efectos de los fármacos , Colforsina/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros/efectos de los fármacos , Humanos , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Canales de Potasio de Rectificación Interna/antagonistas & inhibidores , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio de Rectificación Interna/metabolismo , Proteínas Qa-SNARE/antagonistas & inhibidores , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Proteína 3 de Membrana Asociada a Vesículas/antagonistas & inhibidores , Proteína 3 de Membrana Asociada a Vesículas/química , Proteína 3 de Membrana Asociada a Vesículas/genética , Proteína 3 de Membrana Asociada a Vesículas/metabolismo , Transportador 8 de ZincRESUMEN
AIMS/HYPOTHESIS: Following on from the emerging importance of the pancreas circadian clock on islet function and the development of type 2 diabetes in rodent models, we aimed to examine circadian gene expression in human islets. The oscillator properties were assessed in intact islets as well as in beta cells. METHODS: We established a system for long-term bioluminescence recording in cultured human islets, employing lentivector gene delivery of the core clock gene Bmal1 (also known as Arntl)-luciferase reporter. Beta cells were stably labelled using a rat insulin2 promoter fluorescent construct. Single-islet/cell oscillation profiles were measured by combined bioluminescence-fluorescence time-lapse microscopy. RESULTS: Human islets synchronised in vitro exhibited self-sustained circadian oscillations of Bmal1-luciferase expression at both the population and single-islet levels, with period lengths of 23.6 and 23.9 h, respectively. Endogenous BMAL1 and CRY1 transcript expression was circadian in synchronised islets over 48 h, and antiphasic to REV-ERBα (also known as NR1D1), PER1, PER2, PER3 and DBP transcript circadian profiles. HNF1A and PDX1 exhibited weak circadian oscillations, in phase with the REV-ERBα transcript. Dispersed islet cells were strongly oscillating as well, at population and single-cell levels. Importantly, beta and non-beta cells revealed oscillatory profiles that were well synchronised with each other. CONCLUSIONS/INTERPRETATION: We provide for the first time compelling evidence for high-amplitude cell-autonomous circadian oscillators displayed in human pancreatic islets and in dispersed human islet cells. Moreover, these clocks are synchronised between beta and non-beta cells in primary human islet cell cultures.