ABSTRACT
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.
Subject(s)
CLOCK Proteins/metabolism , Circadian Clocks , Hyperglycemia/metabolism , Insulin/metabolism , Islets of Langerhans/metabolism , CLOCK Proteins/antagonists & inhibitors , CLOCK Proteins/genetics , Cation Transport Proteins/antagonists & inhibitors , Cation Transport Proteins/chemistry , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Cells, Cultured , Circadian Clocks/drug effects , Colforsin/pharmacology , GTP-Binding Protein alpha Subunits, Gq-G11/antagonists & inhibitors , GTP-Binding Protein alpha Subunits, Gq-G11/chemistry , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Gene Expression Profiling , Gene Expression Regulation/drug effects , Genes, Reporter/drug effects , Humans , Insulin Secretion , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Islets of Langerhans/cytology , Islets of Langerhans/drug effects , Potassium Channels, Inwardly Rectifying/antagonists & inhibitors , Potassium Channels, Inwardly Rectifying/chemistry , Potassium Channels, Inwardly Rectifying/genetics , Potassium Channels, Inwardly Rectifying/metabolism , Qa-SNARE Proteins/antagonists & inhibitors , Qa-SNARE Proteins/chemistry , Qa-SNARE Proteins/genetics , Qa-SNARE Proteins/metabolism , RNA Interference , RNA, Small Interfering , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Sodium-Potassium-Exchanging ATPase/chemistry , Sodium-Potassium-Exchanging ATPase/genetics , Sodium-Potassium-Exchanging ATPase/metabolism , Vesicle-Associated Membrane Protein 3/antagonists & inhibitors , Vesicle-Associated Membrane Protein 3/chemistry , Vesicle-Associated Membrane Protein 3/genetics , Vesicle-Associated Membrane Protein 3/metabolism , Zinc Transporter 8ABSTRACT
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.