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High-Resolution Recording of the Circadian Oscillator in Primary Mouse α - and β -Cell Culture

View Article: PubMed Central - PubMed

ABSTRACT

Circadian clocks have been developed in evolution as an anticipatory mechanism allowing for adaptation to the constantly changing light environment due to rotation of the Earth. This mechanism is functional in all light-sensitive organisms. There is a considerable body of evidence on the tight connection between the circadian clock and most aspects of physiology and metabolism. Clocks, operative in the pancreatic islets, have caught particular attention in the last years due to recent reports on their critical roles in regulation of insulin secretion and etiology of type 2 diabetes. While β-cell clocks have been extensively studied during the last years, α-cell clocks and their role in islet function and orchestration of glucose metabolism stayed unexplored, largely due to the difficulty to isolate α-cells, which represents a considerable technical challenge. Here, we provide a detailed description of an experimental approach for the isolation of separate mouse α- and β-cell population, culture of isolated primary α- and β-cells, and their subsequent long-term high-resolution circadian bioluminescence recording. For this purpose, a triple reporter ProGlucagon-Venus/RIP-Cherry/Per2:Luciferase mouse line was established, carrying specific fluorescent reporters for α- and β-cells, and luciferase reporter for monitoring the molecular clockwork. Flow cytometry fluorescence-activated cell sorting allowed separating pure α- and β-cell populations from isolated islets. Experimental conditions, developed by us for the culture of functional primary mouse α- and β-cells for at least 10 days, will be highlighted. Importantly, temporal analysis of freshly isolated α- and β-cells around-the-clock revealed preserved rhythmicity of core clock genes expression. Finally, we describe the setting to assess circadian rhythm in cultured α- and β-cells synchronized in vitro. The here-described methodology allows to analyze the functional properties of primary α- and β-cells under physiological or pathophysiological conditions and to assess the islet cellular clock properties.

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Isolated around-the-clock α- and β-cells express in vivo circadian rhythm for selected core clock genes after fluorescence-activated cell sorting (FACS) separation. Triple transgenic mice (ProGcg-Venus/RIP-Cherry/Per2:Luc) were kept at standard 12 h/12 h light–dark cycles (ZT0 (Zeitgeber) corresponds to 07:00 a.m., and ZT12 corresponds to 07:00 p.m.), with constant access to the drinking water and night-restricted feeding regime for 2 weeks prior to islet collection. Pancreatic islets were isolated around-the-clock every 4 h and subjected to the FACS separation procedure. Temporal profiles of selected core clock transcripts (Clock, Per1, and Per2) were assessed in freshly extracted mRNA at each time point from α- and β-cell populations by quantitative RT-PCR analysis. Transcript levels were normalized to Hprt expression. Data expressed as mean ± SD (N = 2 biological replicates of 6 mice at each time point).
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Figure 5: Isolated around-the-clock α- and β-cells express in vivo circadian rhythm for selected core clock genes after fluorescence-activated cell sorting (FACS) separation. Triple transgenic mice (ProGcg-Venus/RIP-Cherry/Per2:Luc) were kept at standard 12 h/12 h light–dark cycles (ZT0 (Zeitgeber) corresponds to 07:00 a.m., and ZT12 corresponds to 07:00 p.m.), with constant access to the drinking water and night-restricted feeding regime for 2 weeks prior to islet collection. Pancreatic islets were isolated around-the-clock every 4 h and subjected to the FACS separation procedure. Temporal profiles of selected core clock transcripts (Clock, Per1, and Per2) were assessed in freshly extracted mRNA at each time point from α- and β-cell populations by quantitative RT-PCR analysis. Transcript levels were normalized to Hprt expression. Data expressed as mean ± SD (N = 2 biological replicates of 6 mice at each time point).

Mentions: In view of the complexity of the separation procedure by FACS, we next explored if separated α- and β-cell populations maintain their circadian properties, as was previously reported to be the case in isolated intact islets (11, 18). To this end, mRNA levels for selected core clock transcripts have been assessed in sorted islet cells isolated every 4 h during 24 h. The qRT-PCR analysis revealed pronounced rhythmic patterns for Per1 and Per2 genes over 24 h, while the oscillation of Clock transcription was shallow, in agreement with previous reports (Figure 5) (11, 19).


High-Resolution Recording of the Circadian Oscillator in Primary Mouse α - and β -Cell Culture
Isolated around-the-clock α- and β-cells express in vivo circadian rhythm for selected core clock genes after fluorescence-activated cell sorting (FACS) separation. Triple transgenic mice (ProGcg-Venus/RIP-Cherry/Per2:Luc) were kept at standard 12 h/12 h light–dark cycles (ZT0 (Zeitgeber) corresponds to 07:00 a.m., and ZT12 corresponds to 07:00 p.m.), with constant access to the drinking water and night-restricted feeding regime for 2 weeks prior to islet collection. Pancreatic islets were isolated around-the-clock every 4 h and subjected to the FACS separation procedure. Temporal profiles of selected core clock transcripts (Clock, Per1, and Per2) were assessed in freshly extracted mRNA at each time point from α- and β-cell populations by quantitative RT-PCR analysis. Transcript levels were normalized to Hprt expression. Data expressed as mean ± SD (N = 2 biological replicates of 6 mice at each time point).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5383706&req=5

Figure 5: Isolated around-the-clock α- and β-cells express in vivo circadian rhythm for selected core clock genes after fluorescence-activated cell sorting (FACS) separation. Triple transgenic mice (ProGcg-Venus/RIP-Cherry/Per2:Luc) were kept at standard 12 h/12 h light–dark cycles (ZT0 (Zeitgeber) corresponds to 07:00 a.m., and ZT12 corresponds to 07:00 p.m.), with constant access to the drinking water and night-restricted feeding regime for 2 weeks prior to islet collection. Pancreatic islets were isolated around-the-clock every 4 h and subjected to the FACS separation procedure. Temporal profiles of selected core clock transcripts (Clock, Per1, and Per2) were assessed in freshly extracted mRNA at each time point from α- and β-cell populations by quantitative RT-PCR analysis. Transcript levels were normalized to Hprt expression. Data expressed as mean ± SD (N = 2 biological replicates of 6 mice at each time point).
Mentions: In view of the complexity of the separation procedure by FACS, we next explored if separated α- and β-cell populations maintain their circadian properties, as was previously reported to be the case in isolated intact islets (11, 18). To this end, mRNA levels for selected core clock transcripts have been assessed in sorted islet cells isolated every 4 h during 24 h. The qRT-PCR analysis revealed pronounced rhythmic patterns for Per1 and Per2 genes over 24 h, while the oscillation of Clock transcription was shallow, in agreement with previous reports (Figure 5) (11, 19).

View Article: PubMed Central - PubMed

ABSTRACT

Circadian clocks have been developed in evolution as an anticipatory mechanism allowing for adaptation to the constantly changing light environment due to rotation of the Earth. This mechanism is functional in all light-sensitive organisms. There is a considerable body of evidence on the tight connection between the circadian clock and most aspects of physiology and metabolism. Clocks, operative in the pancreatic islets, have caught particular attention in the last years due to recent reports on their critical roles in regulation of insulin secretion and etiology of type 2 diabetes. While β-cell clocks have been extensively studied during the last years, α-cell clocks and their role in islet function and orchestration of glucose metabolism stayed unexplored, largely due to the difficulty to isolate α-cells, which represents a considerable technical challenge. Here, we provide a detailed description of an experimental approach for the isolation of separate mouse α- and β-cell population, culture of isolated primary α- and β-cells, and their subsequent long-term high-resolution circadian bioluminescence recording. For this purpose, a triple reporter ProGlucagon-Venus/RIP-Cherry/Per2:Luciferase mouse line was established, carrying specific fluorescent reporters for α- and β-cells, and luciferase reporter for monitoring the molecular clockwork. Flow cytometry fluorescence-activated cell sorting allowed separating pure α- and β-cell populations from isolated islets. Experimental conditions, developed by us for the culture of functional primary mouse α- and β-cells for at least 10 days, will be highlighted. Importantly, temporal analysis of freshly isolated α- and β-cells around-the-clock revealed preserved rhythmicity of core clock genes expression. Finally, we describe the setting to assess circadian rhythm in cultured α- and β-cells synchronized in vitro. The here-described methodology allows to analyze the functional properties of primary α- and β-cells under physiological or pathophysiological conditions and to assess the islet cellular clock properties.

No MeSH data available.


Related in: MedlinePlus