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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.

No MeSH data available.


Related in: MedlinePlus

Endocrine identity of sorted Venus- and Cherry-positive cell populations, isolated from ProGcg-Venus/RIP-Cherry/Per2:Luc mouse islets by fluorescence-activated cell sorting (FACS). (A,B) Quantitative RT-PCR analysis of (A) insulin (Ins1 and Isn2) and glucagon (Gcg) transcripts; or (B) V-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) β-cell-specific transcript in freshly isolated Venus- and Cherry-positive cells. Data are presented as relative expression of the target gene normalized to the Hprt housekeeping gene (mean ± SEM, N = 3 independent experiments for (A) and N = 4 experiments for (B) with six mice each). (C) Representative bright-field images of cultured Venus- and Cherry-positive cells 48 h after plating. (D) Representative fluorescent images of ProGcg-Venus and Insulin-Cherry transgenic proteins (left panels) colocalizing with glucagon and insulin immunostaining, respectively (merge, right panels). Analysis of purified Venus- and Cherry-positive cell cultures prior the immunostaining showed no cross-contaminations. No Venus-positive cells appeared positive for insulin, and no Cherry-positive cells appeared positive for glucagon by immunostaining. Scale bars = 100 μm. (E) FACS-separated Venus- and Cherry-positive cells were plated in 35 mm dishes (approximately 15,000 cells per dish, as described in Section “Material and Methods”), and subjected to 30 min incubation with 2.8 mM glucose (for basal insulin assessment) or with 7 mM glucose (for basal glucagon assessment). Hormone levels were assessed in the incubation solution with Mouse Insulin and Glucagon ELISA kits (Mercodia). Data are expressed as absolute values (mean ± SD) for N = 6 mice used for two independent islet preparations. (F) Acute secretion assays performed on cultured Venus- and Cherry-positive cells 48 h after plating. Cell supernatants were collected for three successive 30-min periods (30 min basal condition, 30 min stimulated condition, and 30 min re-basal condition, as described in Section “Material and Methods”) after 2 h of cell depletion in KRB solution. Hormone levels were assessed with Mouse Insulin and Glucagon ELISA kits and normalized to the residual hormone content in the end of the experiment. Data are expressed as percentage of glucagon or insulin from cell contents (mean ± SD; N = 2 experiments for Venus-positive cells and N = 4 experiments for Cherry-positive cells (two mice per experiment); * p < 0.05, two-tailed Student’s t-test).
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Figure 4: Endocrine identity of sorted Venus- and Cherry-positive cell populations, isolated from ProGcg-Venus/RIP-Cherry/Per2:Luc mouse islets by fluorescence-activated cell sorting (FACS). (A,B) Quantitative RT-PCR analysis of (A) insulin (Ins1 and Isn2) and glucagon (Gcg) transcripts; or (B) V-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) β-cell-specific transcript in freshly isolated Venus- and Cherry-positive cells. Data are presented as relative expression of the target gene normalized to the Hprt housekeeping gene (mean ± SEM, N = 3 independent experiments for (A) and N = 4 experiments for (B) with six mice each). (C) Representative bright-field images of cultured Venus- and Cherry-positive cells 48 h after plating. (D) Representative fluorescent images of ProGcg-Venus and Insulin-Cherry transgenic proteins (left panels) colocalizing with glucagon and insulin immunostaining, respectively (merge, right panels). Analysis of purified Venus- and Cherry-positive cell cultures prior the immunostaining showed no cross-contaminations. No Venus-positive cells appeared positive for insulin, and no Cherry-positive cells appeared positive for glucagon by immunostaining. Scale bars = 100 μm. (E) FACS-separated Venus- and Cherry-positive cells were plated in 35 mm dishes (approximately 15,000 cells per dish, as described in Section “Material and Methods”), and subjected to 30 min incubation with 2.8 mM glucose (for basal insulin assessment) or with 7 mM glucose (for basal glucagon assessment). Hormone levels were assessed in the incubation solution with Mouse Insulin and Glucagon ELISA kits (Mercodia). Data are expressed as absolute values (mean ± SD) for N = 6 mice used for two independent islet preparations. (F) Acute secretion assays performed on cultured Venus- and Cherry-positive cells 48 h after plating. Cell supernatants were collected for three successive 30-min periods (30 min basal condition, 30 min stimulated condition, and 30 min re-basal condition, as described in Section “Material and Methods”) after 2 h of cell depletion in KRB solution. Hormone levels were assessed with Mouse Insulin and Glucagon ELISA kits and normalized to the residual hormone content in the end of the experiment. Data are expressed as percentage of glucagon or insulin from cell contents (mean ± SD; N = 2 experiments for Venus-positive cells and N = 4 experiments for Cherry-positive cells (two mice per experiment); * p < 0.05, two-tailed Student’s t-test).

Mentions: Insulin and glucagon transcript expression levels were assessed in separated Venus- and Cherry-positive cells by qRT-PCR analysis. As expected, Gcg transcription was the highest in the Venus-positive population, further confirming the α-cell identity, while both insulin transcripts Ins1 and Ins2 were abundant in Cherry-positive cells, indicating their β-cell identity (Figure 4A). Importantly, expression levels of the opposite cell hormone genes (Ins1 and Ins2 in Venus-positive cells, and Gcg in Cherry-positive cells) were more than 10-fold (for Ins1 and Ins2) and 1,000-fold (for Gcg) lower, further confirming the satisfactory purity of the α- and β-cell populations. Furthermore, Cherry-positive cell population expressed β-cell-specific transcription factor MafA at the levels which were 1,000-fold higher compared to this transcript expression in Venus-positive counterparts (Figure 4B).


High-Resolution Recording of the Circadian Oscillator in Primary Mouse α - and β -Cell Culture
Endocrine identity of sorted Venus- and Cherry-positive cell populations, isolated from ProGcg-Venus/RIP-Cherry/Per2:Luc mouse islets by fluorescence-activated cell sorting (FACS). (A,B) Quantitative RT-PCR analysis of (A) insulin (Ins1 and Isn2) and glucagon (Gcg) transcripts; or (B) V-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) β-cell-specific transcript in freshly isolated Venus- and Cherry-positive cells. Data are presented as relative expression of the target gene normalized to the Hprt housekeeping gene (mean ± SEM, N = 3 independent experiments for (A) and N = 4 experiments for (B) with six mice each). (C) Representative bright-field images of cultured Venus- and Cherry-positive cells 48 h after plating. (D) Representative fluorescent images of ProGcg-Venus and Insulin-Cherry transgenic proteins (left panels) colocalizing with glucagon and insulin immunostaining, respectively (merge, right panels). Analysis of purified Venus- and Cherry-positive cell cultures prior the immunostaining showed no cross-contaminations. No Venus-positive cells appeared positive for insulin, and no Cherry-positive cells appeared positive for glucagon by immunostaining. Scale bars = 100 μm. (E) FACS-separated Venus- and Cherry-positive cells were plated in 35 mm dishes (approximately 15,000 cells per dish, as described in Section “Material and Methods”), and subjected to 30 min incubation with 2.8 mM glucose (for basal insulin assessment) or with 7 mM glucose (for basal glucagon assessment). Hormone levels were assessed in the incubation solution with Mouse Insulin and Glucagon ELISA kits (Mercodia). Data are expressed as absolute values (mean ± SD) for N = 6 mice used for two independent islet preparations. (F) Acute secretion assays performed on cultured Venus- and Cherry-positive cells 48 h after plating. Cell supernatants were collected for three successive 30-min periods (30 min basal condition, 30 min stimulated condition, and 30 min re-basal condition, as described in Section “Material and Methods”) after 2 h of cell depletion in KRB solution. Hormone levels were assessed with Mouse Insulin and Glucagon ELISA kits and normalized to the residual hormone content in the end of the experiment. Data are expressed as percentage of glucagon or insulin from cell contents (mean ± SD; N = 2 experiments for Venus-positive cells and N = 4 experiments for Cherry-positive cells (two mice per experiment); * p < 0.05, two-tailed Student’s t-test).
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Figure 4: Endocrine identity of sorted Venus- and Cherry-positive cell populations, isolated from ProGcg-Venus/RIP-Cherry/Per2:Luc mouse islets by fluorescence-activated cell sorting (FACS). (A,B) Quantitative RT-PCR analysis of (A) insulin (Ins1 and Isn2) and glucagon (Gcg) transcripts; or (B) V-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) β-cell-specific transcript in freshly isolated Venus- and Cherry-positive cells. Data are presented as relative expression of the target gene normalized to the Hprt housekeeping gene (mean ± SEM, N = 3 independent experiments for (A) and N = 4 experiments for (B) with six mice each). (C) Representative bright-field images of cultured Venus- and Cherry-positive cells 48 h after plating. (D) Representative fluorescent images of ProGcg-Venus and Insulin-Cherry transgenic proteins (left panels) colocalizing with glucagon and insulin immunostaining, respectively (merge, right panels). Analysis of purified Venus- and Cherry-positive cell cultures prior the immunostaining showed no cross-contaminations. No Venus-positive cells appeared positive for insulin, and no Cherry-positive cells appeared positive for glucagon by immunostaining. Scale bars = 100 μm. (E) FACS-separated Venus- and Cherry-positive cells were plated in 35 mm dishes (approximately 15,000 cells per dish, as described in Section “Material and Methods”), and subjected to 30 min incubation with 2.8 mM glucose (for basal insulin assessment) or with 7 mM glucose (for basal glucagon assessment). Hormone levels were assessed in the incubation solution with Mouse Insulin and Glucagon ELISA kits (Mercodia). Data are expressed as absolute values (mean ± SD) for N = 6 mice used for two independent islet preparations. (F) Acute secretion assays performed on cultured Venus- and Cherry-positive cells 48 h after plating. Cell supernatants were collected for three successive 30-min periods (30 min basal condition, 30 min stimulated condition, and 30 min re-basal condition, as described in Section “Material and Methods”) after 2 h of cell depletion in KRB solution. Hormone levels were assessed with Mouse Insulin and Glucagon ELISA kits and normalized to the residual hormone content in the end of the experiment. Data are expressed as percentage of glucagon or insulin from cell contents (mean ± SD; N = 2 experiments for Venus-positive cells and N = 4 experiments for Cherry-positive cells (two mice per experiment); * p < 0.05, two-tailed Student’s t-test).
Mentions: Insulin and glucagon transcript expression levels were assessed in separated Venus- and Cherry-positive cells by qRT-PCR analysis. As expected, Gcg transcription was the highest in the Venus-positive population, further confirming the α-cell identity, while both insulin transcripts Ins1 and Ins2 were abundant in Cherry-positive cells, indicating their β-cell identity (Figure 4A). Importantly, expression levels of the opposite cell hormone genes (Ins1 and Ins2 in Venus-positive cells, and Gcg in Cherry-positive cells) were more than 10-fold (for Ins1 and Ins2) and 1,000-fold (for Gcg) lower, further confirming the satisfactory purity of the α- and β-cell populations. Furthermore, Cherry-positive cell population expressed β-cell-specific transcription factor MafA at the levels which were 1,000-fold higher compared to this transcript expression in Venus-positive counterparts (Figure 4B).

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 &beta;-cell clocks have been extensively studied during the last years, &alpha;-cell clocks and their role in islet function and orchestration of glucose metabolism stayed unexplored, largely due to the difficulty to isolate &alpha;-cells, which represents a considerable technical challenge. Here, we provide a detailed description of an experimental approach for the isolation of separate mouse &alpha;- and &beta;-cell population, culture of isolated primary &alpha;- and &beta;-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 &alpha;- and &beta;-cells, and luciferase reporter for monitoring the molecular clockwork. Flow cytometry fluorescence-activated cell sorting allowed separating pure &alpha;- and &beta;-cell populations from isolated islets. Experimental conditions, developed by us for the culture of functional primary mouse &alpha;- and &beta;-cells for at least 10&thinsp;days, will be highlighted. Importantly, temporal analysis of freshly isolated &alpha;- and &beta;-cells around-the-clock revealed preserved rhythmicity of core clock genes expression. Finally, we describe the setting to assess circadian rhythm in cultured &alpha;- and &beta;-cells synchronized in vitro. The here-described methodology allows to analyze the functional properties of primary &alpha;- and &beta;-cells under physiological or pathophysiological conditions and to assess the islet cellular clock properties.

No MeSH data available.


Related in: MedlinePlus