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Fractalkine (CX3CL1), a new factor protecting β-cells against TNFα.

Rutti S, Arous C, Schvartz D, Timper K, Sanchez JC, Dermitzakis E, Donath MY, Halban PA, Bouzakri K - Mol Metab (2014)

Bottom Line: CX3CL1 decreased human (but not rat) β-cell apoptosis.Moreover, CX3CL1 decreases basal apoptosis of human β-cells.We further demonstrate that CX3CL1 protects β-cells from the adverse effects of TNFα on their function by restoring the expression and phosphorylation of key proteins of the insulin secretion pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Medicine and Development, Geneva University, Geneva, Switzerland.

ABSTRACT

Objective: We have previously shown the existence of a muscle-pancreas intercommunication axis in which CX3CL1 (fractalkine), a CX3C chemokine produced by skeletal muscle cells, could be implicated. It has recently been shown that the fractalkine system modulates murine β-cell function. However, the impact of CX3CL1 on human islet cells especially regarding a protective role against cytokine-induced apoptosis remains to be investigated.

Methods: Gene expression was determined using RNA sequencing in human islets, sorted β- and non-β-cells. Glucose-stimulated insulin secretion (GSIS) and glucagon secretion from human islets was measured following 24 h exposure to 1-50 ng/ml CX3CL1. GSIS and specific protein phosphorylation were measured in rat sorted β-cells exposed to CX3CL1 for 48 h alone or in the presence of TNFα (20 ng/ml). Rat and human β-cell apoptosis (TUNEL) and rat β-cell proliferation (BrdU incorporation) were assessed after 24 h treatment with increasing concentrations of CX3CL1.

Results: Both CX3CL1 and its receptor CX3CR1 are expressed in human islets. However, CX3CL1 is more expressed in non-β-cells than in β-cells while its receptor is more expressed in β-cells. CX3CL1 decreased human (but not rat) β-cell apoptosis. CX3CL1 inhibited human islet glucagon secretion stimulated by low glucose but did not impact human islet and rat sorted β-cell GSIS. However, CX3CL1 completely prevented the adverse effect of TNFα on GSIS and on molecular mechanisms involved in insulin granule trafficking by restoring the phosphorylation (Akt, AS160, paxillin) and expression (IRS2, ICAM-1, Sorcin, PCSK1) of key proteins involved in these processes.

Conclusions: We demonstrate for the first time that human islets express and secrete CX3CL1 and CX3CL1 impacts them by decreasing glucagon secretion without affecting insulin secretion. Moreover, CX3CL1 decreases basal apoptosis of human β-cells. We further demonstrate that CX3CL1 protects β-cells from the adverse effects of TNFα on their function by restoring the expression and phosphorylation of key proteins of the insulin secretion pathway.

No MeSH data available.


Related in: MedlinePlus

CX3CL1 decreases human β-cell apoptosis. A: BrdU-positive rat β-cells. Sorted rat primary β-cells were cultured on 804G matrix-coated dishes for 24 h in the presence of increasing concentrations of CX3CL1. Proliferation was measured by BrdU incorporation over 24 h (n = 4). B: Rat β-cell death. Sorted rat primary β-cells were cultured on 804G matrix-coated dishes for 24 h in the presence of increasing concentrations of CX3CL1. Cell death is expressed as TUNEL-positive β-cells normalized to Control (absolute value 0.7 ± 0.3%); n = 4. C: Rat β-cell death. Rat islets were dispersed into single cells and cultured (not sorted) on 804G matrix-coated dishes for 24 h in the presence of increasing concentrations of CX3CL1. Cell death is expressed as TUNEL-positive β-cells normalized to Control (absolute value 0.05 ± 0.02%); n = 3. D: Human β-cell death. Human islets were dispersed into single cells and cultured (not sorted) on 804G matrix-coated dishes for 24 h in the presence of increasing concentrations of CX3CL1. Cell death is expressed as TUNEL-positive β-cells normalized to Control (absolute value 11.6 ± 4.5%); n = 3. *p < 0.05 vs. 0 ng/ml CX3CL1 as tested by ANOVA followed by Bonferroni post hoc test.
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fig3: CX3CL1 decreases human β-cell apoptosis. A: BrdU-positive rat β-cells. Sorted rat primary β-cells were cultured on 804G matrix-coated dishes for 24 h in the presence of increasing concentrations of CX3CL1. Proliferation was measured by BrdU incorporation over 24 h (n = 4). B: Rat β-cell death. Sorted rat primary β-cells were cultured on 804G matrix-coated dishes for 24 h in the presence of increasing concentrations of CX3CL1. Cell death is expressed as TUNEL-positive β-cells normalized to Control (absolute value 0.7 ± 0.3%); n = 4. C: Rat β-cell death. Rat islets were dispersed into single cells and cultured (not sorted) on 804G matrix-coated dishes for 24 h in the presence of increasing concentrations of CX3CL1. Cell death is expressed as TUNEL-positive β-cells normalized to Control (absolute value 0.05 ± 0.02%); n = 3. D: Human β-cell death. Human islets were dispersed into single cells and cultured (not sorted) on 804G matrix-coated dishes for 24 h in the presence of increasing concentrations of CX3CL1. Cell death is expressed as TUNEL-positive β-cells normalized to Control (absolute value 11.6 ± 4.5%); n = 3. *p < 0.05 vs. 0 ng/ml CX3CL1 as tested by ANOVA followed by Bonferroni post hoc test.

Mentions: To investigate the potential impact of CX3CL1 on human β-cell survival, cell death was quantified using TUNEL assay on dispersed human islet cells. After 24 h treatment, CX3CL1 decreased human β-cell apoptosis. This decrease in apoptosis was concentration dependent and could only be observed with 5 and 10 nM CX3CL1 (Figure 3D). When cell death of FACS-purified rat β-cells (Figure 3B) or of β-cells of dispersed rat islets (Figure 3C) was evaluated, no significant effect of CX3CL1 could be observed at any concentration studied. These results could suggest that the positive impact of CX3CL1 on human β-cells is species specific. However, there are other potentially confounding factors. Specifically, basal apoptosis (in the absence of CX3CL1) was 11.6 ± 4.5% for human β-cells but only 0.7 ± 0.3% and 0.05 ± 0.02%, respectively, for sorted rat β-cells or β-cells within an unsorted rat islet cell population.


Fractalkine (CX3CL1), a new factor protecting β-cells against TNFα.

Rutti S, Arous C, Schvartz D, Timper K, Sanchez JC, Dermitzakis E, Donath MY, Halban PA, Bouzakri K - Mol Metab (2014)

CX3CL1 decreases human β-cell apoptosis. A: BrdU-positive rat β-cells. Sorted rat primary β-cells were cultured on 804G matrix-coated dishes for 24 h in the presence of increasing concentrations of CX3CL1. Proliferation was measured by BrdU incorporation over 24 h (n = 4). B: Rat β-cell death. Sorted rat primary β-cells were cultured on 804G matrix-coated dishes for 24 h in the presence of increasing concentrations of CX3CL1. Cell death is expressed as TUNEL-positive β-cells normalized to Control (absolute value 0.7 ± 0.3%); n = 4. C: Rat β-cell death. Rat islets were dispersed into single cells and cultured (not sorted) on 804G matrix-coated dishes for 24 h in the presence of increasing concentrations of CX3CL1. Cell death is expressed as TUNEL-positive β-cells normalized to Control (absolute value 0.05 ± 0.02%); n = 3. D: Human β-cell death. Human islets were dispersed into single cells and cultured (not sorted) on 804G matrix-coated dishes for 24 h in the presence of increasing concentrations of CX3CL1. Cell death is expressed as TUNEL-positive β-cells normalized to Control (absolute value 11.6 ± 4.5%); n = 3. *p < 0.05 vs. 0 ng/ml CX3CL1 as tested by ANOVA followed by Bonferroni post hoc test.
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fig3: CX3CL1 decreases human β-cell apoptosis. A: BrdU-positive rat β-cells. Sorted rat primary β-cells were cultured on 804G matrix-coated dishes for 24 h in the presence of increasing concentrations of CX3CL1. Proliferation was measured by BrdU incorporation over 24 h (n = 4). B: Rat β-cell death. Sorted rat primary β-cells were cultured on 804G matrix-coated dishes for 24 h in the presence of increasing concentrations of CX3CL1. Cell death is expressed as TUNEL-positive β-cells normalized to Control (absolute value 0.7 ± 0.3%); n = 4. C: Rat β-cell death. Rat islets were dispersed into single cells and cultured (not sorted) on 804G matrix-coated dishes for 24 h in the presence of increasing concentrations of CX3CL1. Cell death is expressed as TUNEL-positive β-cells normalized to Control (absolute value 0.05 ± 0.02%); n = 3. D: Human β-cell death. Human islets were dispersed into single cells and cultured (not sorted) on 804G matrix-coated dishes for 24 h in the presence of increasing concentrations of CX3CL1. Cell death is expressed as TUNEL-positive β-cells normalized to Control (absolute value 11.6 ± 4.5%); n = 3. *p < 0.05 vs. 0 ng/ml CX3CL1 as tested by ANOVA followed by Bonferroni post hoc test.
Mentions: To investigate the potential impact of CX3CL1 on human β-cell survival, cell death was quantified using TUNEL assay on dispersed human islet cells. After 24 h treatment, CX3CL1 decreased human β-cell apoptosis. This decrease in apoptosis was concentration dependent and could only be observed with 5 and 10 nM CX3CL1 (Figure 3D). When cell death of FACS-purified rat β-cells (Figure 3B) or of β-cells of dispersed rat islets (Figure 3C) was evaluated, no significant effect of CX3CL1 could be observed at any concentration studied. These results could suggest that the positive impact of CX3CL1 on human β-cells is species specific. However, there are other potentially confounding factors. Specifically, basal apoptosis (in the absence of CX3CL1) was 11.6 ± 4.5% for human β-cells but only 0.7 ± 0.3% and 0.05 ± 0.02%, respectively, for sorted rat β-cells or β-cells within an unsorted rat islet cell population.

Bottom Line: CX3CL1 decreased human (but not rat) β-cell apoptosis.Moreover, CX3CL1 decreases basal apoptosis of human β-cells.We further demonstrate that CX3CL1 protects β-cells from the adverse effects of TNFα on their function by restoring the expression and phosphorylation of key proteins of the insulin secretion pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Medicine and Development, Geneva University, Geneva, Switzerland.

ABSTRACT

Objective: We have previously shown the existence of a muscle-pancreas intercommunication axis in which CX3CL1 (fractalkine), a CX3C chemokine produced by skeletal muscle cells, could be implicated. It has recently been shown that the fractalkine system modulates murine β-cell function. However, the impact of CX3CL1 on human islet cells especially regarding a protective role against cytokine-induced apoptosis remains to be investigated.

Methods: Gene expression was determined using RNA sequencing in human islets, sorted β- and non-β-cells. Glucose-stimulated insulin secretion (GSIS) and glucagon secretion from human islets was measured following 24 h exposure to 1-50 ng/ml CX3CL1. GSIS and specific protein phosphorylation were measured in rat sorted β-cells exposed to CX3CL1 for 48 h alone or in the presence of TNFα (20 ng/ml). Rat and human β-cell apoptosis (TUNEL) and rat β-cell proliferation (BrdU incorporation) were assessed after 24 h treatment with increasing concentrations of CX3CL1.

Results: Both CX3CL1 and its receptor CX3CR1 are expressed in human islets. However, CX3CL1 is more expressed in non-β-cells than in β-cells while its receptor is more expressed in β-cells. CX3CL1 decreased human (but not rat) β-cell apoptosis. CX3CL1 inhibited human islet glucagon secretion stimulated by low glucose but did not impact human islet and rat sorted β-cell GSIS. However, CX3CL1 completely prevented the adverse effect of TNFα on GSIS and on molecular mechanisms involved in insulin granule trafficking by restoring the phosphorylation (Akt, AS160, paxillin) and expression (IRS2, ICAM-1, Sorcin, PCSK1) of key proteins involved in these processes.

Conclusions: We demonstrate for the first time that human islets express and secrete CX3CL1 and CX3CL1 impacts them by decreasing glucagon secretion without affecting insulin secretion. Moreover, CX3CL1 decreases basal apoptosis of human β-cells. We further demonstrate that CX3CL1 protects β-cells from the adverse effects of TNFα on their function by restoring the expression and phosphorylation of key proteins of the insulin secretion pathway.

No MeSH data available.


Related in: MedlinePlus