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PINK1 deficiency in β-cells increases basal insulin secretion and improves glucose tolerance in mice.

Deas E, Piipari K, Machhada A, Li A, Gutierrez-del-Arroyo A, Withers DJ, Wood NW, Abramov AY - Open Biol (2014)

Bottom Line: The Parkinson's disease (PD) gene, PARK6, encodes the PTEN-induced putative kinase 1 (PINK1) mitochondrial kinase, which provides protection against oxidative stress-induced apoptosis.This was accompanied by higher basal levels of intracellular calcium leading to increased basal levels of insulin secretion under low glucose conditions.For the first time, these combined results demonstrate that loss of PINK1 function appears to disrupt glucose-sensing leading to enhanced insulin release, which is uncoupled from glucose uptake, and suggest a key role for PINK1 in β-cell function.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK.

ABSTRACT
The Parkinson's disease (PD) gene, PARK6, encodes the PTEN-induced putative kinase 1 (PINK1) mitochondrial kinase, which provides protection against oxidative stress-induced apoptosis. Given the link between glucose metabolism, mitochondrial function and insulin secretion in β-cells, and the reported association of PD with type 2 diabetes, we investigated the response of PINK1-deficient β-cells to glucose stimuli to determine whether loss of PINK1 affected their function. We find that loss of PINK1 significantly impairs the ability of mouse pancreatic β-cells (MIN6 cells) and primary intact islets to take up glucose. This was accompanied by higher basal levels of intracellular calcium leading to increased basal levels of insulin secretion under low glucose conditions. Finally, we investigated the effect of PINK1 deficiency in vivo and find that PINK1 knockout mice have improved glucose tolerance. For the first time, these combined results demonstrate that loss of PINK1 function appears to disrupt glucose-sensing leading to enhanced insulin release, which is uncoupled from glucose uptake, and suggest a key role for PINK1 in β-cell function.

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PINK1 deficiency impairs mitochondrial function and glucose-sensing. (a–d) Representative traces measuring alteration of NADH autofluorescence, rhodamine 123 fluorescence and 2-NBDG uptake in WT control versus PINK1 KO islets, respectively. p < 0.005, Mann–Whitney U-test. (e) GLUT1 and GLUT2 transporter expression in WT and PINK1 KO islets. (f) Alteration of 2-NBDG uptake rate in Scr control versus PINK1 kd cells at basal levels and after DPI or MnTBAP treatment. p < 0.001, p < 0.00001 and p < 0.005, respectively, paired t-test. (g) Alteration of 2-NBDG uptake rate in WT control versus PINK1 KO intact islets at basal levels and after DPI treatment. p < 0.005 and p < 0.001, respectively; Mann–Whitney U-test. (h) Alteration of 2-NBDG uptake rate in PINK1 kd versus Scr control and Parkin kd cells at basal levels and rescue of PINK1 kd uptake rate through re-expression of PINK1-wt. p < 0.005, paired t-test.
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RSOB140051F2: PINK1 deficiency impairs mitochondrial function and glucose-sensing. (a–d) Representative traces measuring alteration of NADH autofluorescence, rhodamine 123 fluorescence and 2-NBDG uptake in WT control versus PINK1 KO islets, respectively. p < 0.005, Mann–Whitney U-test. (e) GLUT1 and GLUT2 transporter expression in WT and PINK1 KO islets. (f) Alteration of 2-NBDG uptake rate in Scr control versus PINK1 kd cells at basal levels and after DPI or MnTBAP treatment. p < 0.001, p < 0.00001 and p < 0.005, respectively, paired t-test. (g) Alteration of 2-NBDG uptake rate in WT control versus PINK1 KO intact islets at basal levels and after DPI treatment. p < 0.005 and p < 0.001, respectively; Mann–Whitney U-test. (h) Alteration of 2-NBDG uptake rate in PINK1 kd versus Scr control and Parkin kd cells at basal levels and rescue of PINK1 kd uptake rate through re-expression of PINK1-wt. p < 0.005, paired t-test.

Mentions: Under normal circumstances, application of glucose to cells induces an increase in glucose metabolism, which, in turn, increases the concentration of available mitochondrial substrates [16]. The substrates are used by mitochondria, and this increases a number of mitochondrial characteristics such as the production of NADH and an increase in mitochondrial membrane potential (ΔΨm) [17]. As PINK1 is known to be essential for maintaining normal mitochondrial function in neurons, we assessed mitochondrial function in the islets. In our experiments, application of increasing concentrations of glucose induced a dose-dependent increase of NADH autofluorescence in WT islets (n = 12; figure 2a). However, the application of the same glucose solutions induced a significantly smaller response in PINK1-deficient islets (n = 10; figure 2a). A lower level of mitochondrial substrates, such as NADH, in PINK1-deficient cells should make them less sensitive to changes in ΔΨm. To confirm this, we assessed alterations in ΔΨm in response to glucose stimuli using rhodamine 123 (Rh123) [18]. Figure 2b shows that application of 5, 10 and 20 mM of glucose induces a step-like increase in ΔΨm in WT islets which registers as a decrease in Rh123 fluorescence. Application of the same glucose solutions to PINK1 KO islets induced a significantly smaller hyperpolarization in ΔΨm. As a control, complete depolarization of the cells was induced with 1 µM FCCP and generated a maximal increase in Rh123 fluorescence. Importantly, application of mitochondrial substrates such as malate or methyl succinate induced a profound mitochondrial hyperpolarization in both cell types (figure 2c), suggesting that the mitochondria in PINK1-deficient islets are not damaged and have a fully functional response when mitochondrial substrates are available. Considering this, our results suggest that PINK1-deficient β-cells have impaired glucose delivery or glycolysis.Figure 2.


PINK1 deficiency in β-cells increases basal insulin secretion and improves glucose tolerance in mice.

Deas E, Piipari K, Machhada A, Li A, Gutierrez-del-Arroyo A, Withers DJ, Wood NW, Abramov AY - Open Biol (2014)

PINK1 deficiency impairs mitochondrial function and glucose-sensing. (a–d) Representative traces measuring alteration of NADH autofluorescence, rhodamine 123 fluorescence and 2-NBDG uptake in WT control versus PINK1 KO islets, respectively. p < 0.005, Mann–Whitney U-test. (e) GLUT1 and GLUT2 transporter expression in WT and PINK1 KO islets. (f) Alteration of 2-NBDG uptake rate in Scr control versus PINK1 kd cells at basal levels and after DPI or MnTBAP treatment. p < 0.001, p < 0.00001 and p < 0.005, respectively, paired t-test. (g) Alteration of 2-NBDG uptake rate in WT control versus PINK1 KO intact islets at basal levels and after DPI treatment. p < 0.005 and p < 0.001, respectively; Mann–Whitney U-test. (h) Alteration of 2-NBDG uptake rate in PINK1 kd versus Scr control and Parkin kd cells at basal levels and rescue of PINK1 kd uptake rate through re-expression of PINK1-wt. p < 0.005, paired t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4042854&req=5

RSOB140051F2: PINK1 deficiency impairs mitochondrial function and glucose-sensing. (a–d) Representative traces measuring alteration of NADH autofluorescence, rhodamine 123 fluorescence and 2-NBDG uptake in WT control versus PINK1 KO islets, respectively. p < 0.005, Mann–Whitney U-test. (e) GLUT1 and GLUT2 transporter expression in WT and PINK1 KO islets. (f) Alteration of 2-NBDG uptake rate in Scr control versus PINK1 kd cells at basal levels and after DPI or MnTBAP treatment. p < 0.001, p < 0.00001 and p < 0.005, respectively, paired t-test. (g) Alteration of 2-NBDG uptake rate in WT control versus PINK1 KO intact islets at basal levels and after DPI treatment. p < 0.005 and p < 0.001, respectively; Mann–Whitney U-test. (h) Alteration of 2-NBDG uptake rate in PINK1 kd versus Scr control and Parkin kd cells at basal levels and rescue of PINK1 kd uptake rate through re-expression of PINK1-wt. p < 0.005, paired t-test.
Mentions: Under normal circumstances, application of glucose to cells induces an increase in glucose metabolism, which, in turn, increases the concentration of available mitochondrial substrates [16]. The substrates are used by mitochondria, and this increases a number of mitochondrial characteristics such as the production of NADH and an increase in mitochondrial membrane potential (ΔΨm) [17]. As PINK1 is known to be essential for maintaining normal mitochondrial function in neurons, we assessed mitochondrial function in the islets. In our experiments, application of increasing concentrations of glucose induced a dose-dependent increase of NADH autofluorescence in WT islets (n = 12; figure 2a). However, the application of the same glucose solutions induced a significantly smaller response in PINK1-deficient islets (n = 10; figure 2a). A lower level of mitochondrial substrates, such as NADH, in PINK1-deficient cells should make them less sensitive to changes in ΔΨm. To confirm this, we assessed alterations in ΔΨm in response to glucose stimuli using rhodamine 123 (Rh123) [18]. Figure 2b shows that application of 5, 10 and 20 mM of glucose induces a step-like increase in ΔΨm in WT islets which registers as a decrease in Rh123 fluorescence. Application of the same glucose solutions to PINK1 KO islets induced a significantly smaller hyperpolarization in ΔΨm. As a control, complete depolarization of the cells was induced with 1 µM FCCP and generated a maximal increase in Rh123 fluorescence. Importantly, application of mitochondrial substrates such as malate or methyl succinate induced a profound mitochondrial hyperpolarization in both cell types (figure 2c), suggesting that the mitochondria in PINK1-deficient islets are not damaged and have a fully functional response when mitochondrial substrates are available. Considering this, our results suggest that PINK1-deficient β-cells have impaired glucose delivery or glycolysis.Figure 2.

Bottom Line: The Parkinson's disease (PD) gene, PARK6, encodes the PTEN-induced putative kinase 1 (PINK1) mitochondrial kinase, which provides protection against oxidative stress-induced apoptosis.This was accompanied by higher basal levels of intracellular calcium leading to increased basal levels of insulin secretion under low glucose conditions.For the first time, these combined results demonstrate that loss of PINK1 function appears to disrupt glucose-sensing leading to enhanced insulin release, which is uncoupled from glucose uptake, and suggest a key role for PINK1 in β-cell function.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK.

ABSTRACT
The Parkinson's disease (PD) gene, PARK6, encodes the PTEN-induced putative kinase 1 (PINK1) mitochondrial kinase, which provides protection against oxidative stress-induced apoptosis. Given the link between glucose metabolism, mitochondrial function and insulin secretion in β-cells, and the reported association of PD with type 2 diabetes, we investigated the response of PINK1-deficient β-cells to glucose stimuli to determine whether loss of PINK1 affected their function. We find that loss of PINK1 significantly impairs the ability of mouse pancreatic β-cells (MIN6 cells) and primary intact islets to take up glucose. This was accompanied by higher basal levels of intracellular calcium leading to increased basal levels of insulin secretion under low glucose conditions. Finally, we investigated the effect of PINK1 deficiency in vivo and find that PINK1 knockout mice have improved glucose tolerance. For the first time, these combined results demonstrate that loss of PINK1 function appears to disrupt glucose-sensing leading to enhanced insulin release, which is uncoupled from glucose uptake, and suggest a key role for PINK1 in β-cell function.

Show MeSH
Related in: MedlinePlus