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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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Assessment of cytosolic calcium levels in PINK1-deficient β-cells. (a) PINK1 expression in MIN6 cells and isolated PINK1 WT and KO islets compared with WT midbrain. *p < 0.05 and **p < 0.005, Mann–Whitney U-test. (b) RT-PCR confirmation of PINK1 knockdown levels obtained via siRNA treatment. p < 0.005, paired t-test. (c) RT-PCR confirmation of PINK1 levels in PINK1 WT and KO islets. p < 0.005, Mann–Whitney U-test. (d) Basal levels of Fura-2 ratio in Scr control versus PINK1 kd siRNA-treated cells. p < 0.005, paired t-test. (e) Basal levels of Fura-2 ratio in WT control versus PINK1 KO islets, p < 0.005, Mann–Whitney U-test. (f) Representative traces showing measurement of Fura-2 ratio alteration induced by glucose stimuli in Scr control versus PINK1 kd cells. (g) Representative traces measuring alteration of Fura-2 ratio in WT control versus PINK1 KO islets, respectively. p < 0.005, paired t-test. (h) Alteration of Fura-2 ratio in Scr control and PINK1 kd cells with increasing glucose stimuli and improvement of PINK1 kd cell response with DPI or MnTBAP treatment. p < 0.005, paired t-test. (i) Alteration of Fura-2 ratio in Scr control and PINK1 kd cells, but not Parkin kd, with increasing glucose stimuli and rescue of PINK1 kd cell response with PINK1-wt re-expression. p < 0.05, paired t-test.
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RSOB140051F1: Assessment of cytosolic calcium levels in PINK1-deficient β-cells. (a) PINK1 expression in MIN6 cells and isolated PINK1 WT and KO islets compared with WT midbrain. *p < 0.05 and **p < 0.005, Mann–Whitney U-test. (b) RT-PCR confirmation of PINK1 knockdown levels obtained via siRNA treatment. p < 0.005, paired t-test. (c) RT-PCR confirmation of PINK1 levels in PINK1 WT and KO islets. p < 0.005, Mann–Whitney U-test. (d) Basal levels of Fura-2 ratio in Scr control versus PINK1 kd siRNA-treated cells. p < 0.005, paired t-test. (e) Basal levels of Fura-2 ratio in WT control versus PINK1 KO islets, p < 0.005, Mann–Whitney U-test. (f) Representative traces showing measurement of Fura-2 ratio alteration induced by glucose stimuli in Scr control versus PINK1 kd cells. (g) Representative traces measuring alteration of Fura-2 ratio in WT control versus PINK1 KO islets, respectively. p < 0.005, paired t-test. (h) Alteration of Fura-2 ratio in Scr control and PINK1 kd cells with increasing glucose stimuli and improvement of PINK1 kd cell response with DPI or MnTBAP treatment. p < 0.005, paired t-test. (i) Alteration of Fura-2 ratio in Scr control and PINK1 kd cells, but not Parkin kd, with increasing glucose stimuli and rescue of PINK1 kd cell response with PINK1-wt re-expression. p < 0.05, paired t-test.

Mentions: To assess whether loss of PINK1 function would potentially have an effect on islet cell function, we sought to determine whether PINK1 was expressed in islet cells. Owing to continuing problems assessing mouse PINK1 by western blot, we assayed MIN6 cells and isolated PINK1 wild-type (WT) and KO islets for PINK1 expression by RT-PCR. PINK1 WT mouse midbrain (known to express high levels of PINK1) was used as a control. Figure 1a demonstrates that PINK1 transcripts are present in both MIN6 cells and isolated WT islets, but these levels are significantly reduced by comparison with transcript levels in the midbrain (n = 4, *p < 0.01 and **p < 0.0006).Figure 1.


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)

Assessment of cytosolic calcium levels in PINK1-deficient β-cells. (a) PINK1 expression in MIN6 cells and isolated PINK1 WT and KO islets compared with WT midbrain. *p < 0.05 and **p < 0.005, Mann–Whitney U-test. (b) RT-PCR confirmation of PINK1 knockdown levels obtained via siRNA treatment. p < 0.005, paired t-test. (c) RT-PCR confirmation of PINK1 levels in PINK1 WT and KO islets. p < 0.005, Mann–Whitney U-test. (d) Basal levels of Fura-2 ratio in Scr control versus PINK1 kd siRNA-treated cells. p < 0.005, paired t-test. (e) Basal levels of Fura-2 ratio in WT control versus PINK1 KO islets, p < 0.005, Mann–Whitney U-test. (f) Representative traces showing measurement of Fura-2 ratio alteration induced by glucose stimuli in Scr control versus PINK1 kd cells. (g) Representative traces measuring alteration of Fura-2 ratio in WT control versus PINK1 KO islets, respectively. p < 0.005, paired t-test. (h) Alteration of Fura-2 ratio in Scr control and PINK1 kd cells with increasing glucose stimuli and improvement of PINK1 kd cell response with DPI or MnTBAP treatment. p < 0.005, paired t-test. (i) Alteration of Fura-2 ratio in Scr control and PINK1 kd cells, but not Parkin kd, with increasing glucose stimuli and rescue of PINK1 kd cell response with PINK1-wt re-expression. p < 0.05, paired t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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RSOB140051F1: Assessment of cytosolic calcium levels in PINK1-deficient β-cells. (a) PINK1 expression in MIN6 cells and isolated PINK1 WT and KO islets compared with WT midbrain. *p < 0.05 and **p < 0.005, Mann–Whitney U-test. (b) RT-PCR confirmation of PINK1 knockdown levels obtained via siRNA treatment. p < 0.005, paired t-test. (c) RT-PCR confirmation of PINK1 levels in PINK1 WT and KO islets. p < 0.005, Mann–Whitney U-test. (d) Basal levels of Fura-2 ratio in Scr control versus PINK1 kd siRNA-treated cells. p < 0.005, paired t-test. (e) Basal levels of Fura-2 ratio in WT control versus PINK1 KO islets, p < 0.005, Mann–Whitney U-test. (f) Representative traces showing measurement of Fura-2 ratio alteration induced by glucose stimuli in Scr control versus PINK1 kd cells. (g) Representative traces measuring alteration of Fura-2 ratio in WT control versus PINK1 KO islets, respectively. p < 0.005, paired t-test. (h) Alteration of Fura-2 ratio in Scr control and PINK1 kd cells with increasing glucose stimuli and improvement of PINK1 kd cell response with DPI or MnTBAP treatment. p < 0.005, paired t-test. (i) Alteration of Fura-2 ratio in Scr control and PINK1 kd cells, but not Parkin kd, with increasing glucose stimuli and rescue of PINK1 kd cell response with PINK1-wt re-expression. p < 0.05, paired t-test.
Mentions: To assess whether loss of PINK1 function would potentially have an effect on islet cell function, we sought to determine whether PINK1 was expressed in islet cells. Owing to continuing problems assessing mouse PINK1 by western blot, we assayed MIN6 cells and isolated PINK1 wild-type (WT) and KO islets for PINK1 expression by RT-PCR. PINK1 WT mouse midbrain (known to express high levels of PINK1) was used as a control. Figure 1a demonstrates that PINK1 transcripts are present in both MIN6 cells and isolated WT islets, but these levels are significantly reduced by comparison with transcript levels in the midbrain (n = 4, *p < 0.01 and **p < 0.0006).Figure 1.

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