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Diabetes Susceptibility Genes Pdx1 and Clec16a Function in a Pathway Regulating Mitophagy in β -Cells

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ABSTRACT

Mitophagy is a critical regulator of mitochondrial quality control and is necessary for elimination of dysfunctional mitochondria to maintain cellular respiration. Here, we report that the homeodomain transcription factor Pdx1, a gene associated with both type 2 diabetes and monogenic diabetes of the young, regulates mitophagy in pancreatic β-cells. Loss of Pdx1 leads to abnormal mitochondrial morphology and function as well as impaired mitochondrial turnover. High-throughput expression microarray and chromatin occupancy analyses reveal that Pdx1 regulates the expression of Clec16a, a type 1 diabetes gene and itself a key mediator of mitophagy through regulation of the E3 ubiquitin ligase Nrdp1. Indeed, expression of Clec16a and Nrdp1 are both reduced in Pdx1 haploinsufficient islets, and reduction of Pdx1 impairs fusion of autophagosomes containing mitochondria to lysosomes during mitophagy. Importantly, restoration of Clec16a expression after Pdx1 loss of function restores mitochondrial trafficking during mitophagy and improves mitochondrial respiration and glucose-stimulated insulin release. Thus, Pdx1 orchestrates nuclear control of mitochondrial function in part by controlling mitophagy through Clec16a. The novel Pdx1-Clec16a-Nrdp1 pathway we describe provides a genetic basis for the pathogenesis of mitochondrial dysfunction in multiple forms of diabetes that could be targeted for future therapies to improve β-cell function.

No MeSH data available.


Overexpression of Clec16a improves mitochondrial respiration and insulin secretion but not mtDNA content after Pdx1 loss of function. A: Fold OCR in si-nontargeting (siNT)- or siPdx1-treated control Clec16a-overexpressing Min6 β-cells (n = 3/group). B: Fold insulin secretion (normalized to total protein content) in siNT- or siPdx1-treated control or Clec16a-overexpressing Min6 β-cells (n = 3/group). C: Relative mtDNA content measured by qPCR (normalized to nuclear DNA expression) in isolated WT and Pdx1+/− islets of 6-week-old mice (n = 4/group). D: Relative mtDNA content measured by qPCR (normalized to nuclear DNA expression) from DNA isolated from siNT- or siPdx1-treated control or Clec16a-overexpressing Min6 β-cells (n = 4/group). *P < 0.05.
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Figure 5: Overexpression of Clec16a improves mitochondrial respiration and insulin secretion but not mtDNA content after Pdx1 loss of function. A: Fold OCR in si-nontargeting (siNT)- or siPdx1-treated control Clec16a-overexpressing Min6 β-cells (n = 3/group). B: Fold insulin secretion (normalized to total protein content) in siNT- or siPdx1-treated control or Clec16a-overexpressing Min6 β-cells (n = 3/group). C: Relative mtDNA content measured by qPCR (normalized to nuclear DNA expression) in isolated WT and Pdx1+/− islets of 6-week-old mice (n = 4/group). D: Relative mtDNA content measured by qPCR (normalized to nuclear DNA expression) from DNA isolated from siNT- or siPdx1-treated control or Clec16a-overexpressing Min6 β-cells (n = 4/group). *P < 0.05.

Mentions: To establish that Clec16a mediates the effect of Pdx1 on mitophagy, we studied the effect of Pdx1 loss of function on mitophagy in Min6 β-cells stably overexpressing Clec16a. Overexpression of Clec16a rescued Nrdp1 protein expression after Pdx1 siRNA treatment, suggesting that Pdx1 affects Nrdp1 expression in a Clec16a-dependent manner (Fig. 4A and B). We next evaluated mitochondrial trafficking and found that restoration of Clec16a expression significantly reduced mitochondrial colocalization with autophagosomes (Fig. 4C) and increased colocalization of autophagosome and lysosome markers (Fig. 4D) in Pdx1-deficient Min6 β-cells. Overexpression of Clec16a also improved defects in mitochondrial respiration and GSIS caused by Pdx1 deficiency (Fig. 5A and B), suggesting that Pdx1 control of mitochondrial metabolism and GSIS is mediated at least in part by its regulation of Clec16a.


Diabetes Susceptibility Genes Pdx1 and Clec16a Function in a Pathway Regulating Mitophagy in β -Cells
Overexpression of Clec16a improves mitochondrial respiration and insulin secretion but not mtDNA content after Pdx1 loss of function. A: Fold OCR in si-nontargeting (siNT)- or siPdx1-treated control Clec16a-overexpressing Min6 β-cells (n = 3/group). B: Fold insulin secretion (normalized to total protein content) in siNT- or siPdx1-treated control or Clec16a-overexpressing Min6 β-cells (n = 3/group). C: Relative mtDNA content measured by qPCR (normalized to nuclear DNA expression) in isolated WT and Pdx1+/− islets of 6-week-old mice (n = 4/group). D: Relative mtDNA content measured by qPCR (normalized to nuclear DNA expression) from DNA isolated from siNT- or siPdx1-treated control or Clec16a-overexpressing Min6 β-cells (n = 4/group). *P < 0.05.
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Figure 5: Overexpression of Clec16a improves mitochondrial respiration and insulin secretion but not mtDNA content after Pdx1 loss of function. A: Fold OCR in si-nontargeting (siNT)- or siPdx1-treated control Clec16a-overexpressing Min6 β-cells (n = 3/group). B: Fold insulin secretion (normalized to total protein content) in siNT- or siPdx1-treated control or Clec16a-overexpressing Min6 β-cells (n = 3/group). C: Relative mtDNA content measured by qPCR (normalized to nuclear DNA expression) in isolated WT and Pdx1+/− islets of 6-week-old mice (n = 4/group). D: Relative mtDNA content measured by qPCR (normalized to nuclear DNA expression) from DNA isolated from siNT- or siPdx1-treated control or Clec16a-overexpressing Min6 β-cells (n = 4/group). *P < 0.05.
Mentions: To establish that Clec16a mediates the effect of Pdx1 on mitophagy, we studied the effect of Pdx1 loss of function on mitophagy in Min6 β-cells stably overexpressing Clec16a. Overexpression of Clec16a rescued Nrdp1 protein expression after Pdx1 siRNA treatment, suggesting that Pdx1 affects Nrdp1 expression in a Clec16a-dependent manner (Fig. 4A and B). We next evaluated mitochondrial trafficking and found that restoration of Clec16a expression significantly reduced mitochondrial colocalization with autophagosomes (Fig. 4C) and increased colocalization of autophagosome and lysosome markers (Fig. 4D) in Pdx1-deficient Min6 β-cells. Overexpression of Clec16a also improved defects in mitochondrial respiration and GSIS caused by Pdx1 deficiency (Fig. 5A and B), suggesting that Pdx1 control of mitochondrial metabolism and GSIS is mediated at least in part by its regulation of Clec16a.

View Article: PubMed Central - PubMed

ABSTRACT

Mitophagy is a critical regulator of mitochondrial quality control and is necessary for elimination of dysfunctional mitochondria to maintain cellular respiration. Here, we report that the homeodomain transcription factor Pdx1, a gene associated with both type 2 diabetes and monogenic diabetes of the young, regulates mitophagy in pancreatic &beta;-cells. Loss of Pdx1 leads to abnormal mitochondrial morphology and function as well as impaired mitochondrial turnover. High-throughput expression microarray and chromatin occupancy analyses reveal that Pdx1 regulates the expression of Clec16a, a type 1 diabetes gene and itself a key mediator of mitophagy through regulation of the E3 ubiquitin ligase Nrdp1. Indeed, expression of Clec16a and Nrdp1 are both reduced in Pdx1 haploinsufficient islets, and reduction of Pdx1 impairs fusion of autophagosomes containing mitochondria to lysosomes during mitophagy. Importantly, restoration of Clec16a expression after Pdx1 loss of function restores mitochondrial trafficking during mitophagy and improves mitochondrial respiration and glucose-stimulated insulin release. Thus, Pdx1 orchestrates nuclear control of mitochondrial function in part by controlling mitophagy through Clec16a. The novel Pdx1-Clec16a-Nrdp1 pathway we describe provides a genetic basis for the pathogenesis of mitochondrial dysfunction in multiple forms of diabetes that could be targeted for future therapies to improve &beta;-cell function.

No MeSH data available.