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

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 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.


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Pdx1 regulates mitophagy in pancreatic β-cells. A: Transmission EM images from 5-month-old WT and Pdx1+/− β-cells. Inset: Focused area of mitochondria on EM images. B: Quantification of mitochondrial morphology (% of total mitochondria) observed in WT and Pdx1+/− β-cells in transmission EM images (∼250 independent mitochondria scored/animal). n = 3 animals/genotype. C: Relative oxygen consumption rate (OCR) measured in isolated WT and Pdx1+/− islets (n = 3/group) of 6- to 8-week-old mice. D: LC3/Mfn2 colocalization in LC3+ puncta quantified from 5-month-old WT and Pdx1+/− β-cells. E: Representative confocal image of 5-month-old WT and Pdx1+/− β-cells stained for insulin (gray), DAPI (DNA [blue]), LC3 (autophagosomes [green]), and Mfn2 (mitochondria [red]). Data are expressed as mean ± SEM. n = 5 mice/group and ∼80 β-cells (>1,100 total LC3+ and Mfn2+ structures) were analyzed per animal. *P < 0.05.
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Figure 1: Pdx1 regulates mitophagy in pancreatic β-cells. A: Transmission EM images from 5-month-old WT and Pdx1+/− β-cells. Inset: Focused area of mitochondria on EM images. B: Quantification of mitochondrial morphology (% of total mitochondria) observed in WT and Pdx1+/− β-cells in transmission EM images (∼250 independent mitochondria scored/animal). n = 3 animals/genotype. C: Relative oxygen consumption rate (OCR) measured in isolated WT and Pdx1+/− islets (n = 3/group) of 6- to 8-week-old mice. D: LC3/Mfn2 colocalization in LC3+ puncta quantified from 5-month-old WT and Pdx1+/− β-cells. E: Representative confocal image of 5-month-old WT and Pdx1+/− β-cells stained for insulin (gray), DAPI (DNA [blue]), LC3 (autophagosomes [green]), and Mfn2 (mitochondria [red]). Data are expressed as mean ± SEM. n = 5 mice/group and ∼80 β-cells (>1,100 total LC3+ and Mfn2+ structures) were analyzed per animal. *P < 0.05.

Mentions: To elucidate the mechanism(s) by which Pdx1 regulates mitochondrial function, we studied Pdx1 heterozygous mice (Pdx1+/−), which develop glucose intolerance and reduced GSIS due to reduced mitochondrial function (6). We evaluated mitochondrial ultrastructure by transmission electron microscopy in wild-type (WT) and Pdx1+/− islets. Pdx1+/− β-cells displayed an increased number of rounded mitochondria with disordered cristae and amorphous structure (Fig. 1A and B). Dysmorphic mitochondria were not observed by EM in non–β islet cells of Pdx1+/− mice (data not shown). As expected, Pdx1+/− mice exhibited glucose intolerance (data not shown) as well as reduced glucose-stimulated and maximal oxygen consumption [after treatment with the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone] in isolated islets (Fig. 1C), suggesting that Pdx1-dependent regulation of mitochondrial structure and function could contribute to impaired glucose control in Pdx1+/− mice.


Diabetes Susceptibility Genes Pdx1 and Clec16a Function in a Pathway Regulating Mitophagy in β -Cells
Pdx1 regulates mitophagy in pancreatic β-cells. A: Transmission EM images from 5-month-old WT and Pdx1+/− β-cells. Inset: Focused area of mitochondria on EM images. B: Quantification of mitochondrial morphology (% of total mitochondria) observed in WT and Pdx1+/− β-cells in transmission EM images (∼250 independent mitochondria scored/animal). n = 3 animals/genotype. C: Relative oxygen consumption rate (OCR) measured in isolated WT and Pdx1+/− islets (n = 3/group) of 6- to 8-week-old mice. D: LC3/Mfn2 colocalization in LC3+ puncta quantified from 5-month-old WT and Pdx1+/− β-cells. E: Representative confocal image of 5-month-old WT and Pdx1+/− β-cells stained for insulin (gray), DAPI (DNA [blue]), LC3 (autophagosomes [green]), and Mfn2 (mitochondria [red]). Data are expressed as mean ± SEM. n = 5 mice/group and ∼80 β-cells (>1,100 total LC3+ and Mfn2+ structures) were analyzed per animal. *P < 0.05.
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Figure 1: Pdx1 regulates mitophagy in pancreatic β-cells. A: Transmission EM images from 5-month-old WT and Pdx1+/− β-cells. Inset: Focused area of mitochondria on EM images. B: Quantification of mitochondrial morphology (% of total mitochondria) observed in WT and Pdx1+/− β-cells in transmission EM images (∼250 independent mitochondria scored/animal). n = 3 animals/genotype. C: Relative oxygen consumption rate (OCR) measured in isolated WT and Pdx1+/− islets (n = 3/group) of 6- to 8-week-old mice. D: LC3/Mfn2 colocalization in LC3+ puncta quantified from 5-month-old WT and Pdx1+/− β-cells. E: Representative confocal image of 5-month-old WT and Pdx1+/− β-cells stained for insulin (gray), DAPI (DNA [blue]), LC3 (autophagosomes [green]), and Mfn2 (mitochondria [red]). Data are expressed as mean ± SEM. n = 5 mice/group and ∼80 β-cells (>1,100 total LC3+ and Mfn2+ structures) were analyzed per animal. *P < 0.05.
Mentions: To elucidate the mechanism(s) by which Pdx1 regulates mitochondrial function, we studied Pdx1 heterozygous mice (Pdx1+/−), which develop glucose intolerance and reduced GSIS due to reduced mitochondrial function (6). We evaluated mitochondrial ultrastructure by transmission electron microscopy in wild-type (WT) and Pdx1+/− islets. Pdx1+/− β-cells displayed an increased number of rounded mitochondria with disordered cristae and amorphous structure (Fig. 1A and B). Dysmorphic mitochondria were not observed by EM in non–β islet cells of Pdx1+/− mice (data not shown). As expected, Pdx1+/− mice exhibited glucose intolerance (data not shown) as well as reduced glucose-stimulated and maximal oxygen consumption [after treatment with the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone] in isolated islets (Fig. 1C), suggesting that Pdx1-dependent regulation of mitochondrial structure and function could contribute to impaired glucose control in Pdx1+/− mice.

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.


Related in: MedlinePlus