The diabetes susceptibility gene Clec16a regulates mitophagy - PubMed (original) (raw)

. 2014 Jun 19;157(7):1577-90.

doi: 10.1016/j.cell.2014.05.016.

Aditi Gupta 2, Marina Bakay 3, Alana M Ferrari 2, David N Groff 2, João Fadista 4, Lynn A Spruce 5, Jake A Kushner 6, Leif Groop 4, Steven H Seeholzer 5, Brett A Kaufman 7, Hakon Hakonarson 8, Doris A Stoffers 9

Affiliations

The diabetes susceptibility gene Clec16a regulates mitophagy

Scott A Soleimanpour et al. Cell. 2014.

Abstract

Clec16a has been identified as a disease susceptibility gene for type 1 diabetes, multiple sclerosis, and adrenal dysfunction, but its function is unknown. Here we report that Clec16a is a membrane-associated endosomal protein that interacts with E3 ubiquitin ligase Nrdp1. Loss of Clec16a leads to an increase in the Nrdp1 target Parkin, a master regulator of mitophagy. Islets from mice with pancreas-specific deletion of Clec16a have abnormal mitochondria with reduced oxygen consumption and ATP concentration, both of which are required for normal β cell function. Indeed, pancreatic Clec16a is required for normal glucose-stimulated insulin release. Moreover, patients harboring a diabetogenic SNP in the Clec16a gene have reduced islet Clec16a expression and reduced insulin secretion. Thus, Clec16a controls β cell function and prevents diabetes by controlling mitophagy. This pathway could be targeted for prevention and control of diabetes and may extend to the pathogenesis of other Clec16a- and Parkin-associated diseases.

Copyright © 2014 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. I-DIRT identifies Clec16a interaction with E3 ubiquitin ligase Nrdp1

(A) Representative Western blot (WB) of parental NIH3T3 fibroblast (12C6-labeled) or stably expressing Flag-Clec16a NIH3T3 fibroblast (13C6-labeled) input protein lysates, supernatant and Flag immunoprecipitation for Flag-Clec16a (n=6). (B) MS/MS map marked with b ions (blue) and y ions (red) for Clec16a specific peptide sequence with calculated m/z ratios. Heavy residue indicated in bold print in peptide sequence. (C) I-DIRT ratios for >800 proteins identified from 6 independent I-DIRT experiments with calculated non-specific value of 0.5. Clec16a and Nrdp1 peaks indicated. Contaminant peptides (human keratin (K), immunoglobulins (Ig), trypsin (T), and albumin (A)) indicated by peaks below non-specific interactors. (D) MS/MS map marked with b ions (blue) and y ions (red) for an Nrdp1 specific peptide sequence with observed and calculated m/z ratios. (E) Amino acid sequence of Nrdp1 with peptides identified by MS (highlighted residues). Trypsin cleavage sites demarcated in blue. (F) Representative WB (3 independent experiments) of lysates of Flag-Clec16a NIH3T3 fibroblasts following anti-Flag IP (left) or anti-Nrdp1 IP (right).

Figure 2

Figure 2. Clec16a controls mitochondrial function through the Nrdp1/Parkin pathway

(A) Model of Cre-mediated recombination of the Clec16a locus. (B) Clec16a expression by qRTPCR of RNA isolated from vehicle (EtOH) or OHT treated WT or _Clec16a_flox fibroblasts (n=3/group). (C) Nrdp1 protein expression by WB of WT or _Clec16a_flox fibroblasts following overnight treatment with vehicle (DMSO) or 10 µM MG132 (n=3/group). (D) Parkin expression by WB in WT or _Clec16a_flox fibroblasts following 3-hr treatment with vehicle (DMSO) or 10 µM FCCP (n=3/group). (E) Parkin subcellular localization by WB in WT or _Clec16a_flox fibroblasts following cell fractionation and enrichment of mitochondrial or cytosolic proteins. SDHA and actin serve as mitochondrial and cytosolic loading controls, respectively (n=3/group). Fold mitochondrial parkin as estimated by densitometry. (F) Mitofusin 2 expression by WB in WT or _Clec16a_flox fibroblasts following 3-hr treatment with vehicle (DMSO) or 10 µM FCCP (n=3/group). (G) Fold oxygen consumption rate (OCR) in WT or _Clec16a_flox fibroblasts, 96 hours after transient overexpression of empty vector (pcDNA3-3xHA) or 3xHA-Nrdp1 (n=3/group). (H) Fold OCR in WT or _Clec16a_flox fibroblasts, 96 hours after transient transfection of non-targeting (siNT) or Parkin (siParkin) siRNA (n=3/group). For all WBs, representative images chosen from among 3 independent experiments. Densitometry, shown adjacent to each WB, represents mean (±SEM) of all 3 independent experiments. qRT-PCR and OCR data expressed as mean (±SEM) of 3 experiments performed in triplicate. *p<0.05 **p<0.01. See also Figure S1.

Figure 3

Figure 3. Pancreas-specific Clec16a loss of function leads to hyperglycemia due to functional defects in β-cells

(A) Clec16a mRNA expression by qRT-PCR from RNA isolated from WT or _Clec16a_Δpanc islets (n=10/group). (B) Clec16a expression by WB in WT or _Clec16a_Δpanc islets (n=4/group). (C) Immunohistochemistry of adjacent sections of WT and _Clec16a_Δpanc pancreata for Clec16a (top) and insulin (bottom). Image shown is representative of IHC performed in 3 mice/group. (D) Blood glucose concentrations measured during IPGTT of 5 week old WT and _Clec16a_Δpanc littermates. (p<0.005 by ANOVA; n=6–15/group) (E) Serum insulin (n=6–15/group) measured during in vivo glucose-stimulated insulin release testing in 8 week old WT and _Clec16a_Δpanc littermates. (F) Fold glucose-stimulated insulin secretion following static incubations in 2.8mM and 16.7 mM glucose performed in isolated WT or _Clec16a_Δpanc islets of 8 week old littermates (n=4/group). (G) Transmission EM images from 3-month-old WT and _Clec16a_Δpanc β-cells. Inset (lower right) – focused area of mitochondria on EM images. (H) Quantification of changes in mitochondrial morphology (% of total mitochondria) observed in WT and _Clec16a_Δpanc β-cells in transmission EM images (~250 independent mitochondria scored/animal). Images captured from 3–6 animals/group. Representative WBs chosen from among 4 independent experiments. Densitometry, shown adjacent to each WB, represents mean (±SEM) of 4 independent experiments. *p<0.05 **p<0.01. See also Figures S2-S4.

Figure 4

Figure 4. Clec16a loss of function results in the accumulation of unhealthy mitochondria

(A) Relative OCR measured in isolated WT and _Clec16a_Δpanc islets (n=6/group). (B) OCR (n=4/group) measured at baseline or following 300 nM FCCP in Min6 β-cells following transfection with non-targeting (siNT) or Clec16a specific siRNA (siClec16a). (C) Fold ATP concentrations (normalized to protein content; n=3/group) in Min6 β-cells following transfection with siNT or siClec16a. (D) Relative mtDNA content measured by qPCR (normalized to nuclear DNA expression) in isolated WT and _Clec16a_Δpanc islets (n=3–4/group). (E) Expression of the mitochondrial proteins succinate dehydrogenase A (SDHA) and mitochondrial complex IV subunit I (mtCOI) in isolated WT and _Clec16a_Δpanc islets by WB. (F) Quantitative RT-PCR of markers of mitochondrial biogenesis (PGC1α, NRF1, TFAM) and ROS-induced genes (ND6 and NF-E2) from RNA isolated from WT and _Clec16a_Δpanc islets (n=5/group). For all WBs, representative images chosen from 3 independent experiments performed. WB densitometry represents mean (±SEM) of 3 independent experiments. *p<0.05 **p<0.01. See also Figures S5-S6.

Figure 5

Figure 5. Clec16a regulates mitophagy

(A) Nrdp1 expression by WB of WT and _Clec16a_Δpanc islets. Representative WB chosen from 4 independent experiments performed. Each sample analyzed comprised islet lysates pooled from 8 mice (32 mice total/group). (B) Parkin expression by WB of WT and _Clec16a_Δpanc islets following 3-hr of vehicle (DMSO) or 10 µM FCCP (n=3/group). (C) Mfn2 and Porin expression by WB in WT and _Clec16a_Δpanc islets following 3-hr of vehicle (DMSO) or 10 µM FCCP (n=3/group). (D) Representative deconvolution image of WT and _Clec16a_flox fibroblasts, 72 hours after overexpression of LC3-GFP and mito-dsRed, treated with vehicle (DMSO) or 150 nM BafA1 for 6 hours. (E) Quantification of LC3-GFP/mito-dsRed co-localization in WT and _Clec16a_flox fibroblasts following vehicle (DMSO) or 150 nM BafA1 for 6 hours (n=5–8/group). (F) Fold LC3-GFP puncta quantified from WT and _Clec16a_flox fibroblasts, 72 hours after overexpression of LC3-GFP, treated with vehicle (DMSO) or 150 nM BafA1 for 6 hours. (G) Representative deconvolution image of WT and _Clec16a_flox fibroblasts treated with 10 µM FCCP for 60 minutes followed by indirect immunofluorescence for Lamp1 (green), LC3 (red), and DAPI (blue). (H) Quantification of LC3+ Lamp1+ co-localization in WT and _Clec16a_flox fibroblasts following 10 µM FCCP for 60 minutes (n=5/group). (I) Quantification of LC3+ Lamp1+ co-localization in non-FCCP treated WT and _Clec16a_flox fibroblasts, 72 hours after transient overexpression of empty vector (pcDNA3–3xHA) or 3xHANrdp1 (n=5–7/group). For Figure 5B and 5C, representative WBs chosen from 3 independent experiments performed. Densitometry, shown adjacent to each WB, represents mean (±SEM) of all experiments performed. *p<0.05 **p<0.01. See also Figures S5-S6 and Movies S1–S3.

Figure 6

Figure 6. Clec16a regulates β-cell endosomal trafficking

(A) Study design for HRP (5 mg/mL) trafficking and degradation by a pulse-chase approach. (B) Quantification of undegraded HRP activity in dispersed WT and _Clec16a_Δpanc islets (n=6/group). (C) Study design for fluorescent-conjugated dextran trafficking and co-localization by pulse-chase in Min6 β-cells. (D) Quantification of FITC-dextran and TRITC-dextran co-localization over time in Min6 β-cells (p<0.05 by ANOVA; n=3/group) 72 hours after transient transfection with non-targeting or Clec16a-specific siRNA. (E) Representative deconvolution image of FITC-dextran and TRITC-dextran treated Min6 β-cells (3 hours post-TRITC-dextran chase) following transient transfection with non-targeting or Clec16a-specific siRNA. (F) Representative deconvolution image following immunofluorescence staining for Rab7 (red) and DAPI (DNA, blue) of Min6 β-cells following transient transfection with non-targeting or Clec16a-specific siRNA. (G) Quantification of undegraded HRP activity in WT and _Clec16a_flox fibroblasts, 72 hours after overexpression of empty vector (pcDNA3–3xHA) or 3xHA-Nrdp1, utilizing HRP pulse-chase approach (n=6/group). *p<0.05 **p<0.01. See also Figure S7 and Movies S1 and S3.

Figure 7

Figure 7. The diabetogenic CLEC16A SNP rs12708716 is associated with reduced islet CLEC16A expression and impaired glucose control

(A) Human islet CLEC16A RNA expression measured by RNA-seq in islet donors stratified by DNA sequencing at position of rs12708716 A/G SNP. **p<0.001. (B) Hemoglobin A1c measurements in islet donors sequenced at position of rs12708716 A/G SNP. (AA n=31/group, AG n=42/group, GG n=8/group). *p<0.05.

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