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Cited by 117Autophagy in Disease and TherapyMitochondrial Function and PathologyMetabolism, Diabetes, and Cancer

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BNIP3L/Nix-induced mitochondrial fission, mitophagy, and impaired myocyte glucose uptake are abrogated by PRKA/PKA phosphorylation

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da Silva Rosa, Simone C., Martens, Matthew D., Field, Jared T., Nguyen, Lucas, Kereliuk, Stephanie M., Hai, Yan, Chapman, Donald, Diehl-Jones, William, Aliani, Michel, West, Adrian R., Thliveris, James, Ghavami, Saeid, Rampitsch, Christof, Dolinsky, Vernon W., Gordon, Joseph W.·2021·Autophagy
DOI 10.1080/15548627.2020.1821548PMC PMC8496715

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Abstract

Abstract Lipotoxicity is a form of cellular stress caused by the accumulation of lipids resulting in mitochondrial dysfunction and insulin resistance in muscle. Previously, we demonstrated that the mitophagy receptor Nix is responsive to lipotoxicity and accumulates in response to diacylglycerols induced by high-fat (HF) feeding. In addition, previous studies have implicated autophagy and mitophagy in muscle insulin sensitivity. To provide a better understanding of these observations, we undertook gene expression array and shot-gun metabolomics studies in soleus muscle from rodents on an HF diet. Interestingly, we observed a modest reduction in several autophagy-related genes including Beclin-1, ATG3, and -5. Moreover, we observed alterations in the fatty acyl composition of cardiolipins and phosphatidic acids. Given the previously reported roles of these phospholipids and Nix in mitochondrial dynamics, we investigated aberrant mitochondrial fission and turn-over as a mechanism of myocyte insulin resistance. In a series of gain-of-function and loss-of-function experiments in rodent and human myotubes, we demonstrate that Nix accumulation triggers mitochondrial depolarization, fragmentation, calcium-dependent activation of DRP1, and mitophagy. In addition, Nix-induced mitochondrial fission leads to myotube insulin resistance through activation of mTOR-p70S6 kinase inhibition of IRS1, which is contingent on phosphatidic acids and Rheb. Finally, we demonstrate that Nix-induced mitophagy and insulin resistance can be reversed by direct phosphorylation of Nix by PKA, leading to the translocation of Nix from the mitochondria and sarcoplasmic reticulum to the cytosol. These findings provide insight into the role of Nix-induced mitophagy and myocyte insulin resistance during an overfed state when overall autophagy-related gene expression is reduced. Furthermore, our data suggests a mechanism by which exercise or pharmacological activation of PKA may overcome myocyte insulin resistance. Graphical Abstract

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Figures

Figure 1. BNIP3L regulates mitochondrial dynamics and mitophagy. (A-B) C2C12 myoblast cells were transfected with Mito-mEmerald to assess mitochondrial morphology (left). Quantification of C2C12 myoblast cells transfected with BNIP3L or empty vector control (right). (C-D) Quantification of C2C12 myoblast cells (C) or L6 myoblasts (D) transfected with BNIP3L or an empty vector control; stained with TMRM. (E) Quantification of C2C12 myoblasts transfected with ER-LAR-GECO, BNIP3L, or an empty vector control. (F) Quantification of C2C12 myoblasts transfected with Mito-CAR-GECO, BNIP3L, or an empty vector control. (G) C2C12 cells were transfected with BNIP3L or empty vector. Protein extracts were analyzed as indicated. (H-J) Quantification of (H) C2C12 myoblasts, (I) C2C12 myotubes, and (J) L6 myoblasts cells transfected with BNIP3L, Mito-pHRed, or an empty vector control. (K) C2C12 cells were transfected with Mito-pHRed, BNIP3L, a dominant negative DNM1L/Drp1 (S36A), or an empty vector control and quantified. (L) C2C12 cells were transfected with Mito-pHRed, BNIP3L, or an empty vector control. Cells were treated with Bafilomycin (6 nM, 3 h) or with the mitochondrial fission inhibitor (mdivi-1, 20 µM, 1 h). (M) Quantification of C2C12 myoblast cells transfected with Mito-pHRed, BNIP3L, mitochondrial targeted BNIP3L fusion constructs BNIP3L-MaoB or BNIP3L-ActA, the ER/SR targeted BNIP3L construct BNIP3L-CYB5, or an empty vector control. (N) C2C12 myoblast cells were transfected with Mito-pHRed, BNIP3L, BNIP3L-CYB5, or an empty vector control. Cells were treated with 2-aminoethoxydiphenyl borate (2APB 10 µM, 1 h), or DMSO as control vehicle and quantified. (O) L6 myotubes were transfected with BNIP3L or empty vector followed by 15 min of insulin stimulation (10 nM). Protein extracts were analyzed as indicated. (P) L6 myotubes were transfected with BNIP3L or an empty vector control. Insulin stimulated glucose uptake (10 nM) was determined by 2NBDG fluorescence and quantified. Data are represented as mean ± S.E.M. *P < 0.05 compared with control, while **P < 0.05 compared with treatment, determined by 1-way or 2-way ANOVA
Figure 1. BNIP3L regulates mitochondrial dynamics and mitophagy. (A-B) C2C12 myoblast cells were transfected with Mito-mEmerald to assess mitochondrial morphology (left). Quantification of C2C12 myoblast cells transfected with BNIP3L or empty vector control (right). (C-D) Quantification of C2C12 myoblast cells (C) or L6 myoblasts (D) transfected with BNIP3L or an empty vector control; stained with TMRM. (E) Quantification of C2C12 myoblasts transfected with ER-LAR-GECO, BNIP3L, or an empty vector control. (F) Quantification of C2C12 myoblasts transfected with Mito-CAR-GECO, BNIP3L, or an empty vector control. (G) C2C12 cells were transfected with BNIP3L or empty vector. Protein extracts were analyzed as indicated. (H-J) Quantification of (H) C2C12 myoblasts, (I) C2C12 myotubes, and (J) L6 myoblasts cells transfected with BNIP3L, Mito-pHRed, or an empty vector control. (K) C2C12 cells were transfected with Mito-pHRed, BNIP3L, a dominant negative DNM1L/Drp1 (S36A), or an empty vector control and quantified. (L) C2C12 cells were transfected with Mito-pHRed, BNIP3L, or an empty vector control. Cells were treated with Bafilomycin (6 nM, 3 h) or with the mitochondrial fission inhibitor (mdivi-1, 20 µM, 1 h). (M) Quantification of C2C12 myoblast cells transfected with Mito-pHRed, BNIP3L, mitochondrial targeted BNIP3L fusion constructs BNIP3L-MaoB or BNIP3L-ActA, the ER/SR targeted BNIP3L construct BNIP3L-CYB5, or an empty vector control. (N) C2C12 myoblast cells were transfected with Mito-pHRed, BNIP3L, BNIP3L-CYB5, or an empty vector control. Cells were treated with 2-aminoethoxydiphenyl borate (2APB 10 µM, 1 h), or DMSO as control vehicle and quantified. (O) L6 myotubes were transfected with BNIP3L or empty vector followed by 15 min of insulin stimulation (10 nM). Protein extracts were analyzed as indicated. (P) L6 myotubes were transfected with BNIP3L or an empty vector control. Insulin stimulated glucose uptake (10 nM) was determined by 2NBDG fluorescence and quantified. Data are represented as mean ± S.E.M. *P < 0.05 compared with control, while **P < 0.05 compared with treatment, determined by 1-way or 2-way ANOVACC BY-NC-ND · PubMed Central
Figure 2. Knockdown of BNIP3L improves mitochondrial function and insulin sensitivity. (A) Western blot analysis of rat soleus muscle exposed to high fat (HF) or low fat (LF) diet for 12-weeks (left). Quantification of BNIP3L expression relative to actin (n = 4)(right). (B) C2C12 myoblast cells were treated overnight with increasing doses of palmitate conjugated to 2% albumin in low-glucose media. Protein extracts were analyzed as indicated. (C) C2C12 myoblast cells were transfected with Mito-pHRed, shBNIP3L, or a scrambled control shRNA. Cells were treated overnight with palmitate (200 µM). (D) Quantification of C2C12 myoblasts transfected with ER-LAR-GECO and treated as in (C). (E) Quantification of C2C12 myoblast cells transfected with Mito-CAR-GECO and treated as in (C). (F) C2C12 myoblast cells were transfected with shBNIP3L or a scrambled control shRNA, followed by palmitate treatment as described in (C). Protein extracts were analyzed as indicated. (G) C2C12 myoblast and (H) human myotube cells were transfected with shBNIP3L or a control shRNA. Cells were treated overnight with palmitate as in (C), stained for TMRM and quantified. (I) L6 myotubes were transfected with shBNIP3L or a control shRNA. Cells were treated overnight with palmitate as in (C). Insulin stimulated glucose uptake (10 nM) was determined by 2NBDG fluorescence and quantified. (J) C2C12 myoblast cells as transfected as in (H), along with DAGR, a diacylglycerol biosensor. Cells were analyzed by using the emission ratio of YFP:CFP (FRET Ratio). (K) Quantification of C2C12 myoblast cells transfected as in (H), along with MYC-BNIP3L and treated as in (C). (L) C2C12 cells were transfected with Mito-pHRed. Cells were pre-treated overnight with etomoxir (100 µM) and palmitate, as described in (C), followed by co-treatment with 2APB (10 µM, 2 h) or DMSO as control vehicle and quantified. Data are represented as mean ± S.E.M. *P < 0.05 compared with control, while **P < 0.05 compared with treatment, determined by t-test, 1-way or 2-way ANOVA
Figure 2. Knockdown of BNIP3L improves mitochondrial function and insulin sensitivity. (A) Western blot analysis of rat soleus muscle exposed to high fat (HF) or low fat (LF) diet for 12-weeks (left). Quantification of BNIP3L expression relative to actin (n = 4)(right). (B) C2C12 myoblast cells were treated overnight with increasing doses of palmitate conjugated to 2% albumin in low-glucose media. Protein extracts were analyzed as indicated. (C) C2C12 myoblast cells were transfected with Mito-pHRed, shBNIP3L, or a scrambled control shRNA. Cells were treated overnight with palmitate (200 µM). (D) Quantification of C2C12 myoblasts transfected with ER-LAR-GECO and treated as in (C). (E) Quantification of C2C12 myoblast cells transfected with Mito-CAR-GECO and treated as in (C). (F) C2C12 myoblast cells were transfected with shBNIP3L or a scrambled control shRNA, followed by palmitate treatment as described in (C). Protein extracts were analyzed as indicated. (G) C2C12 myoblast and (H) human myotube cells were transfected with shBNIP3L or a control shRNA. Cells were treated overnight with palmitate as in (C), stained for TMRM and quantified. (I) L6 myotubes were transfected with shBNIP3L or a control shRNA. Cells were treated overnight with palmitate as in (C). Insulin stimulated glucose uptake (10 nM) was determined by 2NBDG fluorescence and quantified. (J) C2C12 myoblast cells as transfected as in (H), along with DAGR, a diacylglycerol biosensor. Cells were analyzed by using the emission ratio of YFP:CFP (FRET Ratio). (K) Quantification of C2C12 myoblast cells transfected as in (H), along with MYC-BNIP3L and treated as in (C). (L) C2C12 cells were transfected with Mito-pHRed. Cells were pre-treated overnight with etomoxir (100 µM) and palmitate, as described in (C), followed by co-treatment with 2APB (10 µM, 2 h) or DMSO as control vehicle and quantified. Data are represented as mean ± S.E.M. *P < 0.05 compared with control, while **P < 0.05 compared with treatment, determined by t-test, 1-way or 2-way ANOVACC BY-NC-ND · PubMed Central
Figure 3. PRKA phosphorylates BNIP3L at Ser212. (A) Amino acid sequence alignment for human, mouse and rat BNIP3L. (B) Schematic BNIP3L transmembrane domain and the location of Ser212. (C) SIM scan of the wild-type peptide spanning the PRKA site of BNIP3L. The unphosphorylated peptide has 857.28 m/z (z = 2+). (D) Putative phosphorylation showing an increased m/z of 40 that corresponds to PO3 (M = 80.00 Da). (E) SIM scan of mutated peptide (S212A) incubated with kinase, as in (D). (F) MS2 spectra following collision-induced dissociation of the precursor ion in (D, m/z = 897.78). A neutral loss of 49 m/z confirms phosphorylation. (G) SIM scanning showing BNIP3L peptide is not phosphorylated at two residues within the peptide. (H) BNIP3L structure modeled using Phyre2 engine. (I) 3T3 cells were transfected with wild type BNIP3L or a BNIP3L mutant where Ser212 is converted to alanine (S212A) and treated with 10 μM forskolin for 2 h. Protein extracts were immunoblotted, as indicated. (J) C2C12 myoblast cells were treated with 10 μM cilomilast or vehicle for multiple time points. Protein extracts were immunoblotted, as indicated. (K) p-BNIP3L is decreased as total BNIP3L increases in soleus muscle of rodents treated with high fat (HF) or low fat (LF) diet for 12-weeks. *predicted molecular weight, **predicted dimer weight, #aligned with total BNIP3L
Figure 3. PRKA phosphorylates BNIP3L at Ser212. (A) Amino acid sequence alignment for human, mouse and rat BNIP3L. (B) Schematic BNIP3L transmembrane domain and the location of Ser212. (C) SIM scan of the wild-type peptide spanning the PRKA site of BNIP3L. The unphosphorylated peptide has 857.28 m/z (z = 2+). (D) Putative phosphorylation showing an increased m/z of 40 that corresponds to PO3 (M = 80.00 Da). (E) SIM scan of mutated peptide (S212A) incubated with kinase, as in (D). (F) MS2 spectra following collision-induced dissociation of the precursor ion in (D, m/z = 897.78). A neutral loss of 49 m/z confirms phosphorylation. (G) SIM scanning showing BNIP3L peptide is not phosphorylated at two residues within the peptide. (H) BNIP3L structure modeled using Phyre2 engine. (I) 3T3 cells were transfected with wild type BNIP3L or a BNIP3L mutant where Ser212 is converted to alanine (S212A) and treated with 10 μM forskolin for 2 h. Protein extracts were immunoblotted, as indicated. (J) C2C12 myoblast cells were treated with 10 μM cilomilast or vehicle for multiple time points. Protein extracts were immunoblotted, as indicated. (K) p-BNIP3L is decreased as total BNIP3L increases in soleus muscle of rodents treated with high fat (HF) or low fat (LF) diet for 12-weeks. *predicted molecular weight, **predicted dimer weight, #aligned with total BNIP3LCC BY-NC-ND · PubMed Central
Figure 4. Clenbuterol and cilomilast inhibit palmitate-induced mitochondrial defects. (A) 5-day differentiated C2C12 myotubes and L6 myotubes were treated overnight with palmitate (200 µM) conjugated to 2% albumin in low glucose media. Control cells were treated with 2% albumin alone. Myotubes were treated with clenbuterol (500 nM, 2 h), cilomilast (10 μM, 2 h) or DMSO control vehicle and stained with TMRM (red) and Hoechst (blue) and imaged by standard fluorescence microscopy. (B) Quantification of (A). (C) 7-day differentiated human myotubes were treated and stained as in (A). (D) Quantification of (C). (E) 7-day differentiated human myotubes were stained with MitoSOX and quantified. (F) 7-day differentiated human myotubes were treated as in (A). Protein extracts were immunoblotted, as indicated. (G) C2C12 myoblast cells were treated as in (A) and imaged via electron microscopy. Mitochondria are indicated by arrows. (H) C2C12 myoblasts were transfected with Mito-pHRed and treated as in (A). (I) L6 myotube were treated as in (A) and protein extracts were immunoblotted, as indicated. (J) Insulin stimulated glucose uptake (10 nM) was determined by 2NBDG fluorescence in treated L6 myotubes and quantified. (K) C2C12 myoblast cells were transfected with BNIP3L wild type and BNIP3LS212A followed by clenbuterol treatment (500 nM, 2 h). Cells were stained with TMRM and imaged by standard fluorescence microscopy. (L-N) C2C12 myoblasts cells were transfected with BNIP3L, BNIP3LS212A, or BNIP3LS212D with Mito-pHRed (L), Mito-Car-Geco (M), and ER-Lar-Geco (N). (O) L6 myotubes cells were transfected with BNIP3L, BNIP3LS212A, or BNIP3LS212D. Insulin stimulated uptake (10 nM) was determined by 2NBDG fluorescence and quantified. Data are represented as mean ± S.E.M. *P < 0.05 compared with control, while **P < 0.05 compared with treatment, determined by 1-way or 2-way ANOVA
Figure 4. Clenbuterol and cilomilast inhibit palmitate-induced mitochondrial defects. (A) 5-day differentiated C2C12 myotubes and L6 myotubes were treated overnight with palmitate (200 µM) conjugated to 2% albumin in low glucose media. Control cells were treated with 2% albumin alone. Myotubes were treated with clenbuterol (500 nM, 2 h), cilomilast (10 μM, 2 h) or DMSO control vehicle and stained with TMRM (red) and Hoechst (blue) and imaged by standard fluorescence microscopy. (B) Quantification of (A). (C) 7-day differentiated human myotubes were treated and stained as in (A). (D) Quantification of (C). (E) 7-day differentiated human myotubes were stained with MitoSOX and quantified. (F) 7-day differentiated human myotubes were treated as in (A). Protein extracts were immunoblotted, as indicated. (G) C2C12 myoblast cells were treated as in (A) and imaged via electron microscopy. Mitochondria are indicated by arrows. (H) C2C12 myoblasts were transfected with Mito-pHRed and treated as in (A). (I) L6 myotube were treated as in (A) and protein extracts were immunoblotted, as indicated. (J) Insulin stimulated glucose uptake (10 nM) was determined by 2NBDG fluorescence in treated L6 myotubes and quantified. (K) C2C12 myoblast cells were transfected with BNIP3L wild type and BNIP3LS212A followed by clenbuterol treatment (500 nM, 2 h). Cells were stained with TMRM and imaged by standard fluorescence microscopy. (L-N) C2C12 myoblasts cells were transfected with BNIP3L, BNIP3LS212A, or BNIP3LS212D with Mito-pHRed (L), Mito-Car-Geco (M), and ER-Lar-Geco (N). (O) L6 myotubes cells were transfected with BNIP3L, BNIP3LS212A, or BNIP3LS212D. Insulin stimulated uptake (10 nM) was determined by 2NBDG fluorescence and quantified. Data are represented as mean ± S.E.M. *P < 0.05 compared with control, while **P < 0.05 compared with treatment, determined by 1-way or 2-way ANOVACC BY-NC-ND · PubMed Central
Figure 5. P-BNIP3L interacts with YWHAB proteins to determine subcellular location. (A) C2C12 myoblasts were subjected to subcellular fractionation. Protein extracts were immunoblotted for p-BNIP3L and total BNIP3L, as indicated. (B) 293 T cells were transfected with MYC-BNIP3L or HA-YWHAB. Extracts were immunoprecipitated (IP) with a MYC antibody and immunoblotted (IB), as indicated. (C) C2C12 myoblasts were transfected as in (B) and treated with clenbuterol (500 nM, 2 h). Extracts were immunoprecipitated (IP) with HA antibody and immunoblotted (IB), as indicated. (D) C2C12 myoblasts were transfected with HA-YWHAB, MYC-BNIP3L, MYC-BNIP3LS212A or MYC-BNIP3LS212D. Extracts were immunoprecipitated (IP) with HA antibody and immunoblotted (IB), as indicated. (E) C2C12 myoblasts were transfected with either MYC-BNIP3L or HA-YWHAB, followed by clenbuterol treatment (500 nM, 2 h). Extracts were fractioned and protein extracts were immunoblotted, as indicated. (F) C2C12 myoblasts cells were transfected with BNIP3L wild type, HA-YWHAB, and ER-Lar-Geco. (G) C2C12 myoblasts cells were transfected with BNIP3L wild type, HA-YWHAB, and mitoCar-Geco. (H) C2C12 myoblasts cells were transfected with BNIP3L wild type, HA-YWHAB, and Mito-pHRed. (I-J) L6 myotubes were transfected with BNIP3L wild type, HA-YWHAB. Proteins were immunoblotted as indicated (I) and insulin stimulated glucose uptake (10 nM) was determined by 2NBDG fluorescence and quantified (J). Data are represented as mean ± S.E.M. *P < 0.05 compared with control, while **P < 0.05 compared with treatment, determined by 1-way or 2-way ANOVA
Figure 5. P-BNIP3L interacts with YWHAB proteins to determine subcellular location. (A) C2C12 myoblasts were subjected to subcellular fractionation. Protein extracts were immunoblotted for p-BNIP3L and total BNIP3L, as indicated. (B) 293 T cells were transfected with MYC-BNIP3L or HA-YWHAB. Extracts were immunoprecipitated (IP) with a MYC antibody and immunoblotted (IB), as indicated. (C) C2C12 myoblasts were transfected as in (B) and treated with clenbuterol (500 nM, 2 h). Extracts were immunoprecipitated (IP) with HA antibody and immunoblotted (IB), as indicated. (D) C2C12 myoblasts were transfected with HA-YWHAB, MYC-BNIP3L, MYC-BNIP3LS212A or MYC-BNIP3LS212D. Extracts were immunoprecipitated (IP) with HA antibody and immunoblotted (IB), as indicated. (E) C2C12 myoblasts were transfected with either MYC-BNIP3L or HA-YWHAB, followed by clenbuterol treatment (500 nM, 2 h). Extracts were fractioned and protein extracts were immunoblotted, as indicated. (F) C2C12 myoblasts cells were transfected with BNIP3L wild type, HA-YWHAB, and ER-Lar-Geco. (G) C2C12 myoblasts cells were transfected with BNIP3L wild type, HA-YWHAB, and mitoCar-Geco. (H) C2C12 myoblasts cells were transfected with BNIP3L wild type, HA-YWHAB, and Mito-pHRed. (I-J) L6 myotubes were transfected with BNIP3L wild type, HA-YWHAB. Proteins were immunoblotted as indicated (I) and insulin stimulated glucose uptake (10 nM) was determined by 2NBDG fluorescence and quantified (J). Data are represented as mean ± S.E.M. *P < 0.05 compared with control, while **P < 0.05 compared with treatment, determined by 1-way or 2-way ANOVACC BY-NC-ND · PubMed Central
Figure 6. BNIP3L-induced MTOR-RPS6KB activation. (A) C2C12 myoblast cells transfected with MYC-BNIP3L or an empty vector control. Cells were treated with Rapamycin (500 nM, 1 h) or DMSO as vehicle control. Proteins were immunoblotted as indicated. (B) C2C12 myoblast cells transfected with shBNIP3L, or a scramble control shRNA, and treated overnight with palmitate (200 µM) conjugated to 2% albumin in low-glucose media. Proteins were immunoblotted as indicated. (C) C2C12 myoblast cells were transfected as in (A), and were treated with clenbuterol (500 nM), cilomilast (10 μM) or vehicle for 2 h. Proteins were immunoblotted as indicated. (D) C2C12 myoblast cells were transfected as in (A). Cells were treated with mdivi-1 (20 µM) or vehicle for 1 h. Proteins were immunoblotted as indicated. (E) C2C12 myoblast cells were transfected as in (A) and treated with 1-butanol (1%) for 30 min. Proteins were immunoblotted as indicated. (F) C2C12 myoblast cells were transfected as in (A), followed by phosphatidic acid assay and quantification. (G) C2C12 myoblast cells were transfected as in (A), along with the diacylglycerol biosensor DAGR. Cells were treated overnight with palmitate and analyzed by FRET imaging. (H) C2C12 myoblast cells were transfected with MYC-BNIP3L, siRHEB, siPLD6. Proteins were immunoblotted as indicated. (I) C2C12 myoblast cells were transfected with MYC-BNIP3L, RHEB, MYC-BNIP3LS212D, MYC-BNIP3L-ΔTM, along with Mito-pHRed. Cells were imaged by standard fluorescence and quantified. Data are represented as mean ± S.E.M. *P < 0.05 compared with control, while **P < 0.05 compared with treatment, determined by 1-way or 2-way ANOVA
Figure 6. BNIP3L-induced MTOR-RPS6KB activation. (A) C2C12 myoblast cells transfected with MYC-BNIP3L or an empty vector control. Cells were treated with Rapamycin (500 nM, 1 h) or DMSO as vehicle control. Proteins were immunoblotted as indicated. (B) C2C12 myoblast cells transfected with shBNIP3L, or a scramble control shRNA, and treated overnight with palmitate (200 µM) conjugated to 2% albumin in low-glucose media. Proteins were immunoblotted as indicated. (C) C2C12 myoblast cells were transfected as in (A), and were treated with clenbuterol (500 nM), cilomilast (10 μM) or vehicle for 2 h. Proteins were immunoblotted as indicated. (D) C2C12 myoblast cells were transfected as in (A). Cells were treated with mdivi-1 (20 µM) or vehicle for 1 h. Proteins were immunoblotted as indicated. (E) C2C12 myoblast cells were transfected as in (A) and treated with 1-butanol (1%) for 30 min. Proteins were immunoblotted as indicated. (F) C2C12 myoblast cells were transfected as in (A), followed by phosphatidic acid assay and quantification. (G) C2C12 myoblast cells were transfected as in (A), along with the diacylglycerol biosensor DAGR. Cells were treated overnight with palmitate and analyzed by FRET imaging. (H) C2C12 myoblast cells were transfected with MYC-BNIP3L, siRHEB, siPLD6. Proteins were immunoblotted as indicated. (I) C2C12 myoblast cells were transfected with MYC-BNIP3L, RHEB, MYC-BNIP3LS212D, MYC-BNIP3L-ΔTM, along with Mito-pHRed. Cells were imaged by standard fluorescence and quantified. Data are represented as mean ± S.E.M. *P < 0.05 compared with control, while **P < 0.05 compared with treatment, determined by 1-way or 2-way ANOVACC BY-NC-ND · PubMed Central

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  1. 10.1210/me.11.12.1858 · DOI
  2. Olenych SG, 2007, Curr Protoc Cell Biol, 21
  3. Field JT, 2018, Cell Death Discov, 5, 37

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Metadata

Authors
da Silva Rosa, S. C., Martens, M. D., Field, J. T., Nguyen, L., Kereliuk, S. M., Hai, Y., Chapman, D., Diehl-Jones, W., Aliani, M., West, A. R., Thliveris, J., Ghavami, S., Rampitsch, C., Dolinsky, V. W., & Gordon, J. W.
Affiliations
  • Departments of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Canada
  • Children's Hospital Research Institute of Manitoba (CA)
  • Children’s Hospital Research Institute of Manitoba, University of Manitoba, Winnipeg, Canada
  • Human Nutritional Science
  • University of Manitoba (CA)
  • Agriculture and Agri-Food Canada (CA)
Corresponding author
Gordon, Joseph W.
ORCID
Venue
Autophagy
Publisher
Informa UK Limited
Published
2020-10-12
Volume
17
Issue
9
Pages
2257-2272
ISSN
  • 1554-8627 (ISSN-L)
  • 1554-8635
DOI
10.1080/15548627.2020.1821548
PMC
PMC8496715
OpenAlex
W3092101056
Cited by
117
References
69
Citations by year
2026: 13 · 2025: 20 · 2024: 28 · 2023: 19 · 2022: 16 · 2021: 19 · 2020: 2
FWCI
10.04
Type
journal article
Language
en
Open access
Licence
Locations
  • Autophagy (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Figshare (submittedVersion, cc-by)
  • Mspace (University of Manitoba) (submittedVersion)
  • Figshare (cc-by)
Funders
  • Canadian Institutes of Health Research (N/A)
  • Canadian Network for Research and Innovation in Machining Technology, Natural Sciences and Engineering Research Council of Canada (N/A)
  • Children’s Hospital Research Institute of Manitoba (N/A)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolism, Diabetes, and Cancer
Keywords
BNIP3L/NIX, lipotoxicity, mitochondrial fission, mitophagy, glucose uptake, Drp1, mitochondrial depolarization, cardiolipin, phosphatidic acid, Rheb, mTOR signaling, IRS-1, high-fat diet, soleus muscle
MeSH terms
Animals, Autophagy, Cells, Cultured, Glucose, Humans, Membrane Proteins, Phosphorylation, Proto-Oncogene Proteins, Mitochondrial Proteins, Tumor Suppressor Proteins, Muscle Cells, Mitochondrial Dynamics, Mitophagy

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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