Journal: Nature Metabolism

Article Title: LONP1 regulation of mitochondrial protein folding provides insight into beta cell failure in type 2 diabetes

doi: 10.1038/s42255-025-01333-7

Figure Lengend Snippet: a , Schematic diagram illustrating the generation of mouse pseudoislets. b , TUNEL staining for β cell apoptosis performed in mouse pseudoislets dissociated for cytocentrifugation from 5-week-old Ctrl mice and β- Lonp1 KO littermates performed 7 days after adenoviral transduction with RIP2 -driven EV (Ad. RIP2 .EV) and the protease-deficient Lonp1 S855A mutant (Ad. RIP2 . Lonp1 S855A ), with exposure to vehicle (DMSO) or 1 μM of the mtHSP70 inhibitor MKT077 for the final 24 h. Representative images of 3–4 mice per group. The yellow arrows indicate insulin + TUNEL + cells. c , Quantification of TUNEL staining from the studies in b . n = 4 vehicle versus three MKT077 biological replicates. d , Expression of mitochondrial proteins from the soluble and insoluble fractions performed in mouse pseudoislets from 5-week-old Ctrl and β- Lonp1 KO mice 7 days after adenoviral transduction with Ad. RIP2. EV or Ad. RIP2 . Lonp1 S855A using immunoblotting. Representative images of three mice per group. VDAC1 serves as a soluble mitochondrial protein loading control. Vinculin serves as a loading control for both soluble and insoluble fractions. e , Quantification of mitochondrial insoluble proteins (normalized to vinculin) from the studies in d . n = 3 biological replicates per group. f , Expression of mitochondrial proteins from soluble and insoluble fractions of mouse pseudoislets generated from 8-week-old Lonp1 loxP / loxP ; MIP1 -Cre ERT mice 7 days after adenoviral transduction with Ad. RIP2. EV or Ad. RIP2 . Lonp1 S855A . Pseudoislets were co-cultured with vehicle (EtOH) or 2 μM 4-hydroxytamoxifen (4-OHT) to induce recombination in vitro and generate experimental groups (Ctrl or iβ- Lonp1 KO, respectively) before the generation of soluble or insoluble fractions for immunoblotting. Representative images of three biological replicates per group. g , Quantification of mitochondrial insoluble proteins (normalized to vinculin) from the studies in f . n = 3 biological replicates per group. All data are presented as the mean ± s.e.m. c , e , g , *P < 0.05 and * *P < 0.01 were determined using a one-way ANOVA followed by Tukey’s multiple comparisons test. b , Scale bar, 50 μm.

Article Snippet: Drug treatments used for pseudoislet studies included 40 μM 84-B10 (MedChemExpress) for 48 h or 1 μM MKT077 (Cayman Chemical) for 24 h, respectively, as well as the respective vehicle controls (DMSO).

Techniques: TUNEL Assay, Staining, Transduction, Mutagenesis, Expressing, Western Blot, Control, Generated, Cell Culture, In Vitro

Journal: Nature Metabolism

Article Title: LONP1 regulation of mitochondrial protein folding provides insight into beta cell failure in type 2 diabetes

doi: 10.1038/s42255-025-01333-7

Figure Lengend Snippet: ( a ) Representative WB images (left) and quantification (right) of FLAG-epitope tagged LONP1, total LONP1, HMGCS2, and TWINKLE expression in Min6 β-cells 72 h after transfection with pQCXIP vectors expressing a FLAG-tagged empty vector (EV), wild-type LONP1 (WT), or LONP1 S855A mutant (S855A). VINCULIN serves as a loading control. n = 3 independent experiments/group. ( b ) Representative WB of lysates of Min6 β-cells following control anti-IgG immunoprecipitation (IP; middle lane) or anti-LONP1 IP (right lane). n = 4 independent experiments/group. ( c ) Aconitase activity measured in Min6 β-cells exposed to 0.3 μM MKT077 or DMSO for 24 h. n = 3/group. ( d ) TFAM and LONP1 protein levels visualized by WB (Left) and densitometry (Right) of recombinant purified human TFAM and LONP1 to assess LONP1 protease activity in the presence of 40 μM 84-B10 or vehicle control (DMSO). LONP1 protein levels serve as a reference/loading control. n = 3 independent experiments/group. ( e ) Immunofluorescence imaging performed in human β-cell enriched pseudoislets, generated by magnetic sorting for the β cell surface marker NTPDase3, following dissociation for cytocentrifugation and imaging, stained for insulin (red) and DAPI (DNA - blue). Scale bars, 50 μm. Representative image of 4 β-cell enriched pseudoislet preparations each from independent human islet donors. ( f ) Pseudobulk gene expression data of reported transcriptional regulators of LONP1 from β cells of human islet donors with or without T2D by single cell RNA sequencing. Box plots are presented the minimum, first quartile, median, third quartile, maximum, and interquartile range. n = 17 non-diabetic donors, n = 17 donors with T2D. All data in figure are presented mean ± SEM. Statistical analysis: 10 A, 10D, *P < 0.05 by one-way ANOVA followed by Tukey’s multiple comparisons test. 10 C, *P < 0.05 by unpaired two-tailed Student’s t -test.

Article Snippet: Drug treatments used for pseudoislet studies included 40 μM 84-B10 (MedChemExpress) for 48 h or 1 μM MKT077 (Cayman Chemical) for 24 h, respectively, as well as the respective vehicle controls (DMSO).

Techniques: FLAG-tag, Expressing, Transfection, Plasmid Preparation, Mutagenesis, Control, Immunoprecipitation, Activity Assay, Recombinant, Purification, Immunofluorescence, Imaging, Generated, Marker, Staining, Gene Expression, RNA Sequencing, Two Tailed Test

Journal: Nature Metabolism

Article Title: LONP1 regulation of mitochondrial protein folding provides insight into beta cell failure in type 2 diabetes

doi: 10.1038/s42255-025-01333-7

Figure Lengend Snippet: a , Quantification of TUNEL staining for β cell apoptosis in human islets after exposure to BSA or GLT together with DMSO or 40 μM of the LONP1 activator 84-B10 for 48 h. n = 3 independent human islet donors per group. b , Schematic diagram illustrating the generation of human β-cell-enriched pseudoislets. c , Quantification of TUNEL staining for β cell apoptosis in human β-cell-enriched pseudoislets 7 days after adenoviral transduction with RIP2 -driven EV (Ad. RIP2 .EV) or the protease-deficient Lonp1 S855A mutant (Ad. RIP2 . Lonp1 S855A ), followed by exposure to BSA or GLT for the final 48 h, and DMSO or 1 μM MKT077 for the final 24 h. n = 3 independent human islet donors per group. d , Representative immunoblots (left) of mitochondrial proteins from the soluble and insoluble fractions of human β-cell-enriched pseudoislets 8 days after Ad. RIP2. EV or Ad. RIP2 . Lonp1 S855A transduction, exposed to BSA or GLT for the final 72 h. Quantification of mitochondrial insoluble proteins using densitometry (normalized to vinculin) is shown in the graph on the right. VDAC1 serves as a soluble mitochondrial protein loading control. Vinculin serves as a loading control for both soluble and insoluble fractions. n = 4 independent islet donors per group. e , Pseudobulk gene expression of ATF5 from the β cells of human islet donors with or without T2D using scRNA-seq. The box plots present the minimum, first quartile, median, third quartile, maximum and interquartile range. n = 17 donors without T2D, n = 17 donors with T2D. f , RT–qPCR of Atf5 and markers of the UPR mt from RNA isolated from Min6 β cells 72 h after transfection with siATF5 or siCtrl, and exposure to 0.5 mM palmitate or BSA control for the final 48 h. n = 6 per group. All data are presented as the mean ± s.e.m. a , c , d , f , *P < 0.05 and * *P < 0.01 were determined using a one-way ANOVA followed by Tukey’s multiple comparisons test. e , *P < 0.05 was determined using both an unpaired, two-tailed Student’s t -test and FDR < 5% for multiple testing correction.

Article Snippet: Drug treatments used for pseudoislet studies included 40 μM 84-B10 (MedChemExpress) for 48 h or 1 μM MKT077 (Cayman Chemical) for 24 h, respectively, as well as the respective vehicle controls (DMSO).

Techniques: TUNEL Assay, Staining, Transduction, Mutagenesis, Western Blot, Control, Gene Expression, Quantitative RT-PCR, Isolation, Transfection, Two Tailed Test