Journal: Nature Communications
Article Title: THADA inhibition in mice protects against type 2 diabetes mellitus by improving pancreatic β-cell function and preserving β-cell mass
doi: 10.1038/s41467-023-36680-0
Figure Lengend Snippet: a Body weights of WT ( n = 12) and Thada -KO mice ( n = 8) after HFHS diet for the indicated weeks. b Blood glucose levels of HFHS-fed WT ( n = 12) and Thada -KO mice that were fasted overnight ( n = 8). c Fed blood glucose levels of WT ( n = 12) and Thada -KO mice ( n = 8) after HFHS diet for 8 and 12 weeks. d Fed serum insulin levels of HFHS-fed WT and Thada -KO mice ( n = 7). e , f IPGTT and area under the curve of HFHS-fed WT ( n = 12) and Thada -KO mice ( n = 8). g , h ITT and area under baseline of HFHS-fed WT ( n = 7) and Thada -KO mice ( n = 8). i Serum insulin levels at 0, 15, and 30 min after intraperitoneal glucose injection in HFHS-fed WT and Thada -KO mice ( n = 8 for 0 min, n = 12 for 15 and 30 min). j Islets isolated from HFHS-fed WT ( n = 3) and Thada -KO mice ( n = 4) were stimulated with 3.3 and 16.7 mM glucose for 1 h, and insulin secretion was assayed. k Quantification of [Ca 2+ ] i in primary islet cells from NCD-fed WT, HFHS-fed WT and HFHS-fed Thada -KO mice at basal 2.8 mM glucose and after stimulation with 16.7 mM glucose ( n = 16 from five mice each group). l Quantification of [Ca 2+ ] i in primary islet cells from NCD-fed WT, HFHS-fed WT and HFHS-fed Thada -KO mice at basal 2.8 mM glucose and after stimulation with 35 mM KCl ( n = 16 from five mice each group). m Quantification of [Ca 2+ ] i in primary islet cells from NCD-fed WT, HFHS-fed WT and HFHS-fed Thada -KO at basal 5.6 mM glucose and after 10 μM ionomycin stimulation ( n = 14 for NCD-WT, n = 15 for HFHS-WT, n = 16 for HFHS-KO from five mice each group). n Measurements of β-cell area/pancreatic area ratio in HFHS-fed WT and Thada -KO mice ( n = 3). o Measurements of β-cell mass in HFHS-fed WT and Thada -KO mice ( n = 3). p Representative images of islets from HFHS-fed WT and Thada -KO mice stained for insulin (green), Tunel (red), and DAPI (blue). Scale bars, 50 μm. Arrowhead points to Tunel + Insulin + cells. The percentage of Tunel + in Insulin + cells was quantified ( n = 4 mice). At least 3000 Insulin + cells in each mouse were counted for quantification. q Representative images of islets from NCD-fed WT, HFHS-fed WT, and HFHS-fed Thada -KO mice stained for insulin (green), cleaved caspase-3 (red), and DAPI (blue). Scale bar was 20 or 50 μm as indicated. Arrowhead points to cleaved caspase-3 + Insulin + cells. The percentage of cleaved caspase-3 + in Insulin + cells was quantified ( n = 4 mice for NCD-WT and HFHS-WT, n = 3 mice for HFHS-KO). At least 2500 Insulin + cells in each mouse were counted for quantification. The experiments were performed on male mice after HFHS diet for 12 weeks unless otherwise indicated and female mice data were provided in Fig. . Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001; significance is assessed by two-tailed unpaired Student’s t test (a– j , n – p ) or one-way ANOVA followed by Dunnett’s multiple comparison test ( k , l , m , q ). Source data are provided as a Source Data file.
Article Snippet: Stable MIN6 beta-cell line with endogenous Thada -activation was generated by CRISPR/dCas9-SAM system from Genechem (Shanghai, China).
Techniques: Injection, Isolation, Staining, TUNEL Assay, Two Tailed Test, Comparison
![a Increases in [Ca 2+ ] i from control (CON) and Thada -activated ( Thada -SAM) MIN6 beta-cell lines following stimulation with 2.8 mM glucose (2.8 G), 25 mM glucose (25 G) and 30 mM KCl. Representative results of three replicates from each group are provided. b Quantification of the maximum increases in [Ca 2+ ] i after 25 mM glucose ( n = 13 for control and n = 11 for Thada -SAM) or 30 mM KCl stimulation. ( n = 14 for control and n = 11 for Thada -SAM). c , d Quantification of the maximum increases in [Ca 2+ ] i from control ( n = 6) and Thada -SAM β-cells ( n = 5) after 10 μM ionomycin stimulation. Representative results of two replicates from each group are provided. e , f Quantification of the maximum increases in [Ca 2+ ] i from control ( n = 6) and Thada -SAM β-cells ( n = 7) after 2 μM thapsigargin stimulation. Representative results of two replicates from each group are provided. g [Ca 2+ ] i in dispersed islet cells from WT and Thada -KO mice at basal 2.8 mM glucose and after stimulation with 16.7 mM glucose (16.7 G) ( n = 16 from six mice each group). h Quantification of the maximum increases in [Ca 2+ ] i after 16.7 mM glucose stimulation ( n = 16 from six mice each group). i , j Quantification of [Ca 2+ ] i in dispersed islet cells from WT and Thada -KO mice after stimulation with 35 mM KCl at 2.8 mM glucose ( n = 16 from six mice each group). k , l Quantification of [Ca 2+ ] i in dispersed islet cells from WT and Thada -KO mice after 10 μM ionomycin stimulation ( n = 16 from six mice each group). m Immunofluorescence staining for THADA (green), SERCA2 (red), and DAPI (blue) in MIN6 beta-cell line. Scale bars, 10 μm. n Immunofluorescence staining for THADA (green), RyR (red), and DAPI (blue) in MIN6 beta-cell line. Scale bars, 10 μm. o MIN6 cells were immunoprecipitated with either a SERCA2 antibody or an IgG negative control, followed by western blot analysis using THADA and SERCA2 antibodies. p MIN6 cells were immunoprecipitated with either a RyR antibody or an IgG negative control, followed by western blot analysis using THADA and RyR antibodies. q Proximity ligation assay for THADA together with SERCA2 and RyR in MIN6 beta-cell line (NC for negative control). Scale bars, 10 μm. r Increases in [Ca 2+ ] i from control and Thada -activated β-cells pretreated with or without 20 μM S107 or 10 μM CDN1163 (CDN) after 10 μM ionomycin stimulation. Representative results from each group are presented ( n = 10 for control, n = 12 for Thada -SAM, n = 11 for Thada -SAM with S107 and CDN1163). s Control and Thada -SAM β-cells were pretreated with or without CDN1163 and S107 at the indicated concentration for 24 h, then stimulated with 1.4 mM glucose (1.4 G) or 25 mM glucose for 1 h, and insulin secretion was assayed ( n = 4, except for control cells at 25 G and Thada -SAM cells treated with 10 μM S107 at 25 G were n = 3). All western blots and immunostainings (m-q) show representative results from at least three independent experiments. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001; significance is assessed by two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0491/pmc09950491/pmc09950491__41467_2023_36680_Fig5_HTML.jpg)
![a Body weights of WT ( n = 12) and Thada -KO mice ( n = 8) after HFHS diet for the indicated weeks. b Blood glucose levels of HFHS-fed WT ( n = 12) and Thada -KO mice that were fasted overnight ( n = 8). c Fed blood glucose levels of WT ( n = 12) and Thada -KO mice ( n = 8) after HFHS diet for 8 and 12 weeks. d Fed serum insulin levels of HFHS-fed WT and Thada -KO mice ( n = 7). e , f IPGTT and area under the curve of HFHS-fed WT ( n = 12) and Thada -KO mice ( n = 8). g , h ITT and area under baseline of HFHS-fed WT ( n = 7) and Thada -KO mice ( n = 8). i Serum insulin levels at 0, 15, and 30 min after intraperitoneal glucose injection in HFHS-fed WT and Thada -KO mice ( n = 8 for 0 min, n = 12 for 15 and 30 min). j Islets isolated from HFHS-fed WT ( n = 3) and Thada -KO mice ( n = 4) were stimulated with 3.3 and 16.7 mM glucose for 1 h, and insulin secretion was assayed. k Quantification of [Ca 2+ ] i in primary islet cells from NCD-fed WT, HFHS-fed WT and HFHS-fed Thada -KO mice at basal 2.8 mM glucose and after stimulation with 16.7 mM glucose ( n = 16 from five mice each group). l Quantification of [Ca 2+ ] i in primary islet cells from NCD-fed WT, HFHS-fed WT and HFHS-fed Thada -KO mice at basal 2.8 mM glucose and after stimulation with 35 mM KCl ( n = 16 from five mice each group). m Quantification of [Ca 2+ ] i in primary islet cells from NCD-fed WT, HFHS-fed WT and HFHS-fed Thada -KO at basal 5.6 mM glucose and after 10 μM ionomycin stimulation ( n = 14 for NCD-WT, n = 15 for HFHS-WT, n = 16 for HFHS-KO from five mice each group). n Measurements of β-cell area/pancreatic area ratio in HFHS-fed WT and Thada -KO mice ( n = 3). o Measurements of β-cell mass in HFHS-fed WT and Thada -KO mice ( n = 3). p Representative images of islets from HFHS-fed WT and Thada -KO mice stained for insulin (green), Tunel (red), and DAPI (blue). Scale bars, 50 μm. Arrowhead points to Tunel + Insulin + cells. The percentage of Tunel + in Insulin + cells was quantified ( n = 4 mice). At least 3000 Insulin + cells in each mouse were counted for quantification. q Representative images of islets from NCD-fed WT, HFHS-fed WT, and HFHS-fed Thada -KO mice stained for insulin (green), cleaved caspase-3 (red), and DAPI (blue). Scale bar was 20 or 50 μm as indicated. Arrowhead points to cleaved caspase-3 + Insulin + cells. The percentage of cleaved caspase-3 + in Insulin + cells was quantified ( n = 4 mice for NCD-WT and HFHS-WT, n = 3 mice for HFHS-KO). At least 2500 Insulin + cells in each mouse were counted for quantification. The experiments were performed on male mice after HFHS diet for 12 weeks unless otherwise indicated and female mice data were provided in Fig. . Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001; significance is assessed by two-tailed unpaired Student’s t test (a– j , n – p ) or one-way ANOVA followed by Dunnett’s multiple comparison test ( k , l , m , q ). Source data are provided as a Source Data file.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0491/pmc09950491/pmc09950491__41467_2023_36680_Fig7_HTML.jpg)
![a A high-content screening workflow to identify compounds that can reverse THADA-reduced [Ca 2+ ] i . b Primary screen data. Glucose-stimulated [Ca 2+ ] I was normalized to Z score. c Representative images of glucose-stimulated fluo-4 Ca 2+ fluorescence after treatment with vehicle or alnustone for 24 h. Scale bar, 100 μm. d Dose curve of alnustone on the glucose-stimulated [Ca 2+ ] i ( n = 3 biologically independent experiments). e GSIS of Thada -SAM β-cells treated with or without 10 μM alnustone for 24 h ( n = 4 biologically independent experiments). f MIN6 beta-cell lines were treated with or without 33 mM glucose and 0.5 mM palmitate (HGPA) in the presence or absence of 10 μM alnustone for 24 h. Then GSIS was assayed ( n = 4 biologically independent experiments). g – k DIO male mice were injected with vehicle or 10 mg/kg alnustone for 5–7 days, then body weights ( g ), fed blood glucose levels ( h ), IPGTT and the related AUC ( i , j ) as well as serum insulin levels at 0 and 15 min after intraperitoneal glucose injection ( k ) were measured ( n = 5 for vehicle and n = 7 for alnustone). l Model depicting the role of THADA in the pathogenesis of T2DM: THADA expression is induced by glucolipotoxicity in β-cells during T2DM progression, which leads to ER Ca 2+ depletion due to inhibited SERCA2 activity and leaky RyR2 Ca 2+ channel, and activation of the DR5/FADD/caspase-8 pro-apoptotic complex, consequently resulting in impaired insulin secretion and aggravated ER stress-induced β-cell apoptosis. Genetic ablation or inhibition of THADA by alnustone improves glucose tolerance in mice by promoting β-cell function and survival. Image was created with BioRender.com. K ATP : ATP-sensitive K + channel; L-VGCC: L-type voltage-gated Ca 2+ channel; ER: endoplasmic reticulum; SAM: synergistic activation mediator. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001; significance is assessed by two-way ANOVA followed by Tukey’s multiple comparison test ( e ) or two-tailed unpaired Student’s t test ( f – k ). Source data are provided as a Source Data file.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0491/pmc09950491/pmc09950491__41467_2023_36680_Fig8_HTML.jpg)