Mitophagy-mediated adipose inflammation contributes to type 2 diabetes with hepatic insulin resistance

Figures

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  Figure S1.. Trx2 targeting strategy and adipocyte-specific deletion. (A) Schematic diagram of adipocyte-specific Trx2 KO by the loxP-Cre system. (B) Genotyping results of Trx2lox/lox (WT) and Trx2ADKO (Trx2lox/lox;Adipo-Cre; KO) mice. Note that WT mice did not contain Cre. (C) Representative Western blot analysis of Trx2 expression in eWAT, interscapular BAT (iBAT), and pancreas from WT and KO mice. β-Actin was used as a loading control. TK, thymidine kinase.

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  Figure 1.. Adipocyte-specific KO of Trx2 results in hyperglycemia and insulin resistance. (A) Representative immunoblot analysis of Trx2 protein in WAT and BAT of Trx2ADKO mice relative to the WT controls. Trx2 protein levels were quantified and presented as fold changes by taking WT as 1.0. n = 3 male mice. Female mice show similar Trx2 deletion. (B) Representative hematoxylin and eosin images of eWAT and ingWAT from 14-wk-old WT and Trx2ADKO mice (n = 6 male). Diameters of adipocyte in eWAT and ingWAT were quantified. Female mice show similar phenotype in adipocyte size. Scale bars, 100 µm. (C–F) Food intake, oxygen consumption corrected for lean mass, energy expenditure corrected for lean mass, and RER of 14-wk-old WT and Trx2ADKO mice (n = 6). (G and H) Fasting blood glucose and serum insulin levels over time in WT and Trx2ADKO mice (n = 6). (I and J) GTT and ITT in 14-wk-old male WT and Trx2ADKO mice (n = 8). AUC 120 min was calculated. Quantitative data are presented as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus the corresponding value for WT. Significance was analyzed by two-tailed Student’s t test. ns, not significant.

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  Figure S2.. Type 2 diabetic phenotype of adipocyte-specific Trx2-KO mice. (A) Growth curves of male WT and Trx2ADKO mice (n = 8). (B) EchoMRI analysis of fat and lean mass of 14-wk-old male WT and Trx2ADKO mice (n = 8). (C and D) Oxygen consumption corrected for TBW (C) and oxygen consumption corrected for TBW (D). EE, energy expenditure. (E and F) Blood glucose levels in random fed WT and Trx2ADKO male (E) and female mice (F). Both male and female mice exhibited similar phenotypes in blood glucose levels (n = 8 for each group). (G and H) GTT (G) and ITT (H) results of 6-wk-old male WT and Trx2ADKO mice (n = 8). AUC (120 min) was calculated. Quantitative data represent the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with WT controls. Significance was analyzed by two-tailed Student’s t test.

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  Figure S3.. Trx2ADKO mice develop T2DM-related end-organ damage. (A–E) Trx2-KO mice exhibit decreased insulin content and increased β cell apoptosis. (A) Representative hematoxylin and eosin–stained pancreas sections showing pancreatic islets of WT and Trx2ADKO mice at the indicated ages. Scale bars, 20 µm. (B) Nuclei density of six randomly selected pancreatic islets (n = 6 mice). (C) Detection of β cell apoptosis by costaining of TUNEL (green) and insulin (red). Representative images from WT and Trx2ADKO mice at the indicated ages. Scale bars, 20 µm. (D) Quantification of TUNEL-positive β cells (right panel; n = 6 mice). (E) Representative transmission electron micrographs of pancreas tissue from WT and Trx2 ADKO mice (three images/mouse, n = 3 mice/group). Squares correspond to the magnified areas (bottom panel). Scale bars, 1 µm. M, mitochondria. Arrowheads indicate empty granules. (F) Quantification of insulin granules per µm2 islet. (G) Representative transmission electron micrographs of kidney tissue from WT and Trx2ADKO mice (n = 3). White squares correspond to the magnified areas (bottom panel). Red arrowhead indicates podocyte foot process fusion. Scale bars, 1 µm. (H–L)…

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  Figure 2.. HFD enhances type 2 diabetic phenotype in Trx2ADKO mice. (A) Growth curves of WT and Trx2ADKO male mice fed the NCD or HFD (n = 8). (B) Hematoxylin and eosin–stained sections of eWAT from 14-wk-old WT and Trx2ADKO mice fed the NCD or HFD for 8 wk. Diameters of adipocyte in eWAT and ingWAT were quantified. n = 8. Scale bars, 100 µm. (C) Fasting blood glucose levels and serum insulin levels of WT and Trx2ADKO mice fed the NCD or HFD (n = 8). (D and E) GTT (D) and ITT (E) assays in 14-wk-old male WT and Trx2ADKO mice fed the NCD or HFD for 8 wk (n = 8). AUC (120 min) was calculated (bottom panel). Quantitative data are presented as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus the indicated comparisons. Significance was assessed by one-way ANOVA followed by Tukey’s post hoc test.

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  Figure 3.. Adipocyte-specific Trx2 KO promotes hepatic steatosis and hepatic insulin resistance. (A) Representative photograph of livers from 14-wk-old male WT and Trx2ADKO mice fed the NCD or HFD for 8 wk (n = 6). Arrowheads indicate visible lipid deposition. Scale bars, 2 mm. (B) Liver weight and liver weight to body weight ratios of WT and Trx2ADKO mice (KO) mice. (C) Liver TG content of mice. (D) Representative images of BODIPY staining of liver and quadriceps muscle from 14-wk-old male WT and Trx2ADKO mice fed the HFD for 8 wk (n = 6). Squares correspond to the magnified areas (middle panel). Scale bars, 50 µm. (E–H) Serum concentration of TG (E), cholesterol (F), NEFA (G), and adiponectin (H) in WT and Trx2ADKO mice (n = 6). (I–Q) The hyperinsulinemic-euglycemic clamp study from WT and Trx2ADKO mice (n = 7). (I) A steady plasma glucose is shown. (J) Levels of glucose after external infusion. (K) Glucose infusion rate required to maintain euglycemia during the final 40 min of the clamp. (L) Hepatic glucose production and suppression of HGP under basal and insulin-stimulated (clamp) conditions. (M) Insulin-stimulated whole-body glucose uptake. (N) Plasma NEFA concentrations and…

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  Figure 4.. Adipocyte-specific Trx2 KO results in severe mitophagy and mitochondrial dysfunction. (A–C) Serum levels of NEFA (A), adiponectin (B), and leptin (C) in WT and Trx2ADKO mice (n = 6). (D) Diagram of tricarboxylic acid (TCA) cycle and OXPHOS. Cit1 (citrate synthase), Idh1 (isocitrate dehydrogenase), Kgd1 (α-ketoglutarate dehydrogenase), Lsc1 (succinyl-CoA ligase), Sdh1 (succinate dehydrogenase), Fum1 (fumarase), and Mdh1 (mitochondrial malate dehydrogenase). (E) Immunoblot analysis of Trx2 and rate-limiting enzymes of the tricarboxylic acid cycle and OXPHOS complexes I–V from WT and Trx2ADKO mice. Protein levels were quantified and presented as fold changes by taking WT as 1.0. n = 4 mice for each group. (F) Real-time PCR analysis of mitochondrial genes in eWAT from WT and Trx2ADKO mice at 14 and 24 wk (n = 8). (G) Mitochondrial DNA content in eWAT of WT and Trx2ADKO mice (n = 8). (H) ATP content of mitochondria isolated from eWAT of WT and Trx2ADKO mice (n = 6). (I) Representative transmission electron micrographs of eWAT sections from WT and Trx2ADKO mice at 5, 14, and 24 wk (six images/mouse, n = 3 mice/group). Asterisks indicate LDs. Arrowheads indicate mitochondria. Scale…

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  Figure 5.. Trx2 deficiency increases mitochondrial ROS generation and NF-κB activation. (A and B) Immunoblot analysis of proteins involved in mitochondrial dynamics and mitophagy in eWAT of WT and Trx2ADKO mice at 14 and 24 wk (n = 3). β-Actin was used as a loading control. (C) Representative micrographs of eWAT sections showing ROS levels, as assessed by the mitochondrial-specific ROS probe mitoSOX, and quantification of fluorescence intensity in WT and Trx2ADKO mice at 14 and 24 wk (n = 6). AFU, arbitrary fluorescence unit. (D) Representative Western blot showing protein levels of Trx2, Prx3, Glut4, HSL, and PPARγ in eWAT from WT and Trx2ADKO mice at 14 and 24 wk. (E) Immunoblot analysis of IKK–NF-κB signaling molecules (phospho-IKKα/β, IKKβ, phospho-P65, P65, phospho-IκB, IκB, phospho-AKT, and AKT) in eWAT from WT and Trx2ADKO mice at 14 and 24 wk. Protein levels (A, B, D, and E) were quantified and presented as fold changes by taking WT as 1.0. n = 3 mice for each group. (F) Coimmunostaining of phospho-P65 (green) and adipocyte marker FABP4 (red) in eWAT sections from 14-wk-old male WT and Trx2ADKO mice (n = 6). White arrowheads indicate phospho-p65 in the nucleus. (G) Coimmunostaining…

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  Figure S4.. Trx2 deficiency increases ROS in isolated adipocytes and alters mitochondrial energetics and oxidative metabolism. (A–D) TRX2 deficiency reduces lipogenesis during preadipocytes differentiation. (A) Differentiation scheme of preadipocytes isolated from eWAT of WT (Trx2lox/lox) and Trx2ADKO (KO) mice. (B) Representative Western blots confirming a decrease of TRX2 expression in Trx2ADKO adipocytes during differentiation upon Adipo-Cre was activated on day 4. Adeno-Cre infection rapidly induced Trx2 deletion. Protein levels were quantified and are presented as fold changes by taking WT as 1.0. n = 3. (C and D) Representative BODIPY staining images of WT and KO differentiated adipocytes at day 8 (C) and LD sizes (D) from six random fields were quantified (n = 3 biological repeats). (E) Representative micrographs of mitochondrial ROS levels by the mitoSOX staining of WT and Trx2ADKO (KO) differentiated adipocytes at day 4 in the presence or absence of mito-TEMPO (n = 3). (F) Quantification of mitochondrial ROS levels of WT and KO differentiated adipocytes at different time points (n = 3). AFU, arbitrary fluorescence unit. (G) ATP content of mitochondria from WT and KO adipocytes…

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  Figure 6.. Trx2 deficiency promotes mitophagy through NF-κB–dependent p62/SQSTM1 accumulation and mitochondrial recruitment. (A) Representative transmission electron micrographs showing typical mitophagy in differentiated adipocytes of Trx2ADKO mice on day 8 (n = 3 mice/group). Squares correspond to the magnified areas. Arrows indicate mitochondria. Scale bars, 1 µm. (B) Percentage of damaged mitochondria in 10 random fields (n = 20 cells/group). (C) Immunoblot analysis of Trx2, mitophagy receptors p62 and NBR1, and autophagy markers LC3 and Beclin-1 in differentiated adipocytes of WT and Trx2ADKO mice during differentiation. (D) Subcellular distribution of p62, LC3, and Beclin-1 in differentiated adipocytes of WT and Trx2ADKO mice at day 4. Voltage-dependent anion channel was used as a mitochondrial control. (E) Immunoblot analysis of mitophagy-related proteins in mitochondrial fraction of differentiated adipocytes of WT and Trx2ADKO mice at day 4 of differentiation. Inner membrane protein TOMM20 was used as a control. (F) Immunoblot analysis of NF-κB signaling proteins from differentiated adipocytes (day 4) of WT and Trx2ADKO mice in the absence or presence of mito-TEMPO. (G)…

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  Figure 7.. Elevated oxidative stress–NF-κB–p62 pathway in adipose tissues from T2DM patients. (A) The heatmap displayed DEGs among isolated primary human adipocytes from the lean, obesity, and T2DM groups using hierarchical clustering analysis (n = 6/group). (B and C) Statistical analysis of RNA-sequencing gene expression for obesity (B) and T2DM (C) compared with lean as volcano plots. (D) KEGG pathway analysis of DEGs indicated that ROS pathway, NF-κB signaling, and fatty acid metabolism were affected pathway in T2DM adipocytes (arrow). (E) The mRNA level of representative genes related to ROS regulation, inflammation, and fatty acid metabolism in adipocytes (n = 6/group). (F) Heatmap showing the expression of key inflammatory activation related genes in adipocytes between the lean and T2DM groups. (G) GSEA demonstrating enrichment score (ES) of gene sets in RNA-sequencing data of adipocytes. Genes in each gene set were ranked by signal-to-noise ratio according to their differential expression between the lean group and the T2DM group. FDR, false discovery rate. (H–N) Trx2–NF-kB analyses in ND subjects (n = 10) and patients with T2DM-HS (n = 12). (H and I) Relative levels of Trx2 and TNFA…

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  Figure S5.. Mitochondria-specific antioxidant mito-TEMPO ameliorates adipocyte dysfunction and T2DM in Trx2ADKO mice. WT and Trx2ADKO mice at age of 6 wk received injections of mito-TEMPO (0.7 mg/kg/d, i.p. every other day) or vehicle (saline) for 8 wk. Body weight and blood glucose levels were measured at various ages and mouse tissues were harvested at week 14 for analyses. (A and B) Body weight (A) and fasting blood glucose levels (B) of mice at the indicated ages (n = 8 per group). (C) Representative hematoxylin and eosin staining showing overall eWAT morphology and adipocyte size in eWAT (n = 3 per group). (D) ROS analysis was performed by immunofluorescence microscopy of mitoSOX-stained eWAT, and quantification of fluorescence intensity with and without mito-TEMPO treatment. AFU, arbitrary fluorescence unit (n = 3 per group). (E) Immunoblot analysis of eWAT tissues (n = 3 per group). Protein levels were quantified and presented as fold changes by taking WT as 1.0. n = 3 mice for each group. (F and G) Coimmunostaining of phospho-P65 (green) and FABP4 (red) in eWAT sections. Representative images are shown in F with quantifications in G. n = 3 per group. White arrowheads indicate…