PTG‐Dependent Glycogen Metabolic Dysfunction Drives Impaired Adipose Browning: A Novel Mechanism Linking PM 2.5 to Metabolic Disorders

Figures

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  FIGURE 1. Concentrations of PM2.5 at the study site. (A) Daily PM2.5 concentrations in the FA and PM2.5 chamber in the area where the exposure system was located. (B) Average PM2.5 concentrations in the FA and PM2.5 chamber during the exposure period. Data were presented as mean ± SEM.

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  FIGURE 2. Effects of PM2.5 exposure on systemic metabolism and iWAT browning. (A,B) Body weight (A) and body weight gain (B) of mice (n = 8 per group). (C,D) Fat mass (C) and the ratio of fat mass to body weight (D) of mice (n = 8 per group). (E,F) GTT (E) and AUC of GTT (F) of mice (n = 7 per group). (G,H) Heat production of mice (n = 4 per group). Line graph (G) indicates variation patterns of heat production of mice over a 24 h period, column graph (H) indicates the mean values of heat production of mice over a 24 h period. (I,J) Adipose mass (I) and adipose coefficient (J) of mice (n = 8 per group). (K,L) Representative images (K) and quantitative analysis (L) of H&E staining in iWAT sections (n = 5 per group); Scale bar = 50 µm. (M) mRNA expression of thermogenic genes in iWAT (n = 5 per group). (N,O) Representative images (N) and relative IOD (O) of immunohistochemistry in iWAT sections stained with UCP1 antibody (n = 5 per group); Scale bar = 50 µm. (P) Representative bands (top) and quantitative analysis (bottom) of UCP1 in iWAT (n = 4 per group). (Q) mRNA expression of thermogenic genes in 3T3‐L1 adipocytes (n = 4 per group). (R) Representative bands (top) and quantitative…

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  FIGURE 3. Effects of PM2.5 exposure on glycogen metabolism in iWAT. (A) Glycogen content in iWAT of mice (n = 8 per group). (B,C) Representative images (B) and relative IOD (C) of PAS staining in iWAT sections (n = 5 per group); Scale bar = 50 µm. (D) mRNA expression of Ppp1r3c in iWAT (n = 5 per group). (E) Representative bands (top) and quantitative analysis (bottom) of PTG in iWAT (n = 4 per group). (F) Correlation of Ucp1 mRNA levels in iWAT with Ppp1r3c mRNA levels (n = 10). (G) mRNA expression of Ppp1r3c in 3T3‐L1 adipocytes (n = 4 per group). (H) Representative bands (top) and quantitative analysis (bottom) of PTG in 3T3‐L1 adipocytes (n = 3 per group). (I) Correlation of Ucp1 mRNA levels in 3T3‐L1 adipocytes with Ppp1r3c mRNA levels (n = 12). (J) mRNA expression of rate‐limiting enzymes of glycogen metabolism in iWAT (n = 5 per group). (K,L) Representative bands (K) and quantitative analysis (L) of rate‐limiting enzymes of glycogen metabolism in iWAT (n = 4 per group). Data were presented as mean ± SEM, analyzed by unpaired, two‐tailed Student's t‐test (A–E, J, L) or one‐way ANOVA with Tukey's test (G, H). Correlations were examined with the non‐parametric Spearman correlation test…

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  FIGURE 4. Effects of PTG overexpression on PM2.5‐induced glycogen metabolism dysregulation in iWAT. (A) Schematic representation of AAV9‐PTG mice by AAV9 injection. (B) Glycogen content in iWAT (n = 5 per group). (C,D) Representative images (C) and relative IOD (D) of PAS staining in iWAT sections (n = 4 per group); Scale bar = 50 µm. (E) mRNA expression of rate‐limiting enzymes of glycogen synthesis in iWAT (n = 6 per group). (F,G) Representative bands (F) and quantitative analysis (G) of rate‐limiting enzymes of glycogen synthesis in iWAT (n = 4 per group). (H) mRNA expression of rate‐limiting enzymes of glycogen synthesis in 3T3‐L1 adipocytes (n = 3 per group). (I,J) Representative bands (I) and quantitative analysis (J) of rate‐limiting enzymes of glycogen synthesis in 3T3‐L1 adipocytes (n = 3 per group). Data were presented as mean ± SEM, analyzed by two‐way ANOVA with Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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  FIGURE 5. Effects of PTG overexpression on PM2.5‐induced systemic metabolic dysfunctions and impaired iWAT browning. (A,B) Body weight (A) and body weight gain (B) of mice (n = 6 per group). (C,D) Fat mass (C) and the ratio of fat mass to body weight (D) of mice (n = 6 per group). (E,F) GTT (E) and AUC of GTT (F) of mice (n = 6 per group). (G,H) Heat production of mice (n = 6 per group). Line graph (G) indicates variation patterns of heat production of mice over a 24 h period, grouped column graph (H) indicates the mean value at the time points indicated. (I,J) iWAT mass (I) and iWAT coefficient (J) of mice (n = 6 per group). (K,L) Representative images (K) and quantitative analysis (L) of H&E staining in iWAT sections (n = 4 per group); Scale bar = 50 µm. (M,N) Representative thermographic camera images (M) as shown by FLIR image and quantification of temperature changes (N) in iWAT. The white arrow indicates the inguinal area of the abdomen (n = 6 per group). (O) mRNA expression of thermogenic genes in iWAT (n = 6 per group). (P,Q) Representative images (P) and relative IOD (Q) of immunohistochemistry in iWAT sections stained with UCP1 antibody (n = 4 per group); Scale bar = 50 µm. (R,S)…

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  FIGURE 6. Effects of PTG overexpression on PM2.5‐induced mitochondrial dysfunction in iWAT. (A,B) Representative transmission electron microscopy images (A) and quantification of relative mitochondrial number (B) in iWAT sections (n = 3 per group); Scale bar = 200 nm. (C) mRNA expression of Complex I‐IV and ATP synthase subunits‐related genes in iWAT (n = 6 per group). (D,E) Representative bands (D) and quantitative analysis (E) of NDUFB9, SDHA, UQCRFS1, COX5A, and ATPB in iWAT (n = 4 per group). (F,G) OCR (F) and indicators of mitochondrial respiratory function (G) of 3T3‐L1 adipocytes (n = 3 per group). (H,I) Representative bands (H) and quantitative analysis (I) of NDUFB9, SDHA, UQCRFS1, COX5A, and ATPB in 3T3‐L1 adipocytes (n = 3 per group). Data were presented as mean ± SEM, analyzed by two‐way ANOVA with Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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  FIGURE 7. VEGFB mediated the protective effects of PTG overexpression against PM2.5‐impaired iWAT browning. (A) Target analysis of Ppp1r3c and Ucp1 by GeneMANIA. (B) Representative bands (top) and quantitative analysis (bottom) of VEGFB in iWAT (n = 4 per group). (C) Representative bands (top) and quantitative analysis (bottom) of VEGFB in 3T3‐L1 adipocytes (n = 3 per group). (D) Representative bands (top) and quantitative analysis (bottom) of VEGFB in 3T3‐L1 adipocytes (n = 3 per group). (E) mRNA expression of thermogenic genes in 3T3‐L1 adipocytes (n = 3 per group). (F) Representative bands (top) and quantitative analysis (bottom) of UCP1 in 3T3‐L1 adipocytes (n = 3 per group). (G) mRNA expression of Complex I‐IV and ATP synthase subunits‐related genes in 3T3‐L1 adipocytes (n = 3 per group). (H,I) Representative bands (H) and quantitative analysis (I) of NDUFB9, SDHA, UQCRFS1, COX5A, and ATPB in 3T3‐L1 adipocytes (n = 3 per group). Data were presented as mean ± SEM, analyzed by two‐way ANOVA with Tukey's multiple comparisons test (B,C, E–I) or one‐way ANOVA with Tukey's test (D). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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  FIGURE 8. ADRB3 acted as an upstream regulator of PTG in PM2.5‐inhibited iWAT browning. (A) Representative bands (top) and quantitative analysis (bottom) of PTG and VEGFB in iWAT (n = 4 per group). (B) Representative bands (top) and quantitative analysis (bottom) of PTG and VEGFB in 3T3‐L1 adipocytes (n = 3 per group). (C) mRNA expression of thermogenic genes in iWAT (n = 6 per group). (D) Representative bands (top) and quantitative analysis (bottom) of UCP1 in iWAT (n = 4 per group). (E) mRNA expression of thermogenic genes in 3T3‐L1 adipocytes (n = 3 per group). (F) Representative bands (top) and quantitative analysis (bottom) of UCP1 in 3T3‐L1 adipocytes (n = 3 per group). (G) Schematic illustration of proposed mechanisms. This schematic illustrates the pathway linking PM2.5 exposure to impaired iWAT browning through PTG‐dependent glycogen metabolism dysregulation. Data were presented as mean ± SEM, analyzed by two‐way ANOVA with Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.