LOX-mediated Adipose Tissue Stiffening Promotes Weight Rebound

Figures

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  Fig. 1. AT stiffening emerges in obesity and correlates with high expression of LOX in clinical samples. A: Schematic diagram of abdominal subcutaneous AT stiffness measurement. B: AT stiffness of lean and obese patients measured by ultrasonic elastography. C: LOX expression in blood serum of lean and obese patients. D: Correlation analysis between abdominal subcutaneous AT stiffness and LOX expression. E:) LOX expression in subcutaneous AT of obese patients during obesity, weight loss, and weight maintenance (data source: http://www.ncbi.nlm.nih.gov/geo/GSE35411). (n.s., no significance; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001). AT, Achilles tendon; LOX, lysyl oxidase.

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  Fig. 2. Construction of animal models (Control, Weight gain, Obesity, and Weight rebound group) and metabolic analysis. A: Animal models feeding plan. B: Body weight change curve of different groups. Data are presented as mean ± SD (n = 5). C: Quantitative analysis of body fat percentage. Data are presented as mean ± SD (n = 5). D: Mouse peritoneal glucose tolerance test (upper limit of blood glucose detected by blood glucose meter is 33.3 mmol/L) Data are presented as mean ± SD (n = 5). E: Results of blood lipid metabolism. Data are presented as mean ± s.d. (n = 5). F: Quantification of AT stiffness measured by AFM. Data are presented as mean ± s.d. (n = 5). G: Immunohistochemical staining. H: Quantitative analysis of LOX in epididymal AT across all groups (the scale bar represents 150 μm). Data are presented as mean ± SD (n = 5). (n.s., no significance; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001). AFM, atomic force microscopy; HDL: High density lipoprotein cholesterol; LDL: Low density lipoprotein cholesterol; LOX, lysyl oxidase; TC: Total cholesterol; TG: Triglycerides.

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  Fig. 3. AT stiffness dynamically changes in different stages in Weight rebound model and is consistent with the trend of LOX changes. A: The construction of Weight rebound model and the explanation of different stages (Stage 1: Initial stage; Stage 2: Obesity stage; Stage 3: Weight loss stage; Stage 4: Rebound stage). B: Quantification of AT stiffness measured by AFM. Data are presented as mean ± SD (n = 3). C: HE staining. D: Quantification of adipocyte size (the scale bar represents 150 μm). Data are presented as mean ± SD (n = 3). E: Masson staining. F: Quantification of collagen content results (the scale bar represents 150 μm). Data are presented as mean ± SD (n = 3). G: Immunohistochemical staining. H: Quantitative analysis of LOX in epididymal AT of mice (the scale bar represents 150 μm). Data are presented as mean ± SD (n = 3). I: Western blotting analysis of LOX proteins in epididymal AT of mice at different stages. Data are presented as mean ± SD (n = 3). (n.s., no significance; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001). AFM, atomic force microscopy; AT, Achilles tendon; LOX, lysyl oxidase.

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  Fig. 4. Matrix stiffness induces high LOX expression and increased lipid accumulation. A: Schematic diagram of GelMA hydrogel preparation. B: Hydrogel stiffness tested by rheometer. Data are presented as mean ± SD (n = 3). C: Cell viability of 3T3-L1 cells after 24 h culturing on the hydrogels. Data are presented as mean ± SD (n = 3). D: Immunofluorescence staining of LOX in 3T3-L1 cells after culturing on the hydrogels with different stiffness (the scale bar represents 50 μm). E: Quantification of LOX level from D. Data are presented as mean ± SD (n = 3). F: Secreted LOX level measured by ELISA. Data are presented as mean ± SD (n = 3). G: BODIPY staining for differentiation of 3T3-L1 cells after culturing on the hydrogels with different stiffness (the scale bar represents 20 μm). H: Quantification of lipid accumulation from G. Data are presented as mean ± SD (n = 3). (n.s., no significance; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001). GelMA, methacryloyl gelatin; LOX, lysyl oxidase.

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  Fig. 5. LOX inhibition/knockdown impairs lipid accumulation ability of 3T3-L1 cells on stiff matrix (∼3 kPa). A: BODIPY staining for differentiation of 3T3-L1 cells with/without BAPN inhibition on stiff matrix (the scale bar represents 50 μm). B: Quantification of lipid accumulation from A. Data are presented as mean ± SD (n = 3). C: Oil-red staining for differentiation of 3T3-L1 cells with/without BAPN inhibition on stiff matrix (the scale bar represents 20 μm). D: Quantification of oil-red staining for differentiation of 3T3-L1 cells with/without BAPN inhibition on stiff matrix. Data are presented as mean ± SD (n = 3). E: BODIPY staining for differentiation of 3T3-L1 cells with/without siRNA knockdown on stiff matrix (the scale bar represents 50 μm). F: Quantification of lipid accumulation from E. Data are presented as mean ± SD (n = 3). G: Oil-red staining for differentiation of 3T3-L1 cells with/without siRNA knockdown on stiff matrix (the scale bar represents 20 μm). H: Quantification of oil-red staining for differentiation of 3T3-L1 cells with/without siRNA knockdown on stiff matrix. Data are presented as mean ± SD (n = 3). (n.s., no significance; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P <…

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  Fig. 6. LOX inhibition slows down the weight rebound process in vivo. A: Mouse weight change curve. Data are presented as mean ± SD (n = 3). B: AT stiffness (Con: Control; WR: Weight Rebound; and +B: +BAPN). Data are presented as mean ± SD (n = 3). C: AT stiffness at different stages (Stage 2: Obesity stage; Stage 3: Weight loss stage; and Stage 4: Rebound stage) in the Control and Weight rebound group (Con: Control; WR: Weight Rebound; and +B: +BAPN). Data are presented as mean ± SD (n = 3). D: Masson staining of AT slices (the scale bar represents 150 μm). E: Quantitative results of collagen content. Data are presented as mean ± SD (n = 3). F: LOX immunohistochemical staining of AT slices (the scale bar represents 150 μm). G: LOX quantification results. Data are presented as mean ± SD (n = 3). H: Western blotting analysis. I: quantification of LOX proteins in epididymal adipose tissue of mice after treatment with/without BAPN. Data are presented as mean ± SD (n = 3). (n.s., no significance; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001) J: Schematic of the “Loop Breaker” mechanism. The diagram illustrates that pharmacological inhibition of LOX activity (red cross) leads to matrix softening. This…