Silver nanoparticles inhibit beige fat function and promote adiposity

Figures

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  Figure 1. AgNPs blocked differentiation of beige adipocytes. (A) Experimental schematics of AgNP administration along the differentiation process of adipocytes. On the last day of differentiation, cells were harvested and analyzed. (B) Representative images of Oil red O staining (left) and quantification (right) in C3H10T1/2 adipocytes. (C–E) Quantitative PCR analysis of the influence of Ag20NPs on the differentiation and thermogenic and mitochondrial gene programs in C3H10T1/2 adipocytes (C) under different Ag20NPs doses or (D) on different days of differentiation, or (E) in primary beige adipocytes. Data are presented as mean ± SEM and *P < 0.05, **P < 0.01 compared to control group.

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  Figure 2. AgNPs disrupted mitochondrial and thermogenic functions of beige adipocytes. (A) Experimental schematics of AgNP administration on differentiated adipocytes. (B) Basal oxygen consumption in primary beige adipocytes. (C) Thermogenic and mitochondrial gene programs in C3H10T1/2 adipocytes under different Ag20NP doses. (D) AgNPs blocked forskolin (FSK)-induced elevation in the thermogenic (left) and mitochondrial (right) gene programs in C3H10T1/2 adipocytes. (E) AgNPs blocked forskolin (FSK)-induced elevation in PGC1α and UCP1 protein levels. (F) Ag20NP treatment inhibited thermogenic and mitochondrial gene programs in primary beige adipocytes. Data are presented as mean ± SEM and *P < 0.05, **P < 0.01 compared to control group. #P < 0.05 compared to FSK group.

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  Figure 3. AgNPs suppressed brown gene programs in subcutaneous fat and whole body energy expenditure in mice. Mice under chronic treatment were treated with Ag20NPs or vehicle once a month for three months and sacrificed one month after last injection. (A) The thermogenic and mitochondrial gene programs in subcutaneous fat (iWAT). (B) Representative images of UCP1 immunostaining in iWAT. (C and D) Mice were analyzed in CLAMS for (C) oxygen consumption or (D) carbon dioxide consumption. n = 6 per group. Data are presented as mean ± SEM and *P < 0.05, **P < 0.01 compared to control group.

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  Figure 4. AgNP exposure led to increased adiposity in mice. Mice under chronic treatment were treated with Ag20NPs or vehicle once a month for three months and sacrificed one month after last injection. (A) Body weights; (B) Fat mass; (C) Weight of subcutaneous fat (iWAT), brown fat (BAT) and epididymal white fat (eWAT); (D) Representative images of H&E staining of adipocytes from iWAT, BAT and eWAT; (E) Serum levels of total cholesterol, HDL-C, triglycerides; (F) Liver TG contents; (G) Representative images of liver H&E staining and (H) percentage change of glucose induction after 15 min glucose challenge in glucose tolerance test. n = 6 per group. Data are presented as mean ± SEM and *P < 0.05, **P < 0.01 compared to control group.

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  Figure 5. AgNPs-ROS-ERK signaling mediates AgNP impact on adiposity. (A) Representative fluorescent images of Mitotracker staining on differentiated C3H10T1/2 adipocytes treated with or without Ag20NPs. (B) Intensity of Mitotracker fluorescent signals was assessed by flow cytometry. (C and D) Differentiated C3H10T1/2 adipocytes were treated with vehicle, Ag20NPs or Ag20NPs plus antioxidant NAC. The reactive oxygen species (ROS) production in cells were assessed by DCF assay. Representative fluorescent images and quantification were shown in (C) and changes in brown gene programs were shown in (D). (E) Western blot assessing the phosphorylated and total MAPK signaling pathways of differentiated C3H10T1/2 adipocytes treated with vehicle, Ag20NPs or Ag20NPs plus NAC. (F) Differentiated C3H10T1/2 adipocytes were treated with vehicle, Ag20NPs or Ag20NPs plus specific ERK inhibitor FR180204 and changes in brown gene programs were assessed. Data are presented as mean ± SEM and *P < 0.05, **P < 0.01 compared to control group. #P < 0.05, ##P < 0.01 compared to Ag20NPs group.

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  Figure 6. Impact of AgNPs on beige adipocytes and mouse physiology. After AgNPs exposure, nanoparticles enter the cytoplasm and reside in lysosomes, releasing ROS into the lysosomal acidic environment. The elevated, cellular ROS levels cause oxidative stress and increase ERK phosphorylation, which, in turn, suppress thermogenic and mitochondrial gene programs including PGC1α and UCP1. AgNPs exposure caused mitochondrial dysfunction leads to decreased energy expenditure and increased adiposity in mice.

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  Figure S1. Characterization of AgNPs. (A and B) Representative TEM images of (A) Ag20NPs and (B) Ag200NPs. (C) Hydrodynamic sizes of Ag20NPs and Ag200NPs. (D) UV-Visible spectra of Ag20NPs and Ag200NPs.

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  Figure S2. No influences of AgNPs on adipocyte viability. (A) Ag20NPs and Ag200NPs of different doses were added during the differentiation process of C3H10T1/2 adipocytes and MTT analysis was performed at the end of differentiation for cell viability. (B) Differentiated C3H10T1/2 adipocytes were treated with Ag20NPs and Ag200NPs of different doses and MTT analysis was performed for cell viability. Data are presented as mean ± SEM.

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  Figure S3. AgNPs blocked beige adipocytes differentiation in a dose- and time-dependent manner. (A and B) C3H10T1/2 adipocytes were treated with Ag200NPs during the differentiation process. On the last day of differentiation, cells were harvested and analyzed for differentiation and thermogenic and mitochondrial gene programs (A) under different Ag200NP doses or (B) on different days of differentiation. (C and D) Primary epididymal and brown adipocytes were isolated and treated with Ag20NPs as in (A) and differentiation and thermogenic and mitochondrial gene programs were analyzed. Data are presented as mean ± SEM and *P < 0.05, **P < 0.01 compared to control group.

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  Figure S4. Gene expression analyses of Ag200NP-treated beige adipocytes and Ag20NP-treated primary brown adipocytes. (A) The thermogenic and mitochondrial gene programs of differentiated C3H10T1/2 adipocytes treated with vehicle or Ag200NPs of different doses were analyzed. (B) Primary brown adipocytes were isolated, differentiated, and treated with vehicle or Ag20NPs. The thermogenic and mitochondrial gene programs were assessed. Data are presented as mean ± SEM and **P < 0.01 compared to control group.

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  Figure S5. The effects of acute or chronic AgNPs exposure to mice. (A and B) Mice were treated with intragastric administration of 125 mg/kg Ag20NPs and analyzed after 48 h for (A) silver ion levels in iWAT, BAT, eWAT and liver and (B) thermogenic and mitochondrial gene programs in iWAT. (C and D) Mice were injected subcutaneously with 5 mg/kg Ag20NPs in iWAT and analyzed after 48 h for (C) silver ion levels in iWAT, BAT, eWAT, liver, and gastrocnemius muscle and (D) thermogenic and mitochondrial gene programs in iWAT. (E) Serum levels of ALT and AST after iWAT injection of Ag20NPs for 48 h. (F-H) Mice were treated chronically with Ag20NPs once a month for three months and sacrificed one month after last injection for differentiation and thermogenic and mitochondrial gene programs in (F) BAT and (G) eWAT, as well as (H) locomotor activity and food intake. n = 4 per group for acute treatment and n = 6 per group for chronic treatment. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 compared to control group.

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  Figure S6. Phenotypes of chronic Ag20NPs-treated mice. Mice were treated chronically with Ag20NPs once a month for three months and sacrificed one month after last injection for (A) lean mass, (B) inflammatory gene expressions in eWAT, (C) liver weights, and (E and F) performance in glucose and insulin tolerance test. n = 6 per group. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 compared to control group.