New Synthesized Activating Transcription Factor 3 Inducer SW20.1 Suppresses Resistin-Induced Metabolic Syndrome

Abstract

Obesity is an emerging concern globally with increasing prevalence. Obesity is associated with many diseases, such as cardiovascular disease, dyslipidemia, and cancer. Thus, effective new antiobesity drugs should be urgently developed. We synthesized SW20.1, a compound that induces activating transcription factor 3 (ATF3) expression. The results of Oil Red O staining and quantitative real-time polymerase chain reaction revealed that SW20.1 was more effective in reducing lipid accumulation in 3T3-L1 preadipocytes than the previously synthesized ST32db, and that it inhibited the expression of the genes involved in adipogenesis and lipogenesis. A chromatin immunoprecipitation assay indicated that SW20.1 inhibited adipogenesis and lipogenesis by binding to the upstream promoter region of resistin at two sites (-2861/-2854 and -241/-234). In mice, the intraperitoneal administration of SW20.1 reduced body weight, white adipocyte weight in different regions, serum cholesterol levels, adipogenesis-related gene expression, hepatic steatosis, and serum resistin levels. Overall, SW20.1 exerts antiobesity effects by inhibiting resistin through the ATF3 pathway. Our study results indicate that SW20.1 is a promising therapeutic drug for diet-induced obesity.

Keywords: SW20.1; activating transcription factor 3; obesity; resistin.

Figure 1

Characterization of the activating transcription factor 3 (ATF3) expression ability of SW20.1 in preadipocytes (3T3-L1 cells). (a) Chemical structure of SW20.1 and ST32db. (b) ATF3 gene expression induced by SW20.1 and other derivatives assayed through the quantitative real-time polymerase chain reaction (qRT-PCR). (c) Time- and dose-dependent mRNA ATF3 expression measured using qRT-PCR. Statistical comparisons were performed using unpaired t-test (b,c). Data are presented as mean ± standard error; * p < 0.05 and **** p < 0.0001 compared with the control.

Figure 2

SW20.1 suppresses lipid accumulation and adipogenesis/lipogenesis. (a) Oil Red O staining of differentiated 3T3-L1 adipocytes treated with dimethyl sulfoxide, SW20.1, or ST32db for 8 days. (b) Relative qualification of the lipid content. (c) Analysis of expression of genes involved in adipogenesis, such as genes encoding leptin, resistin, peroxisome proliferator-activated receptor γ2 (PPARγ2), CCAAT/enhancer-binding protein α (C/EBPα), and adiponectin. (d) Analysis of the expression of genes involved in lipogenesis, such as the genes encoding carbohydrate-responsive element binding protein (ChREBP), stearoyl-CoA 9-desaturase (SCD1), and fatty acid binding protein 4 (FABP4). (e) Analysis of the expression of genes involved in beta-oxidation, such as the genes encoding peroxisome-proliferator-activated receptor-γ coactivator- 1alpha (PGC1α), PPARα, and heat shock protein 70 (HSP70). Statistical comparisons were performed using one-way ANOVA (b) and unpaired t-test (c–e). Data are presented as mean ± standard error; * p < 0.05; *** p < 0.001, and **** p < 0.0001.

Figure 3

Molecular mechanism of SW20.1. (a) Activating transcription factor 3 (ATF3) expression was reduced by shRNA-ATF3. (b) Resistin expression was recovered after the knockdown of ATF3 through shRNA-ATF3. (c) Sequences of four potential binding sites for ATF3 in resistin promoter, including sites 1 (−2861/−2854), 2 (−2716/−2709), 3 (−930/−923), and 4 (−241/−234). (d,e) Results of chromatin immunoprecipitation experiments with ATF3-specific antibody and primers to amplify the sites 1 and 4 of the resistin locus, which contained one predicted ATF3 binding site in 3T3-L1 preadipocytes. Statistical comparisons were performed using an unpaired t-test (a,b,d,e). Data are presented as mean ± standard error; * p < 0.05; ** p < 0.01.

Figure 3

Molecular mechanism of SW20.1. (a) Activating transcription factor 3 (ATF3) expression was reduced by shRNA-ATF3. (b) Resistin expression was recovered after the knockdown of ATF3 through shRNA-ATF3. (c) Sequences of four potential binding sites for ATF3 in resistin promoter, including sites 1 (−2861/−2854), 2 (−2716/−2709), 3 (−930/−923), and 4 (−241/−234). (d,e) Results of chromatin immunoprecipitation experiments with ATF3-specific antibody and primers to amplify the sites 1 and 4 of the resistin locus, which contained one predicted ATF3 binding site in 3T3-L1 preadipocytes. Statistical comparisons were performed using an unpaired t-test (a,b,d,e). Data are presented as mean ± standard error; * p < 0.05; ** p < 0.01.

Figure 4

Results of treatment with or without SW20.1 or ST32db in wild-type mice with high-fat-diet (HFD)-induced obesity and metabolic syndrome. (a–c) Body weight, food intake, and body imaging changes of mice with HFD-induced obesity during 10 weeks of treatment with SW20.1 or ST32db (three times per week). (d) Weights of inguinal white adipose tissue (WAT), retroperitoneal WAT, mesenteric WAT, epididymal WAT, interscapular brown adipose tissue (BAT), epididymal BAT, and perivascular adipose tissue. (e) Glucose tolerance test results. (f) Insulin tolerance test results. (g,h) Cholesterol and triglyceride serum levels. (i) Inguinal WAT and liver hematoxylin and eosin staining results (scale bar = 100 μm). (j,k) Size and diameter of adipocytes in each group. Statistical comparison between the three groups were performed using repeated two-way analysis of variance (ANOVA) (a), one-way ANOVA (d,g,h,j,k), and unpaired t-test to compare pairwise groups at different time points (e,f). Data are presented as the mean ± standard error; ns, non-significant; * p < 0.05, ** p < 0.01, and **** p < 0.0001 for SW20.1 compared with the control and # p < 0.05 for SW20.1 compared with ST32db (control and ST32db group, n = 5; SW20.1 group, n = 4).

Figure 4

Results of treatment with or without SW20.1 or ST32db in wild-type mice with high-fat-diet (HFD)-induced obesity and metabolic syndrome. (a–c) Body weight, food intake, and body imaging changes of mice with HFD-induced obesity during 10 weeks of treatment with SW20.1 or ST32db (three times per week). (d) Weights of inguinal white adipose tissue (WAT), retroperitoneal WAT, mesenteric WAT, epididymal WAT, interscapular brown adipose tissue (BAT), epididymal BAT, and perivascular adipose tissue. (e) Glucose tolerance test results. (f) Insulin tolerance test results. (g,h) Cholesterol and triglyceride serum levels. (i) Inguinal WAT and liver hematoxylin and eosin staining results (scale bar = 100 μm). (j,k) Size and diameter of adipocytes in each group. Statistical comparison between the three groups were performed using repeated two-way analysis of variance (ANOVA) (a), one-way ANOVA (d,g,h,j,k), and unpaired t-test to compare pairwise groups at different time points (e,f). Data are presented as the mean ± standard error; ns, non-significant; * p < 0.05, ** p < 0.01, and **** p < 0.0001 for SW20.1 compared with the control and # p < 0.05 for SW20.1 compared with ST32db (control and ST32db group, n = 5; SW20.1 group, n = 4).

Figure 4

Results of treatment with or without SW20.1 or ST32db in wild-type mice with high-fat-diet (HFD)-induced obesity and metabolic syndrome. (a–c) Body weight, food intake, and body imaging changes of mice with HFD-induced obesity during 10 weeks of treatment with SW20.1 or ST32db (three times per week). (d) Weights of inguinal white adipose tissue (WAT), retroperitoneal WAT, mesenteric WAT, epididymal WAT, interscapular brown adipose tissue (BAT), epididymal BAT, and perivascular adipose tissue. (e) Glucose tolerance test results. (f) Insulin tolerance test results. (g,h) Cholesterol and triglyceride serum levels. (i) Inguinal WAT and liver hematoxylin and eosin staining results (scale bar = 100 μm). (j,k) Size and diameter of adipocytes in each group. Statistical comparison between the three groups were performed using repeated two-way analysis of variance (ANOVA) (a), one-way ANOVA (d,g,h,j,k), and unpaired t-test to compare pairwise groups at different time points (e,f). Data are presented as the mean ± standard error; ns, non-significant; * p < 0.05, ** p < 0.01, and **** p < 0.0001 for SW20.1 compared with the control and # p < 0.05 for SW20.1 compared with ST32db (control and ST32db group, n = 5; SW20.1 group, n = 4).

Figure 5

SW20.1 suppresses adipogenesis in high-fat-diet (HFD)-fed mice. (a) Serum protein levels of resistin determined using enzyme-linked immunosorbent assay in HFD-fed mice. (b) Expression of genes involved in adipogenesis in the inguinal white adipose tissue (iWAT). (c) Expression of lipogenesis genes in iWAT. Statistical comparisons were performed using one-way analysis of variance (a) and unpaired t-test (b,c). Data are expressed as mean ± standard error; ns, non-significant; * p < 0.05 (n = 3 per group).

Figure 5

SW20.1 suppresses adipogenesis in high-fat-diet (HFD)-fed mice. (a) Serum protein levels of resistin determined using enzyme-linked immunosorbent assay in HFD-fed mice. (b) Expression of genes involved in adipogenesis in the inguinal white adipose tissue (iWAT). (c) Expression of lipogenesis genes in iWAT. Statistical comparisons were performed using one-way analysis of variance (a) and unpaired t-test (b,c). Data are expressed as mean ± standard error; ns, non-significant; * p < 0.05 (n = 3 per group).