Salsalate Activates Skeletal Muscle Thermogenesis and Protects Mice from High-Fat Diet Induced Metabolic Dysfunction

Figures

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  Fig. 1. Salsalate treatment attenuates body weight gain in mice fed with HFD. (a–e) The 8-week-old male mice were fed with HFD without or with salsalate for 40 days. Body weight (a–b), food intake (c–d) and body temperature (e) were monitored throughout the experiments. (f–g) The hepatic ALT (f) and AST (g) activities were measured. Data are expressed as mean ± SEM (n = 5). *P < 0.05 and **P < 0.01 by two-tailed Student's t-test.

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  Fig. 2. Salsalate inhibits lipid synthesis in liver. (a) Liver morphology. (b) Liver weight. (c) The mRNA levels of trophic factors. (d) Liver H&E staining. (e–f) Liver triglyceride (e) and plasma triglyceride (f) were analyzed. (g–h) Liver cholesterol (g) and plasma cholesterol (h) were measured. (i–j) Plasma HDL (i) and LDL/VLDL (j) were detected. Data are expressed as mean ± SEM (n = 5). *P < 0.05 and **P < 0.01 by two-tailed Student's t-test.

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  Fig. 3. Salsalate attenuates white adipose tissue biogenesis. (a) Morphology of epididymal white adipose (eWAT) tissues. (b) eWAT weight. (c) H&E staining of eWAT. (d) Average adipocyte diameter. (e) The mRNA levels of leptin and adiponectin in eWAT. (f–g) Plasma leptin (f) and adiponectin (g) were measured. Data are expressed as mean ± SEM (n = 5). *P < 0.05 and **P < 0.01 by two-tailed Student's t-test.

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  Fig. 4. Salsalate improves systemic glucose tolerance and insulin signal transduction in eWAT. (a) Glucose tolerance tests. (b) Blood glucose levels. Blood samples from mice with free access to water and food were collected by tail vein bleeding. (c) Plasma insulin concentrations. (d) Insulin tolerance tests. (e–g) The insulin signaling transduction in liver (e), skeletal muscle (f) and eWAT (g) were analyzed. Data are expressed as mean ± SEM (n = 5). *P < 0.05, **P < 0.01 and ***P < 0.001 by two-tailed Student's t-test.

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  Fig. 5. Salsalate impedes hepatic gluconeogenesis. (a) Salsalate inhibits glucose production from pyruvate. (b–c) The mRNA (b) and protein (c) levels of PGC-1α, G6Pase and PEPCK. (d) Densitometric quantification of the immunoblots in (c). (e) Salsalate down-regulates gluconeogenic gene expression in the starved HepG2 cells. (f) Salsalate inhibits glucose production in the starved HepG2 cells. Data are expressed as mean ± SEM (n = 5). *P < 0.05 and **P < 0.01 by two-tailed Student's t-test.

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  Fig. 6. Salsalate promotes glucose and lipid transport and oxidation in skeletal muscle. (a–c) The mRNA levels of genes involved in adipogenesis and heat production in BAT (a) and iWAT (b–c). (d–f) The mRNA levels of genes involved fatty acid transport (d), hydrolysis (e) and oxidation (f). (g) The expression of genes responsible for glucose transport. (h) The expression of genes in glycolysis and tricarboxylic acid cycle. (i) The expression of genes for mitochondrial uncoupling. Data are expressed as mean ± SEM (n = 5). *P < 0.05 and **P < 0.01 by two-tailed Student's t-test.

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  Fig. 7. Salsalate stimulates mitochondria capacity and SERCA2 expression in skeletal muscles. (a) Average mitochondrial numbers based on transmission electron micrographs at the magnification of 5 k. (n = 5). (b) Representative transmission electron micrographs at the magnification of 20 k. The scale bar represents 0.5 μm. (c) The expression of mitochondrial electron transport chain genes. (d) Western blot showing expression of different subunits of electron transport chain proteins. (e) Densitometric quantification of the immunoblots in (d). (f) Succinate dehydrogenase (SDH) staining. The scale bar represents 100 μm. (g) The expression of Sln, Serca1a and Serca2a in skeletal muscle. (h) Western blot showing expression of SERCA1 and SERCA2 in skeletal muscle. (i) Salicylate improves Serca2a expression in C2C12 cells. (j) Western blot showing the increased expression of SERCA2 by salicylate in C2C12 cells. Data are expressed as mean ± SEM (n = 5). *P < 0.05, **P < 0.01 and ***P < 0.001 by two-tailed Student's t-test. ##P < 0.05 by one-way ANOVA with Bonferroni's post hoc test.