Effects Of Short-chain Fatty Acids On Glucose Metabolism And Inflammatory Signals In Skeletal Muscle

Objective:Obese individuals have disorders in glycolipid metabolism,chronic low-grade inflammation and increased levels of various inflammatory factors.Skeletal muscle is the main organ for the body to absorb postprandial glucose,which plays an important role in maintaining blood sugar homeostasis.Intestinal microflora maintains metabolic balance.It has been reported that short-chain fatty acids,the final product of intestinal microflora,can improve insulin sensitivity and alleviate insulin resistance.Previously,our research team found that AMPK promotes skeletal muscle glucose uptake through non-insulin-dependent pathway.Axin1 and Tiam1 participate in AMPK-mediated skeletal muscle glucose transport.Based on this,we explored the mechanism of short-chain fatty acids affecting skeletal muscle glucose uptake in vitro and in vivo,as well as their effects on inflammatory signals,in order to provide new ideas for the treatment of metabolic diseases such as obesity.Content:In the first part,C2C12 skeletal muscle cells were incubated with different concentrations of sodium acetate,sodium propionate and sodium butyrate in vitro,respectively.The effect of short-chain fatty acids on carbohydrate metabolism of C2C12 skeletal muscle cells were investigated by measuring the phosphorylation level of AMPK and the protein levels of Axin1 and Tiam1.In vivo,the effect of short-chain fatty acids on glycometabolism were investigated by measuring the phosphorylation of AMPK and the protein levels of Axin1 and Tiam1 in gastrocnemius of mice fed with Clostridium butyricum.In the second part,the inflammation model of C2C12 cells was established with palmitic acid and TNF? in vitro.The effects of short-chain fatty acids on inflammation signal in C2C12 cells were explored by measuring the phosphorylation of JNK,NF-?B and IKK?/?.In vivo,obese mice induced by high-fat diet were given Clostridium butyricum.The effects of short-chain fatty acids on inflammation signal of skeletal muscle were investigated by detecting the phosphorylation of JNK,NF-?B and IKK?/? in gastrocnemius muscle.Methods:In the first part: C2C12 cells were incubated with different concentrations of sodium acetate,sodium propionate and sodium butyrate for 24 hours in vitro.Cell viability was measured by MTS assay,and glucose uptake was measured by 2-NBDG method.Phosphorylation of AMPK and total protein levels of Axin1 and Tiam1 were detected by Western Blot.Mice fed with chow diet were given Clostridium butyricum every other day by intragastric administration in vivo.After 8 weeks,gastrocnemius muscle was taken.Western blot was used to detect the AMPK phosphorylation level and the total protein levels of Axin1 and Tiam1 of the gastrocnemius muscle.In the second part: In vitro,C2C12 cells were incubated with 500?M PA and 4 m M sodium acetate,sodium propionate and sodium butyrate,respectively.The phosphorylation of JNK and NF-?B were detected by Western Blot.C2C12 cells were incubated with 10ng/ml TNF? and 4m M sodium acetate,sodium propionate and sodium butyrate,respectively.The phosphorylation of IKK?/? was detected by Western Blot.In vivo,after 14 weeks of feeding on a high-fat diet,Clostridium butyricum was administered by every other day for 8 weeks.The gastrocnemius muscle was taken.The phosphorylation of JNK,NF-?B and IKK?/? were detected by Western Blot.Results : Part1:1.Sodium acetate,sodium propionate and sodium butyrate had no effect on the viability of C2C12 cells under treatments with 1m M,2m M,4m M,8m M and 16 m M.2.Sodium acetate promoted glucose uptake in C2C12 cells under treatments with 1m M,2m M,4m M,8m M and 16 m M.Sodium propionate promoted glucose uptake in C2C12 cells under treatments with 1m M,2m M,4m M,8m M and 16 Mm.Sodium butyrate promoted glucose uptake in C2C12 cells under treatments with 1m M,2m M and 4m M.3.Compared with the control group,sodium acetate activated AMPK under treatments with 1m M and 4m M without affecting the total protein of AMPK.Sodium propionate activated AMPK under treatments with 4m M without affecting the total protein of AMPK.Sodium butyrate activated AMPK under treatments with 4m M,8m M and 16 m M without affecting the total protein of AMPK.4.Compared with the control group,sodium acetate up-regulated the expression of Axin1 under treatments with 1m M,2m M,4m M and 8Mm.Sodium propionate up-regulated the expression of Axin1 under treatments with 4m M.Sodium butyrate up-regulated the expression of Axin1 under treatments with 4m M,8m M and 16 m M.5.Compared with the control group,sodium acetate up-regulated the expression of Tiam1 under treatments with 1m M,2m M,4m M,8m M and 16 m M.Sodium propionate up-regulated the expression of Tiam1 under treatments with 1m M,2m M,4m M and 8m M.Sodium butyrate up-regulated the expression of Tiam1 under treatments with 4m M and 8m M.6.Compared with the control group,the phosphorylation level of AMPK and the total protein levels of Axin1 and Tiam1 increased in Clostridium butyricum group.Part2:1.Sodium butyrate significantly inhibited PA-induced phosphorylation of JNK,whereas sodium acetate and sodium propionate had no effect.2.Sodium acetate,sodium propionate and sodium butyrate significantly inhibited PA-induced phosphorylation of NF-?B.3.Sodium butyrate significantly inhibited the phosphorylation of IKK?/? induced by TNF?,whereas acetic acid and propionic acid had no effect.4.Clostridium butyricum significantly inhibited phosphorylation of JNK,NF-?B and IKK?/? induced by high-fat diet in mice.5.Sodium acetate,sodium propionate and sodium butyrate can alleviate the lipid deposition induced by PA.Conclusion:1.Short-chain fatty acids may promote skeletal muscle glucose uptake by activating AMPK and up-regulating the expression of Axin1 and Tiam1.2.Short-chain fatty acids significantly inhibit the expression of inflammatory molecules and alleviate chronic inflammation caused by insulin resistance in skeletal muscle.