| Objective: Caloric restriction(CR)followed by refeeding,a phenomenon known as catch-up growth(CUG),results in excessive lipid deposition and insulin resistance in skeletal muscle,but the underlying mechanisms remain elusive.We observed the changes of VEGF-B signaling pathway,lipid deposition and insulin sensitivity in skeletal muscle of CUG rats to explore the role of VEGF-B signaling pathway on insulin resistance of CUG.Methods:1.6-week-old male Sprague-Dawley rats were randomly divided into two groups after one week of adaptation to the housing conditions: NC(normal chow)group and RN(caloric restriction(CR)and refeeding with a normal chow)group.The NC rats were fed normal chow ad libitum for 4,8,and 12 weeks,respectively.The RN rats were composed of a CR subgroup(maintained on food restriction for 4 weeks at 60 % of that eaten by weight-matched NC rats)and two refeeding subgroups(refed with 100 % of that eaten by weight-matched NC rats for 4 and 8 weeks,respectively,after CR for 4 weeks).We measured the food intake and body weight of the rats every day to calculate daily energy intake.2.Serum and skeletal muscle indicators were detected in animal experiments at 4,8,and 12 weeks,respectively: total body fat mass percentage,fasting plasma glucose,fasting plasma insulin,fasting plasma lipid,glucose infusion rate required to maintain euglycemia during hyperinsulinemic-euglycemic clamp and insulin-stimulated muscle glucose uptake,skeletal muscle TG and total DAG,PKCθ membrane translocation,insulin-stimulated p-AKT,VEGF-B signaling pathway related genes m RNA and protein level(VEGF-B,VEGFR1,NRP1 FATP3,FATP4).3.Rat primary skeletal muscle microvascular endothelial cells were sorted and cultured using magnetic beads method,and then factor Ⅷ related antigen was detected.Cells were pre-incubated with the anti-VEGFR1 antibody,or anti-NRP1 antibody,and then VEGF-B was added into the medium.The m RNA levels of Fatp3 and Fatp4 were detected by RT-PCR.Cells were transfected with si RNA targeting Fatp3 or Fatp4,and then VEGF-B was added into the medium.Cellular fatty acid uptake were detected by immunofluorescence staining.Endothelial cells were cultured on trans-well insert membranes to mimicked the vascular endothelial layer.Cells were pre-incubated with the anti-VEGFR1 antibody,or anti-NRP1 antibody,and then VEGF-B was added into the medium.Fluorescence of fatty acids in trans-well lower layer medium was detected by fluorescence microplate reader.Results:1.RN rats were given approximately 60 % of normal diet for 4 weeks and then refed for 8 weeks with 100 % of the diet of the weight-matched NC rats.Body weight increased moderately in the NC group;however,in the RN group it slightly increased during CR and rapidly increased after refeeding.2.The total body fat mass percentage,fasting plasma insulin levels,and fasting plasma lipid levels were significantly higher,and the glucose infusion rate during hyperinsulinemic-euglycemic clamp was markedly decreased,and fasting blood glucose levels had no significant difference after refeeding with normal diet compared with the age-matched control.3.Muscle TG and total DAG levels dramatically increased,and PKCθ membrane translocation increased was significantly increased,and insulin-stimulated p-AKT was markedly decreased,and insulin-stimulated muscle glucose uptake dramatically decreased after refeeding with normal diet compared with the age-matched controls.4.The m RNA and protein levels of VEGF-B,VEGFR1,NRP1,FATP3 and FATP4 in skeletal muscle were significantly increased at the end of CR and after refeeding compared with their controls.5.VEGF-B could up-regulate the m RNA levels of Fatp3 and Fatp4 in skeletal muscle microvascular endothelial cells,and this effect was blocked by VEGFR1 or NRP1 antibodies.Treating endothelial cells with VEGF-B significantly increased cellular fatty acid uptake.Conversely,silencing Fatp3 or Fatp4 reduced cellular fatty acid uptake,and counteracted the effect of VEGF-B.VEGF-B strongly promoted fatty acid flux across the endothelial monolayer,and this flux was blocked by the antibodies against VEGFR1 or NRP1.Conclusions:1.The continuous activation of VEGF-B signaling pathway might be an important mechanism for initiating and maintaining the increaseed skeletal muscle fatty acid uptake of CUG rats,causing skeletal muscle lipid deposition and subsequent insulin resistance.2.VEGF-B induced fatty acid uptake and transport in skeletal muscle microvascular endothelial cells by regulating FATP3 and FATP4.Objective: We measured the aberrant changes in epigenetic modifications at the Vegfb promoter in the skeletal muscle of CUG rats to explore the role of epigenetic mechanism on insulin resistance of CUG.Methods:1.6-week-old male Sprague-Dawley rats were randomly divided into two groups after one week of adaptation to the housing conditions : NC(normal chow)group and RN(caloric restriction(CR)and refeeding with a normal chow)group.The NC rats were fed normal chow ad libitum for 4,8,and 12 weeks,respectively.The RN rats were composed of a CR subgroup(maintained on food restriction for 4 weeks at 60 % of that eaten by weight-matched NC rats)and two refeeding subgroups(refed with 100 % of that eaten by weight-matched NC rats for 4 and 8 weeks,respectively,after CR for 4 weeks).We measured the food intake and body weight of the rats every day to calculate daily energy intake.2.Skeletal muscle indicators were detected in animal experiments at 4,8,and 12 weeks,respectively: histone methylation(H3K4me3,H3K9me2,and H3K27me3)and acetylation(H3K9ac,H3K14 ac,and H3K18ac)levels at the Vegfb promoter,histone acetyltransferases(p300,CBP,GCN5,and PCAF)m RNA levels,histone acetyltransferases p300 protein level,p300 level at the Vegfb promoter.3.Knocking down p300 using si RNA or inhibiting p300 activity via its specific inhibitor(C646)in L6 cells,H3K14 ac and H3K18 ac levels at the Vegfb promoter were detected by Ch IP,and VEGF-B m RNA and protein levels were detected by RT-PCR and WB.Overexpressed p300 in L6 cells by GV141 vector transfection anddetected the above indicators.Results:1.H3K14 ac and H3K18 ac modifications at the Vegfb promoter were significantly increased in skeletal muscle at the end of CR and after refeeding compared with their controls.H3K9me2 and H3K27me3 modifications at the Vegfb promoter were only decreased after refeeding.H3K4me3 modifications at the Vegfb promoter were only increased after refeeding.No significant difference of H3K9 ac at the Vegfb promoter during CUG.2.Histone acetyltransferases p300 m RNA and protein levels in skeletal muscle were significantly elevated at the end of CR and after refeeding compared with their controls.However,CBP,GCN5 and PCAF m RNA levels did not change in the CUG rats compared with the normal group.Moreover,the recruitment of p300 to the Vegfb promoter were significantly increased in the skeletal muscle of CUG rats.3.H3K14 ac and H3K18 ac levels at the Vegfb promoter as well as VEGF-B m RNA and protein levels were significantly decreased after treatment with p300 si RNA or C646 in L6 cells.At the same time,H3K14 ac and H3K18 ac levels at the Vegfb promoter as well as VEGF-B m RNA and protein levels were significantly increased after p300 overexpression in L6 cells.Conclusions:1.Significantly increased histone acetylation modifications might be the leading cause of lasting VEGF-B activation in CUG skeletal muscle.2.p300 regulated VEGF-B expression in L6 cells by acetylating H3K14 and H3K18. |
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