| BackgroundSeveral metabolic diseases such as type 2 diabetes, metabolic syndrome have been one of the most serious public health problems, their central pathogenesis and pathophysiology involve insulin resistance (IR). And the dysfunction of insulin signaling is the important cause of IR, therefore, to enhance the activity of insulin signaling is a key strategy to prevent and treat IR. In additional to phosphatidylinositol-3 kinase (PI3K) insulin signal pathway, scientist found a new insulin signaling named P-arrestin-2-dependent signaling several years ago. Upon stimulation by insulin,?-arrestin 2 scaffolds Src and Akt to insulin receptor (InsR), causing the formation of a new?-arrestin 2 signal complex, which enhances insulin-mediated glucose metabolism. The novel insulin signaling provides new insight into the understanding of IR, and will be a potential molecular target against IR. At present, thiazolidinediones (TZD) is a typical drug to improve IR, which can stimulate peroxisome proliferator-activate receptors-y, and advance the activity of PI3K pathway. Although TZD has been widely used to treat IR relative diseases, it leads to some side effect. For example, it causes retention of water and sodium, leads to adipose tissue accumulation, results in weight gain, and increases risk of cardiovascular disease events in type 2 diabetes patients, including fatal cardiovascular cases. So it will be an important direction to develop a new insulin sensitizing reagent, which will fail to bring about lipid increases and cardiovascular cases. In clinical practice, Pollen Typhae, which can activate blood and dissolve stasis in Traditional Chinese Medicine, is used to treat type 2 diabetes, metabolic syndrome, obesity, which are associated with IR. In vitro, we found that Pollen Typhae total flavone (PTF), extracts from Pollen Typhae, increased glucose uptake and glucose consumption, and decreased lipid accumulation in 3T3-L1 adipocytes in the present of insulin, which shows that PTF could improve IR. However, PTF lacks systemic study and the potential molecular mechanism is not clear. Objective1. To investigate the effect of PTF on glucose consumption and glucose uptake in C2C12 myotubes, and to determine the relationship between effect and insulin.2. To observe the effect of PTF on glucose consumption and glucose uptake in high-concentration-palmitic-acid(PA)-induced and high-glucose-as well as high-insulin-induced insulin resistant C2C12 myotubes.3. In order to explore molecular mechanism of PTF in improving insulin sensitivity, it's good nice to investigate the effect of PTF on protein expression of signal molecules in PI3K pathway and?-arrestin-2-dependent signaling.4. To further explore molecular mechanism of PTF in improving insulin sensitivity, it's necessary to check the effect of PTF on mRNA expression of signal molecular genes in?-arrestin-2-dependent signaling.Methods1. Effect of PTF on the activity of C2C12 myotubes was determined by the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) assay.2. Glucose consumption and glucose uptake in C2C12 myotubes were measured by the glucose oxidase method and the transport of 2-Deoxy-D-[2,6-3H] glucose (2-DOG) into the cells respectively.3. To simulate the pathological features of lipid metabolic disturbance and high glucose as well as high insulin in insulin resistant patients, insulin resistant C2C12 myotubes were induced by high concentration PA and high glucose as well as high insulin respectively based on 2-DOG uptake.4. Western Blot were adopted to measure protein expression of relative signal molecules and their phosphorylation in both PI3K pathway and?-arrestin-2-dependent signaling.5. Co-immunoprecipitation was used to check the formation P-arrestin 2 signal complex, with determining the constituent proteins in the complex by Western Blot.6. The activity of PI3K was checked by enzyme linked immunosorbent assay (ELISA).7. Real time polymerase chain reaction (Real-time PCR) was adopted to measure mRNA expression of?-arrestin 2 gene?Src gene and Akt2 gene. Results1. Effect of PTF on cellular viability in C2C12 myotubesC2C12 myotubes were incubated with PTF at different dose for 24 hours, cellular viability was negative correlated with dose of PTF. Compared to control group, cellular viability was obviously decreased by over 5.65%(P<0.01) when the dose of PTF was no less than 1.0 g/L, but had no changes (P>0.05) at concentrations between 0.05g/L-1.0g/L.2. Effect of PTF on glucose consumption in C2C12 myotubesC2C12 myotubes were incubated with PTF at different dose (0.1 g/L,0.25 g/L, 0.5 g/L) and different time (8 h,16 h,24 h), PTF increased glucose consumption in an insulin-dependent manner in C2C12 myotubes. Compared to insulin-treated group, only 0.5 g/L PTF could signifcantly improve cellular glucose consumption by 10.62% in the present of insulin (P<0.05) at the time of 8 h; while at the time of 16 h, both 0.25 g/L and 0.5 g/L PTF could obviously increased cellular glucose consumption by 10.61%(P<0.05) and 17.09%(P<0.01) repectively in an insulin-dependent manner; and at the time of 24 h, PTF at all dose remarkably enhanced insulin-treated cellular glucose consumption by 11.60%(P<0.01),12.77%(P<0.01) and 16.23%(P<0.01) respectively.3. Effect of PTF on glucose uptake in C2C12 myotubesC2C12 myotubes were incubated with PTF at different dose (0.1 g/L,0.25 g/L, 0.5 g/L) and different time (8h,16h,24h), PTF increased glucose uptake in an insulin-dependent manner in C2C12 myotubes. Compared to insulin-treated group, only 0.5 g/L PTF could evidently promote cellular glucose uptake by 13.38%in the present of insulin (P<0.05) at the time of 8 h; while at the time of 16 h, both 0.25 g/L and 0.5 g/L PTF could dramaticlly improve cellular glucose uptake by 18.51% (P<0.01) and 34.04%(P<0.01) repectively in an insulin-dependent manner; and at the time of 24 h, PTF at all dose prominently enhanced insulin-treated cellular glucose uptake by 11.41%(P<0.05),20.79%(P<0.01) and 27.36%(P<0.01) respectively.4. Effect of wortmannin and PP2 on glucose uptake induced by PTF in C2C12 myotubesCompared to insulin-treated group, glucose uptake was remarkably reduced (P<0.01) in the presence of insulin in C2C12 myotubes which were incubated with wortmannin (5 uM) for 16 h, an inhibitor of PI3K, but wortmannin couldn't inhibit the improvement in glucose uptake induced by PTF dependent of insulin (P>0.05); in the same way, PP2 (5 uM), the inhibitor of Src, which treated C2C12 myotubes for 16 h, significantly decreased insulin-stimulated glucose uptake (P<0.01) when compared to insulin-treated group, and also inhibited the enhancement in glucoes uptake induced by PTF in the presence of insulin (P<0.01).5. Establishment of insulin resistant model in C2C12 myotubesCompared to normal insulin-treated group, C2C12 myotubes which were incubated with 0.5 mM PA for 16 h, were significantly decreased in both glucose consumption and glucose uptake by 19.29%(P<0.01) and 20.27%(P<0.01) in the presence of insulin; glucoes uptake in C2C12 myotubes cultured with 25 mM glucose and 1?M insulin for 16 h was also markedly reduced (P<0.01) compared to low dose glucose (5 mM)+insulin (1 nM) group.6. Effect of PTF on glucose consumption and 2-DOG uptake in insulin resistant C2C12 myotubesCompared to insulin resistant model in C2C12 myotubes, PTF (0.5 g/L) which treated PA-induced insulin resistant cells for 16 h notably increased glucose consumption (P<0.01) and glucose uptake (P<0.01) in an insulin-dependent manner, which was similar to normal insulin-treated group (P>0.05). In the same way, PTF hardly reversed decrease in glucose uptake in high-glucose-and high-insulin-induced insulin resistant cells (P<0.01).7. Effect of PTF on protein expression of signal molecules in insulin signalingCompared to control group, PTF, with which C2C12 myotubes were incubated for 16 h, prominently increased the protein expression of P-arrestin 2?Src and Akt, which were the 1.65 fold (P<0.01),1.72 fold (P<0.01) and 1.24 fold (P<0.05) of control group respectively, and 1.66 fold (P<0.01),1.58 fold (P<0.01) and 1.49 fold (P<0.01) of insulin-treated group respectively.Besides, compared to insulin-treated group, PTF greatly enhanced respectively the phosphorylation of InsR?at Tyrl 150/1151, Src at Tyr416, Akt at Thr308 and Akt at Ser473 in an insulin-dependent manner (all P<0.01).Upon stimulation by insulin,?-arrestin 2 scaffolded Src and Akt to InsR to form a?-arrestin 2 signal complex, and PTF with which C2C12 myotubes were incubated for 16 h significantly promoted the formation of complex in the presence of insulin.PTF incubated C2C12 myotubes for 16 h had no effect on the protein expression of PI3K-p85 and the activity of PI3K when compared to control group and insulin-treated group repectively (P>0.05). 8. Effect of wortmannin and PP2 on phosphorylation of Akt induced by PTFWortmannin, with which C2C12 myotubes were incubated for 16 h, could remarkably inhibit phosphorylation of Akt at Thr308 and Ser473 in the presence of insulin by 55.95%(P<0.01) and 44.56%(P<0.01) respectively when compared to insulin-treated group, but failed to inhibit the increases in phosphorylation of Akt at Thr308 and Ser473 induced by PTF in an insulin-dependent manner (P>0.05).In the same way, PP2, with which C2C12 myotubes were incubated for 16 h, could also significantly reduce phosphorylation of Akt at Thr308 and Ser473 in an insulin-dependent manner by 51.18%(P<0.01) and 46.83%(P<0.01) respectively when compared to insulin-treated group, and also inhibit the improvement in phosphorylation of Akt at Thr308 and Ser473 induced by PTF in the presence of insulin, by reducing 65.74%(P<0.01) and 66.06%(P<0.01).9. Effect of PTF on gene expression of signal molecules in insulin signalingPTF, with which C2C12 myotubes were incubated for 16 h, could obviously increase the mRNA expression of Src gene and Akt2 gene, which were 1.18 fold (P<0.05) and 1.26 fold (P<0.01) of control group respectively.Conclusion1. PTF increased glucose consumption and glucose uptake in an insulin-dependent manner in C2C12 myotubes with no cytotoxicity.2. The efficacy of PTF enhancing glucose uptake in an insulin-dependent manner in C2C12 myotubes was inhibited by PP2, an inhibitor of Src, but not wortmannin, an inhibitor of PI3K.3. Both high concentration PA and high glucose as well as high insulin could induce C2C12 myotubes to generate IR.4. PTF could reverse IR induced by high concentration PA and high glucose as well as high insulin respectively in C2C12 myotubes.5. PTF enhanced mRNA expression of Src gene and Akt2 gene as well as their protein expression, while PTF increased protein expression of (3-arrestin 2 with no effect on its gene expression.6. PTF improved phosphorylation of InsR(3 at Tyr 1150/115?Src at Tyr416 and Akt at Thr308/Ser473 in an insulin-dependent manner, and promoted?-arrestin 2 to scaffold Akt and Src to InsR to form signal complex in the presence of insulin.7. Phosphorylation of Akt at Thr308/Ser473 induced by PTF could be inhibited by PP2, but not wortmannin.8. PTF had no effect on cellular protein expression of PI3K-p85 and the activity of PI3K regardless of the presence of insulin.In a word, PTF increased glucose consumption and glucose uptake in an insulin-dependent manner in C2C12 myotubes through enhancing the activity of?-arrestin-2-dependent signaling. |
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