Effects Of Thymosin Beta4on Tumor Necrosis Factor-α-induced Adipokines Expression And Insulin Resistance In Human Adipocytes

Objectives:(1) To investigate the effects and mechanisms of Thymosin beta4(Tβ4) on tumor necrosis factor (TNF)-α-induced adipokines expression in mature human visceral adipocytes.(2) To investigate the effects of Tβ4on TNF-α-stimulated dysfunction of insulin signal transduction in mature human visceral adipocytes.Methods:(1) The human visceral preadipocytes were induced and differentiated into mature adipocytes, which were determined by the Oil Red O staining.(2) The well-differentiated adipocytes were treated by TNF-α in various concentrations (0.5-20ng/ml) for different periods(6-36h), respectively. The mRNA levels of adiponectin and MCP-1from different groups were measured by RT-PCR assay.(3) Well-differentiated human adipocytes were pretreated with or without various concentrations(50-500ng/ml) of Tβ4for2h, and then were stimulated for12or24h by the addition of10ng/ml TNF-α. The mRNA expression levels of adiponectin and MCP-1were measured by RT-PCR. The protein content of adiponectin and MCP-1in conditioned medium were measured by ELISA.(4) The human adipocytes were treated with TNF-α (10ng/ml) for different periods (30-75min), the levels of NF-κB phosphorylation and NF-κB activation from different groups were detected by western-blot assay.(5) Well-differentiated human adipocytes were pretreated with100ng/ml Tβ4for various periods(2-18h), and then were treated by addition of TNF-α (10ng/ml) for30min. Total proteins, cytosolic proteins and nuclear proteins were extracted, respectively. The levels of NF-κB nuclear translocation and phosphorylation were determined by the western-blot assay.(6) The mature human adipocytes were pretreated with100ng/ml T(34for2h, and were subsequently incubated for24h by adding TNF-α (10ng/ml) or placebo. The intracellular levels of glutathione (GSH), CuZn superoxide dismutase (SOD), and MnSOD were assayed using ELISA Kits.(7) Human adipocytes were pretreated with100ng/ml Tβ4for2h, and were incubated for30min by adding TNF-α (10ng/ml) or placebo. The levels of JNK and IRS-1(Ser307) phosphorylation were assayed by Western-blot assay.(8) The human adipocytes were pretreated with Tβ4(100ng/ml) for2h and were subsequently incubated for48h by adding TNF-α (10ng/ml) or not, then stimulated with100nM insulin or placebo for30min. The levels of AKT activation from various groups were evaluated by the western-blot assay.Results:(1) Almost all human preadipocytes (90-95%) were differentiated into mature adipocytes.(2) TNF-α decreased adiponectin transcription and inceased MCP-1transcription in human adipocytes in a dose and time-dependent fashion.(3) The levels of adiponectin mRNA were higher in all Tβ4+TNF-α groups compared to TNF-α group, however, MCP-1mRNA were lower in all Tβ4+TNF-α groups when compared with that in TNF-α group, indicated that Tβ4could attenuate adipokines expression induced by TNF-α. Then, Tβ4also partially recovered adiponectin secretion and ameliorated the MCP-1secretion stimulated by TNF-a.(4) TNF-α could upregulate the levels of NF-κB phosphorylation and NF-κB activation in human adipocytes.(5) The levels of NF-κB phosphorylation and NF-κB activation were lower in Tβ4+TNF-α groups when compared with that in TNF-α group, suggested that Tβ4efficiently inhibits NF-κB nuclear translocation and phosphorylation induced by TNF-α..(6) Compared with the control group, the amount of GSH and CuZnSOD/MnSOD activity were significantly decreased in TNF-α treatment group. The amount of GSH and activity of CuZnSOD/MnSOD from Tβ4+TNF-α group were significantly higher than TNF-α treatment group, indicating that Tβ4could improve the oxidative stress induced by TNF-α.(7) The leves of IRS-1(Ser307) phosphorylation and JNK phosphorylation were higher in TNF-α group comparted to control group, Tβ4could attenuate TNF-α-induced IRS-1(Ser307) and JNK phosphorylation in human adipocytes.(8) Insulin treatment resulted in increasing AKT phosphorylation. The level of AKT phosphorylation was much lower in TNF-α+Insulin group than insulin group; the level of AKT phosphorylation in Tβ4+TNF-α+Insulin group was much more higher than TNF-α+insulin group. There was no significant difference in the level of AKT phosphorylation among other four without insulin groups.Conclusions:(1) Tβ4could effectively inhibit TNF-α-induced NF-κB activation and oxidative stress, and thus improve the abnormal expression of inflammatory cytokines induced by TNF-α in human adipocytes.(2) Tβ4can downregulate JNK and IRS-1(Ser307) phosphorylation induced by TNF-a in human adipocytes, and thus effectively restore the AKT activation responsed to insulin stimulation after TNF-α treatment, indicating that Tβ4can be able to improve TNF-α-induced insulin resistance.