Figure 4. Sustained activation of AMPKα and reduction of CCR5 evokes S6 kinase activity and insulin responsiveness.

(a) CCR5^−/− hypothalamic neurons have higher AMPKα activity–phosphor-AMPKα^T172 in culture (N = 4). (b) Insulin stimulation evoked a transient increase of AMPKα activation in WT hypothalamic neurons (N = 3 in each groups). (c) Reduction of CCR5 by shRNA in N2A cells and control N2A cells were treated with AICAR or Compound C (CC) for 15 min. The activities of AMPKα, S6K, and IRS-1, CCR5 and GLUT4 expression levels were analyzed by protein blot. The phosphorylation of IRS-1^T612 was reduced in CCR5 knockdown cells whereas p-IRS-1^S302 and p-S6K^T421 were increased. S6K activity was not affected by AICAR treatment but was reduced by Compound C. (d) N2A cells treated with either AICAR or by overexpression AMPKα and AMPKα-TD (AMPKα-T172D) had higher levels of phospho-AMPKαT172. (d, e) The activation of IRS-1/2 by insulin was diminished in cells overexpressing AMPKα and AMPKα-TD, and improved by CC. (d, f) S6 kinase activity was enhanced by constitutively expressed AMPKα and AMPKα-TD. (N = 3; *p < 0.05; **p < 0.01 compare to control; ^#p < 0.05, compared to AMPK expressing cells. NS: not significant difference).