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. 2012 Sep 13;61(10):2461–2471. doi: 10.2337/db11-1278

FIG. 6.

FIG. 6.

Hypothalamic S6K1 modulates Crh expression via MC4R. A and B: Mice receiving intracerebroventricular injection of adenovirus expressing constitutively active S6K1 (+CAS6K1) or GFP (−CAS6K1), or daily intracerebroventricular injection of 1 μL of 0.1 mmol/L SHU9119 (+SHU) or 1 μL PBS (−SHU) via cannula implantation, were maintained on a control (+leu) or leucine-deficient (−leu) diet for 7 days. Data are means ± SEM of at least two independent experiments (n = 6 for each group). Statistical significance was determined by one-way ANOVA followed by the SNK test. *P < 0.01, for the effect of −leu vs. control diet within the same intracerebroventricular group; #P < 0.01, for the effect of with vs. without SHU or Ad-CAS6K1 under −leu diet. C–G: Primary cultured hypothalamic neurons infected with adenovirus expressing S6K1-specific shRNA (+shS6K1) or control scramble shRNA (−shS6K1) in the absence or presence of 0.1 μmol/L SHU9119 (+SHU) or PBS (−SHU), or infected with Ad-S6K1 (+CAS6K1) or Ad-GFP (−CAS6K1), were then incubated in −leu medium in the absence or presence of 0.3 μmol/L MTII (+MTII) or PBS (−MTII). The titer for ShS6K1 and CAS6K1 in C are from 103 to 107 pfu/60 cm2 cells. Data are representative of at least two independent experiments or are mean ± SEM for at least two independent experiments. Statistical significance was determined by ANOVA followed by the SNK test. *P < 0.05, for the effect of with vs. without Ad-ShS6K1 or Ad-CAS6K1 under the same treatment; #P < 0.05, for the effect of with vs. without Ad-ShS6K1 after SHU treatment in D and E, or for the effect of with vs. without Ad-CAS6K1 after MTII treatment in F and G. A: Hypothalamic Crh mRNA. B: Hypothalamic MC4R and p-S6 proteins (left, Western blot; right, quantitative measurements of MC4R protein relative to actin). C: MC4R protein. D: S6K1 protein (top, Western blot; bottom, quantitative measurements of S6K1 protein relative to actin). E: Crh mRNA. F: p-S6 protein (top, Western blot; bottom, quantitative measurements of p-S6 protein relative to their total protein). G: Crh mRNA.