(A) AAV PKC-θ shRNA knocked down expression of PKC-θ in neuronal culture (lanes 1 and 3). (B) Attenuated diet-induced obesity in mice (n = 8–10/group), following bilateral arcuate infusion of 0.5 μl AAV PKC-θ shRNA or scrambled oligo AAV administration (data are mean ± SEM; *P < 0.05 relative to scrambled oligo control for body weight change). (C) Improved response to an i.p. glucose tolerance test (mean ± SEM; n = 8–10/group) in mice on the HFS diet, following AAV PKC-θ shRNA versus scrambled oligo AAV administration (*P < 0.05 relative to scrambled oligo AAV injections). (D) Improved insulin signaling (enhanced p-AKT 30 minutes following a peripheral injection of 5 U/kg) in mice (n = 4–5/group) on the HFS diet, following AAV PKC-θ shRNA versus scrambled oligo AAV administration (data are mean ± SEM; *P < 0.05 relative to control). (E) GFP immunoreactivity in the arcuate nucleus demonstrates transfection and expression of viral proteins at the site of injection. Original magnification, ×10 (left panel); ×20 (right panel). (F) Western blot analysis demonstrates a significant reduction (*P < 0.05) in PKC-θ protein levels, 2.5 weeks after AAV PKC-θ shRNA administration, only in the arcuate after AAV PKC-θ shRNA versus scrambled oligo AAV administration (quantification [mean ± SEM] from 2 separate Western blots; n = 4–5/group), (G) with no changes in PKC-θ in the remaining hypothalamus (quantification [mean ± SEM] from 2 separate Western blots; n = 4–5/group). (H and I) PKC-δ, a PKC isoform with high sequence homology to PKC-θ was not affected by viral injections (quantification [mean ± SEM] from 2 separate Western blots; n = 4–5/group). (J) The AAV PKC-θ shRNA versus scrambled oligo AAV administration had no effect on muscle levels of PKC-θ (quantification [mean ± SEM] from 2 separate Western blots; n = 4–5/group).