We evaluated GK expression in total hypothalamic protein extracts at 48 h post-transduction using Western blot analysis of hypothalamic samples from rats injected with Ad-shβGal-EGFP or Ad-shGK-EGFP. As shown in Fig. 4A, GK expression was found to be significantly decreased (75%) in rats injected with AdshGK-EGFP with respect to Ad-shβGal-EGFP-injected rats. We also evaluated whether GLUT2 expression, which is also present in tanycytes12, was altered by GK inhibition. We detected no significant differences in protein levels between both animal groups, demonstrating the specificity of the GK shRNA (Fig. 4B). Similarly, GKRP, which is also expressed in tanycytes15, was not affected by GK inhibition (Fig. 4C). EGFP was detected at similar levels in rats transduced with both adenoviruses (Fig. 4D). The intensity of the bands was normalized to β-actin and expressed as a percentage of the ratio obtained with the control. Thus, only GK expression was significantly decreased following injection of the adenovirus carrying the shGK-RNA, and no compensatory effects on GLUT2 or GKRP expression occur under these conditions. Finally, immunohistochemistry analysis in the basal hypothalamus in both Ad- shGK and Ad-shβgal using anti-GK and anti-vimentin antibodies demonstrated a minor degree of GK fluorescence in GK knockdown animals. In these animals, GK-associated fluorescence was only decreased in vimentin-positive cells; no differences were detected in AN neurons (Supplementary Fig. S3).Figure 4GK inhibition 48 h after AdshGK injection into the 3V. (A) Western blot assays of hypothalamic protein extracts in control (Ad-shβGal-injected rats) and Ad-shGK-injected rats using anti-GK, anti-GLUT2, anti-GKRP, and anti-GFP (transduction control) antibodies. An anti-β-actin antibody was used as loading control. Images are representative of three different experiments. (B) Densitometric analysis of each protein relative to β-actin evaluated in Ad-shGK-injected rats, as a percentage of this protein in Ad-shβgal-EGFP-injected rats. Statistics: t-test with Welch correction *p < 0.05.