Fig. 1: IR in tanycytes is necessary for insulin signalling in the ARC.

a, Knockout strategy to inactivate IR specifically in tanycytes. Animals 10–12 weeks old received i.c.v. injection of either control virus AAV-Dio2-GFP or AAV-Dio2-mKate2 (IR^GFP-Tan, IR^mKate2) or Cre-recombinase-carrying virus AAV-Dio2-iCRE-GFP or AAV-Dio2-Cre (IR^ΔTan). b, Representative images of pAKT signal in basal and lateral tanycytes of unstimulated (0 min) and 5 and 10 min post i.v. injection of 0.5 IU kg⁻¹ insulin in NCD-fed control animals IR^GFP-Tan (NCD) (top panel), tanycyte-specific IR KO animals (IR^ΔTan) (NCD) (middle panel) and HFD-fed control animals IR^GFP-Tan (HFD) (lower panel). pAKT was quantified in DAPI-positive tanycyte layer. White and yellow dashed lines indicate quantified ROI in basal and lateral tanycytes, respectively. c,d, Mean intensity of pAKT signal in DAPI-positive nuclei of basal (c) and lateral tanycytes (d). c, 5 min: P(IR^GFP-Tan (NCD) versus IR^ΔTan (NCD)) = 0.047, P(IR^GFP-Tan (NCD) versus IR^GFP-Tan (HFD)) = 0.0041; 10 min: P(IR^GFP-Tan (NCD) versus IR^ΔTan (NCD)) = 0.0144, P(IR^GFP-Tan (NCD) versus IR^GFP-Tan (HFD)) = 0.0205. d, P(IR^GFP-Tan (NCD) versus IR^ΔTan (NCD)) = 0.0994, P(IR^GFP-Tan (NCD) versus IR^GFP-Tan (HFD)) = 0.0151; 10 min: P(IR^GFP-Tan (NCD) versus IR^ΔTan (NCD)) = 0.0031, P(IR^GFP-Tan (NCD) versus IR^GFP-Tan (HFD)) = 0.0309. b–d, n(0 min) = 3/IR^GFP-Tan (NCD), 4/IR^ΔTan (NCD), 4/IR^GFP-Tan (HFD); n(5 min) = 5 mice per group; n(10 min) = 5/IR^GFP-Tan (NCD), 6/IR^ΔTan (NCD), 4/IR^GFP-Tan (HFD). e, Knockout strategy to inactivate IR in endothelial cells. The 10–12-week-old IR^fl/fl Slco1c1-CreERT2^wt/wt and IR^fl/fl Slco1c1-CreERT2^tg/wt littermates received tamoxifen (10 mg d⁻¹, 5 d). f, Representative images of pAKT signal in lectin-positive brain cortices of unstimulated mice (0 min) and 5 min post i.v. injection of 0.5 IU kg⁻¹ insulin in IR^wt/wt and IR^ΔBVEC mice. g, pAKT in lectin-positive microvessels, normalized to IR^wt/wt control animals. P(5 min) = 0.0007. f,g, n(0 min) = 3/IR^wt/wt, 4/IR ^ΔBVEC; n(5 min) = 4/IR^wt/wt, 6/IR ^ΔBVEC (unpaired, two-sided Student’s t-test). h, Representative images of pAKT in the ARC of unstimulated mice (0 min) and 5, 10, 20 and 30 min post i.v. injection of 0.5 IU kg⁻¹ insulin in NCD-fed control animals IR^GFP-Tan (NCD) (top panel), tanycyte-specific IR KO animals IR^ΔTan (NCD) (middle panel) and HFD-fed control animals IR^GFP-Tan (HFD) (lower panel). i, Quantification of pAKT-positive cells per ARC hemisphere. 5 min: P(IR^GFP-Tan (NCD) versus IR^ΔTan (NCD)) = 0.104, P(IR^GFP-Tan (NCD) versus IR^GFP-Tan (HFD)) = 0.0341; 10 min: P(IR^GFP-Tan (NCD) versus IR^ΔTan (NCD)) = 0.0014, P(IR^GFP-Tan (NCD) versus IR^GFP-Tan (HFD)) = 0.0069; 20 min: P(IR^GFP-Tan (NCD) versus IR^ΔTan (NCD)) = 0.007, P(IR^GFP-Tan (NCD) versus IR^GFP-Tan (HFD)) = 0.0023. h,i, n(0 min) = 3/IR^GFP-Tan (NCD), 4/IR^ΔTan(NCD), 4/IR^GFP-Tan (HFD); n(5 min) = 5 mice per group; n(10 min, 20 min, 30 min) = 5/IR^GFP-Tan (NCD), 6/IR^ΔTan(NCD), 4/IR^GFP-Tan (HFD). j, Representative images of pAKT in ARC of unstimulated mice (0 min) and 5 and 15 min post i.v. injection of 0.5 IU kg⁻¹ insulin in tamoxifen-treated IR^wt/wt and IR^ΔBVEC mice. k, Quantification of pAKT-positive cells per hemisphere of ARC in treated IR^wt/wt and IR^ΔBVEC mice. j,k, n(0 min) = 4/IR^wt/wt, 3/IR^ΔBVEC; n(5 min) = 3/IR^wt/wt, 5/IR^ΔBVEC; n(5 min) = 3/IR^wt/wt, 4/IR^ΔBVEC. c,d,i, One-way ANOVA, Tukey post hoc test. Data are represented as the mean ± s.e.m. b,f,h,k, Scale bar, 100 µm. KO, knockout; p.o., per oral; ROI, region of interest.

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