Supplementary Fig. 1. DVC infusion: DVC infusion: saline / MK801 /

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1 Supplementry Fig. 1 y 13 y DVC nnul?on ± A-GluN1 shrna / A-MM ± LV-GlyT1 shrna / LV-MM Vsulr theteriz?on Experiment Clmp DVC nnul?on Miroilysis Experiment ivgtt DVC infusion: sline / - min i.v. gluose (.5 g/kg) DVC infusion: DVC infusion: sline / MK1 / sline / / +MK1 / MK1 / 7CKNA +7CKNA -9 min - 9 [3-3 H]-gluose (. µci min -1 ) SST (3 µg kg -1 min -1 ) Insulin (1. mu kg -1 min -1 ) Gluose (s neee) Miroilysis infusion: ECF Reovery Bseline DVC infusion: sline/ -7 min - 3 Gluose uptke (mg /kg/min) Sline MK1 7CKNA A-MM A-GluN1 shrna Plsm gluose (mg/l) 1 1 Bsl n Clmp e Plsm insulin (ng/ml) Sline Bsl n Clmp f Boy weight (kg) Supplementry Figure 1. Metoli effets of hemil inhii?on of DVC GlyT1 in helthy rts. () Experimentl protools for experiments shown in Fig. 1. In intrvenous gluose tolerne tests (ivgtt), infusions of sline or into the DVC ommene t t=-min n were mintine for the uron of the experiment. In lmps experiments, pre-infusions of sline, MK1, or 7CKNA into the DVC ommene t t=-9min. Infusions of sline, MK1,, +MK1, or +7CKNA into the DVC ommene t t=-min n were mintine for the uron of the lmps. () Experimentl protool for miroilysis stuies shown in Fig. 1i. () Gluose uptke, () sl n lmp plsm gluose levels uring lmps with DVC infusion of sline (n=11), (n=9), MK1 (n=9), +MK1 (n=5), +7CKNA (n=5), A-MM+ (n=5), or A-GluN1 shrna+ (n=5), (e) sl n lmp plsm insulin levels uring DVC infusion of sline (n=) or (n=), n (f) oy weights on the morning of lmp experiments efore DVC infusion of sline (n=11) or (n=9). Dt re shown s the men + SEM.

2 Supplementry Fig. e Gluose infusion rte (mg /kg/min) ICV- sline ICV- Gluose uptke (mg /kg/min) 1 Sline SHAM HVAG * Gluose prou?on (mg /kg/min) f Plsm gluose (mg/ml) * 1 1 Gluose uptke (mg /kg/min) 3 1 Bsl n Clmp Supplementry Figure. Metoli effets of hemil inhii?on of GlyT1 in the th ventrile of mie n in the DVC of hep? vgotomize rts. () Gluose infusion rtes, () rtes of lmp gluose prouon, () gluose uptke, n () lmp plsm gluose levels uring lmp experiments with ICV- th ventrile infusion of (n=5) or sline (n=) in C57BL/ mie; *P<. vs sline etermine y t-test. (e) Gluose uptke, n (f) sl n lmp plsm gluose levels uring lmp experiments with DVC infusion of in vgotomize (n=7) or shm-operte (n=5) rts or DVC infusion of sline in vgotomize (n=7) or shm-operte rts (n=5) rts. Dt re shown s the men + SEM. Plsm gluose (mg/l)

3 Supplementry Fig. 3 i ii iii v Supplementry Figure 3. Brin regions inlue in the GlyT1 protein nlysis. This inlue the DVC region (lue), the leh (L) lterl region (yellow) ontining spinl trigeminl tr. (sp5), spinl 5nu, ul prt (Sp5C), spinl 5nu, interpolr (Sp5I), the right (R) lterl region (purple) ontining sp5, Sp5C, Sp5I n the oiom region (green) ontining pyrmil tr. (py). i: Sggitl imge represenng the rt rin. ii: Vgl tringle overlying the DVC lote in the ul prt of the rin. iiiv: three oronl imges represenng the proximl, meil n istl regions of the ul rin ining ll the regions inlue in the nlysis of the GlyT1 protein. iv

4 Supplementry Fig. 1 Bsl n Clmp. Gluose uptke (mg /kg/min) LV-MM LV-GlyT1 shrna + MK1 Plsm gluose (mg/l) + Boy weight (kg) Supplementry Figure. Metoli effets of moleulr inhii?on of DVC GlyT1 in helthy rts. () Gluose uptke, () sl n lmp plsm gluose levels uring lmps, n () oy weights on the morning of lmp experiments in rts injete with DVC LV-MM (n=7), LV-GlyT1 shrna (n=7), or LV-GlyT1 shrna with DVC MK1 infusion (n=). Dt re shown s the men + SEM.

5 Supplementry Fig. 5 Gluose uptke (mg /kg/min) 3 HFD Sline Glyine 7CKNA Plsm gluose (mg/l) 1 1 Bsl n Clmp Gluose uptke (mg /kg/min) 1 3 HFD Iv DMSO Iv + + Plsm gluose (mg/l) 1 1 Bsl n Clmp + + Supplementry Figure 5. Metoli effets of the DVC n iv infusion of in 3-HFD rts. () Gluose uptke n () sl n lmp plsm gluose levels uring lmps with DVC infusion of sline (n=5), glyine (n=), (n=5), glyine+7ckna (n=5), or +7CKNA (n=5) in 3- HFD rts, () Gluose uptke n () sl n lmp plsm gluose levels uring lmps with iv infusion of % DMSO (n=7) or (n=7) in 3- HFD rts. Dt re shown s the men + SEM.

6 Supplementry Fig. Gluose uptke (mg /kg/min) RC HFD Sline Plsm gluose (mg/l) 1 1 Bsl n Clmp Gluose uptke (mg /kg/min) HFD + + DVC LV-MM DVC LV-GlyT1 shrna Plsm gluose (mg/l) 1 1 Bsl n Clmp + + Supplementry Figure. Metoli effets of hemil n moleulr inhii?on of DVC GlyT1 in oese rts. () Gluose uptke n () sl n lmp plsm gluose levels uring lmps in RC-fe rts with DVC infusion of sline (n=5) or (n=5) n in HFD-fe rts with DVC infusion of sline (n=7) or (n=7), () Gluose uptke n () sl n lmp plsm gluose levels uring lmps in HFD-fe rts injete with DVC LV-GlyT1 shrna (n=) or LV-MM (n=9). Dt re shown s the men + SEM.

7 MM shrna Supplementry Fig GlyT19 GlyT17 MM shrna GlyT19 7 GlyT17 Correspons to Fig. Correspons to Fig MM shrna shrna MM GlyT17 7 GlyT Correspons to Fig. Correspons to Fig. 13 Supplementry Figure 7. Sns of unroppe Western lots. Corresponing to representve imges shown in (): Fig. ; (): Fig. ; (): Fig. ; (): Fig. 7