Resveratrol activates duodenal Sirt1 to reverse insulin resistance in rats through a neuronal network

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1 Resveratrol activates duodenal Sirt1 to reverse insulin resistance in rats through a neuronal network Clémence D. Côté, Brittany A. Rasmussen, Frank A. Duca, Melika Zadeh-Tahmasebi, Joseph A. Baur, Mira Daljeet, Danna M. Breen, Beatrice M. Filippi & Tony K.T. Lam Supplementary Table 1. Diet content of the regular chow and the lard-oil enriched high fat diet.

2 Supplementary Table 2. Plasma insulin and glucose concentrations of the groups receiving duodenal and/or MBH infusions during the basal and clamp conditions. Chow + saline (N = 7) HFD + saline (N = 10) HFD + resveratrol (N = 6) HFD + SIRT1 shrna + resveratrol (N = 6) HFD + resveratrol + EX527 (N = 5) HFD + resveratrol + tetracaine (N = 5) HFD + MBH insulin + duodenal resveratrol (basal clamp) (N = 6) Basal Insulin (ng/ml) 0.9 ± ± ± ± ± ± ± 0.3 Glucose (mm) 6.7 ± ± ± ± ± ± ± 0.3 Clamp Insulin (ng/ml) 2.4 ± ± ± ± ± ± ± 0.2 Glucose (mm) 6.8 ± ± ± ± ± ± ± 0.4! Data are means ± SEM (Basal: min; Clamp: ). HFD, high fat diet; MBH, mediobasal hypothalamus.

3 Supplementary Table 3. Fat pad and relative fat pad mass in 28 d chow-fed and HFD-fed rats. Fat pad Chow HFD Epididymal (g) 5.3 ± ± 0.9 Retroperitoneal (g) 4.2 ± ± 1.2* Visceral (g) 3.1 ± ± 0.9* Total (g) 12.6 ± ± 2.8* Relative fat pad mass (%) * (*P < 0.05 vs. chow) Data are mean ± SEM

4 Supplementary Fig. 1 Supplementary Figure 1 Schematic diagram of the working hypothesis. Intraduodenal resveratrol activates a duodenal Ampk! Sirt1 pathway to trigger a vagal gut-brain neuronal axis to remotely reverse hypothalamic insulin resistance.

5 Supplementary Fig. 2 Supplementary Figure 2 Intraduodenal resveratrol infusion improves insulin sensitivity via duodenal Sirt1 without affecting the rate of glucose uptake, and Sirt1 knockdown is restricted to the duodenum. (a,b) HGP percent suppression from basal (a) and glucose uptake (b) during the hyperinsulinemic euglycemic clamp in normal chow-fed rats with intraduodenal saline (n = 7) or DMSO (n = 5), and HFD-fed rats with intraduodenal saline (n = 10) or resveratrol (n = 7), or i.v. resveratrol (n = 5) (c,d,e) Sirt1 protein expression normalized to β actin and representative western blots of two total western blots in the jejunum, ileum, and liver of 3 d duodenal LVmismatch (n = 6) or LV-Sirt1 shrna (n = 10) injected rats (determined by unpaired t-test). (f,g) HGP percent suppression from basal (f) and the rate of glucose uptake (g) during the hyperinsulinemic euglycemic clamp in HFD-fed injected with 3 d intraduodenal LVmismatch with intraduodenal saline (n = 5) or resveratrol (n = 6) or LV-Sirt1 shrna with intraduodenal saline (n = 5) or resveratrol (n = 6). (h,i,j) The glucose infusion rate (h), HGP percent suppression from basal (i) and the rate of glucose uptake (j) during the hyperinsulinemic euglycemic clamp in normal chow-fed rats injected with either intraduodenal LV-mismatch (n = 5) or LV-Sirt1 shrna (n = 5) for 14 d (h,i,j, *P < 0.05, **P < determined by unpaired t-test). Values are shown as mean ± s.e.m. Data analyzed by one-way ANOVA with Tukey s post hoc test unless otherwise noted. **P < 0.01

6 Supplementary Fig. 3 Supplementary Figure 3 Duodenal resveratrol activates duodenal Sirt1, Ampk, and Pka to improve insulin sensitivity independent of changes in glucose uptake. (a) Duodenal mucosal NADH levels in saline (n = 6) or resveratrol (n = 7) treated HFD-fed rats. (b,c,d) The glucose infusion rate (b) HGP percent suppression from basal (c) and the rate of glucose uptake (d) during the hyperinsulinemic euglycemic clamp in normal chow- and HFD-fed rats infused with intraduodenal saline (n = 7, 10) or with EX527 alone (n = 5, 5), and HFD-fed rats infused with resveratrol alone (n = 7) or EX527 and resveratrol (n = 5). (e) Left: Representative western blot of one total western blot and quantitative analysis of pampk protein expression normalized to Ampk in HEK293 cells treated with DMSO (n = 5) or 1 h resveratrol (n = 5) Right: Ampk activity in HEK293 cells treated with DMSO (n = 5) or 1 h resveratrol (n = 5) (*P < 0.05, **P < determined by unpaired t-test). (f,g,h) The glucose infusion rate (f) HGP percent suppression from basal (g) and glucose uptake (h) during the hyperinsulinemic euglycemic clamp in normal chow-fed rats infused with intraduodenal saline (n = 7) or compound C (n = 5) and in HFD-fed rats infused with intraduodenal saline (n = 10), compound C (n = 5), resveratrol (n = 7), resveratrol with compound C (n = 6), SRT1720 (n = 5), and SRT1720 with compound C (n = 7). (i,j) The glucose infusion rate (i) HGP percent suppression from basal (j, left) and glucose uptake (j, right) during the hyperinsulinemic euglycemic clamp in normal chow-fed rats with intraduodenal saline (n = 7) or with Rp-CAMPS alone (n = 5) and in HFD-fed rats with saline (n = 10) or with resveratrol (n = 7) or with Rp-CAMPS (n = 6) or with resveratrol + Rp-CAMPS (n = 5). Values are shown as mean ± s.e.m. Data analyzed by one-way ANOVA with Tukey s post hoc test unless otherwise noted. ** P < 0.01; *P < 0.05

7 Supplementary Fig. 4 Supplementary Figure 4 Intraduodenal resveratrol requires a vagal gut-brain neuronal axis to remotely improve hypothalamic insulin sensitivity to lower GP, independent of changes in glucose uptake. (a,b) HGP percent suppression from basal (a) and glucose uptake (b) during the hyperinsulinemic euglycemic clamp in normal chow-fed rats with intraduodenal infusion of either saline (n = 7) or tetracaine alone (n = 5) and in HFD-fed rats with intraduodenal infusion of saline (n = 10), resveratrol (n = 7) or tetracaine (n = 5) or resveratrol + tetracaine (n = 5) or resveratrol + NTS infusion of either saline (n = 5) or MK801 (n = 5), or resveratrol after Sham (n = 5) or HVAG (n = 6) (c,d) HGP percent suppression from basal (c) and the glucose uptake (d) during the pancreatic (basal insulin) euglycemic clamp with MBH insulin and duodenal saline in normal chow-fed rats (n = 7), and MBH insulin and duodenal saline (n = 5), MBH saline and duodenal resveratrol (n = 5), MBH insulin and duodenal resveratrol (n = 6), MBH S961 (n = 5) or insulin + S961 (n = 6) and duodenal resveratrol in HFD-fed rats. Values are shown as mean ± s.e.m. Data analyzed by one-way ANOVA with Tukey s post hoc test. ** P < 0.01

8 Supplementary Fig. 5 Supplementary Figure 5 Intraduodenal resveratrol improves insulin sensitivity in 28 d HFD-fed obese insulin resistant rats. (a,b) Body weight gain (a) and percent body weight gain (b) of 28 day chow-fed (n = 5) and HFD-fed (n = 6) rats (*P < 0.05 determined by unpaired t test at each time point). (c,d) HGP percent suppression from basal (c) and the rate of glucose uptake (d) during the hyperinsulinemic euglycemic clamp in 28 d normal chow-fed rats with intraduodenal saline (n = 5) and 28 d HFD-fed rats with intraduodenal saline (n = 8) or resveratrol (n = 6). Values are shown as mean ± s.e.m. Unless noted, data analyzed by one-way ANOVA with Tukey s post hoc test. **P < 0.01