Volume 27, Issue 1, Pages 101-117.e5 (January 2018) Metformin Alters Upper Small Intestinal Microbiota that Impact a Glucose-SGLT1- Sensing Glucoregulatory Pathway  Paige V. Bauer, Frank A. Duca, T.M. Zaved Waise, Brittany A. Rasmussen, Mona A. Abraham, Helen J. Dranse, Akshita Puri, Catherine A. O’Brien, Tony K.T. Lam  Cell Metabolism  Volume 27, Issue 1, Pages 101-117.e5 (January 2018) DOI: 10.1016/j.cmet.2017.09.019 Copyright © 2017 Elsevier Inc. Terms and Conditions

Cell Metabolism 2018 27, 101-117.e5DOI: (10.1016/j.cmet.2017.09.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Upper Small Intestinal Glucose Sensing Regulates GP (A) Working hypothesis. (B) Experimental procedure and clamp protocol. (C–F) Graphs showing (C) the glucose infusion rate, (D) glucose production, (E) percent suppression of clamp versus basal glucose production, and (F) glucose uptake in rats following upper small intestinal infusion of saline (n = 7), glucose (n = 9), galactose (n = 6), fructose (n = 6), or sucralose (n = 6) during the pancreatic clamps. Data in all graphs represent the mean + SEM; ∗∗∗p < 0.001 versus saline, fructose and sucralose, as assessed by ANOVA with Tukey's post hoc test. See also Figure S1 and Tables S1 and S2. 3-OMG, 3-O-methyl glucose; SGLT1, sodium glucose co-transporter 1; GLP-1, glucagon-like peptide-1; GLP-1R, glucagon-like peptide-1; LV-SGLT1 shRNA, lentiviral SGLT1 shRNA; Ex-9, exendin-9; and SRIF, somatostatin. Cell Metabolism 2018 27, 101-117.e5DOI: (10.1016/j.cmet.2017.09.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Role of SGLT1 in Glucose Sensing (A and B) Graphs showing the glucose infusion rate (A) and glucose production (B) during the clamp of rats that received upper small intestinal saline (n = 7), glucose (n = 9), phlorizin (n = 5), glucose and phlorizin (n = 8), 3-OMG (n = 7), and 3-OMG and phlorizin (n = 6). Data in all graphs represent the mean + SEM; ∗∗∗p < 0.001 versus saline, phlorizin, glucose and phlorizin, and 3-OMG and phlorizin, as assessed by ANOVA with Tukey's post hoc test. (C) Quantitative analysis (top; n = 5 [LV-MM]; n = 5 [LV-SGLT1 shRNA]) and representative western blot (bottom) of SGLT1 protein expression in the upper small intestinal mucosa of rats infected with LV-MM or LV-SGLT1 shRNA after the clamp with a saline infusion. Data represents the mean + SEM; ∗p < 0.05. (D and E) Graphs showing the glucose infusion rate (D) and glucose production (E) of rats infected with upper small intestinal LV-MM or LV-SGLT1 shRNA that received upper small intestinal saline (n = 5, 5) or glucose (n = 6, 6) during the clamp. Data in all graphs represent the mean + SEM; ∗∗∗p < 0.01 versus all other groups, as assessed by ANOVA with Tukey's post hoc test. See also Figure S2 and Table S2. SGLT1, sodium glucose co-transporter 1; 3-OMG, 3-O-methyl glucose; LV-MM, lentiviral mismatch; LV-SGLT1 shRNA, lentiviral SGLT1 shRNA. Cell Metabolism 2018 27, 101-117.e5DOI: (10.1016/j.cmet.2017.09.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Role of GLP-1 in Glucose Sensing (A and B) Graphs showing the glucose infusion rate (A) and glucose production (B) during the clamp of rats that received upper small intestinal infusion of saline (n = 7), glucose (n = 9), MK-329 (n = 6), glucose and MK-329 (n = 6), Exendin-9 (n = 5), or glucose and Exendin-9 (n = 5). Data in all graphs represent the mean + SEM; ∗∗∗p < 0.001 versus saline, MK-329, Exendin-9, and Exendin-9 and glucose, as assessed by ANOVA with Tukey's post hoc test. (C and D) Graphs showing the glucose infusion rate (C) and glucose production (D) during the clamp of RC or 3-day HFD-fed rats that received saline (n = 7, 7) or glucose (n = 9, 5). Data in all graphs represent the mean + SEM; ∗∗∗p < 0.01 versus all other groups, as assessed by ANOVA with Tukey's post hoc test. (E) Portal active GLP-1 levels immediately following the clamps in RC- and HFD-fed rats that received an upper small intestinal infusion of saline (n = 6, 6) or glucose (n = 6, 6). Data in all graphs represent the mean + SEM; ∗p < 0.05 versus all other groups, as assessed by ANOVA with Tukey's post hoc test. (F) Upper small intestinal mucosal mRNA expression of SGLT1 in untreated rats fed RC (n = 6) or HFD (n = 6) and HFD rats pretreated 1 day prior with metformin (n = 5). Data in all graphs represent the mean + SEM; ∗p < 0.05 versus all other groups, as assessed by ANOVA with Tukey's post hoc test. See also Figure S3. Regular chow (RC); high-fat diet (HFD); high-fat diet with metformin pretreatment (HFD + MET). Cell Metabolism 2018 27, 101-117.e5DOI: (10.1016/j.cmet.2017.09.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Metformin Pretreatment Restores Glucose Sensing via SGLT1 (A) Experimental outline. (B and C) Graphs showing the glucose infusion rate (B) and glucose production (C) of HFD rats pretreated with saline or metformin (1 day prior) that received upper small intestinal saline or glucose. Saline-pretreated rats with saline infusion (n = 6), saline-pretreated rats with glucose infusion (n = 6), metformin-pretreated rats with saline infusion (n = 6), and metformin-pretreated rats with glucose infusion (n = 9). Data in all graphs represent the mean + SEM; ∗∗∗p < 0.001 versus metformin-pretreated rats with saline infusion, as assessed by t-test. (D) The mRNA expression of SGLT1 in the upper small intestinal mucosa of LV-MM (n = 5) or LV-SGLT1 shRNA (n = 5) rats that received metformin pretreatment and an upper small intestinal glucose infusion during the clamp. Data represents the mean + SEM; ∗p < 0.05. (E and F) Graphs showing the (E) glucose infusion rate and (F) glucose production of HFD rats injected with LV-MM (n = 5) or LV-SGLT1 shRNA (n = 7) that received metformin pretreatment and upper small intestinal glucose infusion during the clamp. Data represent the mean + SEM; ∗∗∗p < 0.001. See also Figure S4. USI, upper small intestine; LV, lentivirus; HFD, high-fat diet; SGLT1, sodium glucose co-transporter 1; LV-MM, lentiviral mismatch; and LV-SGLT1 shRNA, lentiviral SGLT1 shRNA. Cell Metabolism 2018 27, 101-117.e5DOI: (10.1016/j.cmet.2017.09.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Metformin Alters Microbiota (A) Principal coordinate analysis (PCoA) of Bray-Curtis distances between RC, HFD, and HFD+MET samples. The percentage of variation explained by the plotted principal coordinates is indicated in the axis labels. Each dot represents an upper small intestinal community from one rat. (B) Relative abundance of genera that are significantly altered by HFD feeding and/or metformin treatment in the upper small intestinal microbiota of RC, HFD, and HFD+MET rats (expressed as a fraction of the total upper small intestinal community). Data represent the mean + SEM; ∗padj < 0.05, ∗∗∗padj < 0.005 RC versus HFD; #padj < 0.05 HFD versus HFD + MET; $$$padj < 0.005 RC versus HFD + MET. (C) Heatmap of the relative abundance at the species level. Each row corresponds to one sample. ∗padj < 0.05 RC versus HFD; #padj < 0.05 HFD versus HFD + MET; $padj < 0.05 RC versus HFD + MET. See also Figure S5 and Table S3. Regular chow (RC), n = 8; high fat diet (HFD), n = 6; high fat diet pretreated with metformin (HFD + MET), n = 6. Cell Metabolism 2018 27, 101-117.e5DOI: (10.1016/j.cmet.2017.09.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 Transplant of Upper Small Intestinal Microbiota from Metformin-Treated Rats Restores Glucose Sensing via SGLT1 (A and B) Working hypothesis (A) and experimental outline (B) of microbiota transplant. (C and D) Graphs showing the glucose infusion rate (C) and glucose production (D) of HFD rats transplanted with saline- or metformin-treated upper small intestinal microbiota and given a saline or glucose upper small intestinal infusion during the clamp (1 day following the microbiota transfer). Saline-infused rats receiving saline-treated microbiota (n = 6), glucose-infused rats receiving saline-treated microbiota (n = 6), saline-infused rats receiving metformin-treated microbiota (n = 6), and glucose-infused rats receiving metformin-treated microbiota (n = 10). Data represent the mean + SEM; ∗∗∗p< 0.001 versus HFD rats transplanted with metformin-treated upper small intestinal microbiota and given a saline upper small intestinal infusion, as assessed by t-test. (E) Portal active GLP-1 levels immediately following the clamps in HFD rats transplanted with saline- or metformin-treated upper small intestinal microbiota and given a glucose upper small intestinal infusion during the clamp (n = 6 and 7, respectively). Data represent the mean + SEM; ∗p < 0.05. (F) The mRNA expression of SGLT1 in the upper small intestinal mucosa of HFD rats transplanted with saline- or metformin-treated microbiota following the clamp (n = 6). Data represent the mean + SEM; ∗p < 0.05. (G) Glucose production of HFD rats injected with LV-MM or LV-SGLT1 shRNA that were subsequently transplanted with metformin-treated microbiota and given a glucose upper small intestinal infusion (n = 6 and 7, respectively). Data represent the mean + SEM; ∗∗p < 0.01. See also Figure S6. USI, upper small intestine; SGLT1, sodium glucose co-transporter 1; GLP-1R, glucagon-like peptide-1 receptor; LV, lentivirus; HFD, high-fat diet; LV-MM, lentiviral mismatch; LV-SGLT1 shRNA, lentiviral SGLT1 shRNA. Cell Metabolism 2018 27, 101-117.e5DOI: (10.1016/j.cmet.2017.09.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 Metformin-Treated Microbiota Transplant Alters Microbiota (A) Principal coordinate analysis (PCoA) of Bray-Curtis distances between samples from HFD rats transplanted with saline-treated microbiota and metformin-treated microbiota. The percentage of variation explained by the plotted principal coordinates is indicated in the axis labels. Each dot represents an upper small intestinal community from one rat. (B) Relative abundance of genera that are significantly altered in HFD rats transplanted with metformin-treated microbiota, relative to HFD rats transplanted with saline-treated microbiota (expressed as a fraction of the total upper small intestinal community). See also Figure S7 and Table S4. Data represent the mean + SEM; ∗padj < 0.05, ∗∗padj < 0.01, ∗∗∗padj < 0.005. (C) Heatmap of the relative abundance at the species level. Each row corresponds to one sample. HFD rats transplanted with saline-treated upper small intestinal microbiota (HFD + SAL-M), n = 8; HFD rats transplanted with metformin-treated upper small intestinal microbiota (HFD + MET-M), n = 6. ∗padj < 0.05. Cell Metabolism 2018 27, 101-117.e5DOI: (10.1016/j.cmet.2017.09.019) Copyright © 2017 Elsevier Inc. Terms and Conditions