Lactobacillus gasseri in the Upper Small Intestine Impacts an ACSL3-Dependent Fatty Acid-Sensing Pathway Regulating Whole-Body Glucose Homeostasis

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1 Article Lactobacillus gasseri in the Upper Small Intestine Impacts an ACSL3-Dependent Fatty Acid-Sensing Pathway Regulating Whole-Body Glucose Homeostasis Graphical Abstract Authors Paige V. Bauer, Frank A. Duca, T.M. Zaved Waise,..., Mozhgan Rasti, Catherine A. O Brien, Tony K.T. Lam Correspondence tony.lam@uhnres.utoronto.ca In Brief Bauer et al. report that a glucoregulatory pre-absorptive ACSL3-dependent fatty acid-sensing pathway in the upper small intestine is compromised by a high-fat diet. Fatty acid sensing and glucose homeostasis are restored by probiotic Lactobacillus gasseri administration or by transplantation of microbiota from chowfed animals. Highlights d Upper small intestinal fatty acid-acsl3 sensing impacts glucose homeostasis d d d HFD decreases Lactobacillus gasseri (LG) and disrupts ACSL3-fatty acid sensing Regular chow microbiota transplant restores LG and ACSL3- lipid sensing Lactobacillus gasseri administration restores ACSL3-lipid sensing in HFD rodents Bauer et al., 18, Cell Metabolism 7, March, 18 ª 18 Elsevier Inc.

2 Cell Metabolism Article Lactobacillus gasseri in the Upper Small Intestine Impacts an ACSL3-Dependent Fatty Acid-Sensing Pathway Regulating Whole-Body Glucose Homeostasis Paige V. Bauer, 1, Frank A. Duca, 1 T.M. Zaved Waise, 1 Helen J. Dranse, 1 Brittany A. Rasmussen, 1, Akshita Puri, Mozhgan Rasti, 1 Catherine A. O Brien,,,5 and Tony K.T. Lam 1,,3,,7, * 1 Toronto General Hospital Research Institute, UHN, MaRS Centre, Toronto Medical Discovery Tower, Room 1-75, 11 College Street, Toronto, ON M5G 1L7, Canada Department of Physiology, University of Toronto, Toronto, ON M5S 1A8, Canada 3 Department of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada Princess Margaret Cancer Centre, UHN, Toronto, ON M5G M9, Canada 5 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5S 1A8, Canada Banting and Best Diabetes Centre, University of Toronto, Toronto, ON M5G C, Canada 7 Lead Contact *Correspondence: tony.lam@uhnres.utoronto.ca SUMMARY Long-chain acyl-coa synthetase (ACSL)-dependent upper small intestinal lipid metabolism activates pre-absorptive pathways to regulate metabolic homeostasis, but whether changes in the upper small intestinal microbiota alter specific fatty acid-dependent pathways to impact glucose homeostasis remains unknown. We here first find that upper small intestinal infusion of Intralipid, oleic acid, or linoleic acid pre-absorptively increases glucose tolerance and lowers glucose production in rodents. High-fat feeding impairs pre-absorptive fatty acid sensing and reduces upper small intestinal Lactobacillus gasseri levels and ACSL3 expression. Transplantation of healthy upper small intestinal microbiota to high-fat-fed rodents restores L. gasseri levels and fatty acid sensing via increased ACSL3 expression, while L. gasseri probiotic administration to nontransplanted high-fat-fed rodents is sufficient to restore upper small intestinal ACSL3 expression and fatty acid sensing. In summary, we unveil a glucoregulatory role of upper small intestinal L. gasseri that impacts an ACSL3-dependent glucoregulatory fatty acid-sensing pathway. INTRODUCTION Glucose intolerance is a risk factor for type diabetes (Roglic and World Health Organization, 1). An elevation of glucose production (GP) by the liver contributes to glucose intolerance and is the result of impaired insulin secretion and action, as well as defects in glucose effectiveness and nutrient-sensing pathways in the gut and brain (Defronzo, 9; Lam, 1; Rossetti et al., 199). Metformin and bariatric surgery are effective therapies that improve glucose tolerance by lowering GP in diabetes (Batterham and Cummings, 1; Hundal et al., ), and the mechanisms are linked to a small intestinal nutrient sensingdependent neuronal network (Breen et al., 1; Duca et al., 15b; Jiao et al., 13). Metformin and bariatric surgery also changes gut microbiota (Forslund et al., 15; Tremaroli et al., 15; Wu et al., 17), suggesting the microbiota interact with intestinal nutrient sensing to impact glucose homeostasis. The gut microbiota rapidly changes in response to a high-fat diet (HFD) (David et al., 1; Turnbaugh et al., 9), and the microbiota regulates body weight and adiposity (Backhed et al., ; Ridaura et al., 13; Turnbaugh et al., ). Despite progress in identifying functional and compositional alterations in the fecal metagenome of type diabetic individuals (Karlsson et al., 13; Qin et al., 1), most studies have overlooked the role of the microbiota residing in the upper small intestine, an important site for nutrient sensing and metabolic regulation (Duca et al., 15a). For example, the transfer of feces from healthy individuals to individuals with metabolic syndrome via a duodenal catheter alters the fecal microbiota of recipients in association with improvements in insulin sensitivity (Vrieze et al., 1). This microbiota transfer could alter the microbial community in the upper small intestine, contributing to this insulin-sensitizing effect. Similarly, pre- and probiotics improve glucose regulation and robustly alter distal intestinal and fecal microbiota of rodents and humans (Balakumar et al., 1; Cani et al., a; Ejtahed et al., 1; Everard and Cani, 13); however, these substances must first pass the upper small intestine where they could elicit their glucoregulatory effects. This is in line with the fact that most probiotics are derived from Lactobacillus (Asemi et al., 13; Balakumar et al., 1; Ejtahed et al., 1; Yadav et al., 7), which is a prominent inhabitor of the small intestine (Gu et al., 13; Wirth et al., 1). The upper small intestine contacts ingested nutrients that initiate negative feedback pathways to regulate glucose homeostasis (Duca et al., 15a). For example, lipid sensing (achieved via Intralipid infusion) in the upper small intestine activates a gutbrain axis to lower GP and plasma glucose levels (Wang et al., 8). This effect is dependent upon formation of long-chain fatty acyl-coenzyme A (CoA) via long-chain acyl-coa synthetase 57 Cell Metabolism 7, , March, 18 ª 18 Elsevier Inc.

3 Figure 1. Upper Small Intestinal Infusion of Intralipid, Oleic Acid, or Linoleic Acid, Increases Glucose Tolerance in RC but Not HFD Rodents (A) Experimental procedure and intravenous glucose tolerance test (i.v.gtt) protocol. (B G) Percent change in plasma glucose levels (inset: integrated area under the curve [AUC]) (B, D, and F) and absolute plasma insulin levels (C, E, and G) during the i.v.gtt in regular chow rats that received an upper small intestinal infusion of saline (n = ), Intralipid (n = ), PBS with bile salts (n = ), oleic acid (n = 8), or linoleic acid (n = ). (H J) Percent change in plasma glucose levels (inset: integrated AUC) during the i.v.gtt in 3-day high-fat diet rats that received an upper small intestinal infusion of saline (n = 7), Intralipid (n = ) (H), PBS + bile salts (n = ), oleic acid (n = ) (I), or linoleic acid (n = ) (J). *p <.5, **p <.1, ***p <.1 determined by t test. Data are shown as the mean ± SEM. See also Table S1 and Figure S1. i.v.gtt, intravenous glucose tolerance test; AUC, area under the curve; HFD, high-fat diet. (ACSL), as general inhibition of ACSL isoforms in the upper small intestine negates lipid sensing (Wang et al., 8). This glucoregulatory lipid-sensing pathway is abolished in HFD rodents (Cheung et al., 9), but whether HFD feeding affects upper small intestinal ACSL expression via changes in microbiota, and whether this postulated change in ACSL expression affects lipid sensing, is unknown. Interestingly, inoculation of germ-free mice with the microbiota of healthy conventionally raised mice alters the expression of hundreds of genes involved in glucose and lipid metabolism in the upper and distal gut (Derrien et al., 11; El Aidy et al., 13), including ACSL expression (El Aidy et al., 13), suggesting that the microbiota could alter intestinal ACSL expression. Based on these findings, we tested the hypothesis that upper small intestinal microbiota alter upper small intestinal ACSL-dependent glucoregulatory fatty acid-sensing pathways in rodents. RESULTS Upper Small Intestinal Fatty Acid-Sensing Mechanisms Increase Glucose Tolerance and Lower GP in Healthy but Not High-Fat-Fed Rodents To begin addressing the potential interaction between the upper small intestinal microbiota and glucoregulatory lipid-sensing pathways, we first investigated whether gut lipid sensing regulates whole-body glucose tolerance under physiological conditions. Intralipid (lipid emulsion) was infused for a total of 5 min into the upper small intestine of conscious, unrestrained, regular chow (RC)-fed rats to activate pre-absorptive lipid-sensing pathways as described previously (Wang et al., 8), and glucose tolerance was assessed with an intravenous glucose tolerance test (i.v.gtt) (Figure 1A). Intralipid versus saline infusion increased glucose tolerance (Figures 1B and S1A) independently of a rise in plasma insulin levels (Figure 1C). To examine the effect of individual fatty acids within Intralipid, oleic acid or linoleic acid (prevalent fatty acids found in human circulation) was dissolved in PBS with bile salts (containing.1% [wt/vol] lecithin and 1% [wt/vol] sodium taurocholate) and infused for a total of 5 min into the upper small intestine during the i.v.gtt. Both oleic acid and linoleic acid improved glucose tolerance (Figures 1D, 1F, S1B, and S1C) independently of a rise in plasma insulin levels (Figures 1E and 1G) when compared with an infusion of PBS with bile salts alone. Next, surgically recovered rats were given a saturated fat-enriched HFD (Table S1) for 3 days, which has previously been shown to induce hyperphagia and hepatic insulin resistance in rodents (Cote et al., 15), before being subjected to the i.v.gtt with a gut infusion of Intralipid, oleic acid, or linoleic acid. Rats exhibited hyperphagia in response to the HFD diet (Figure S1D) but had comparable body weight (Figure S1E), which is consistent with reports documenting that 3-day Cell Metabolism 7, , March,

4 Figure. Upper Small Intestinal Oleic Acid or Linoleic Acid Infusion Lowers GP through a Gut Peptide-Dependent Neuronal Network in RC but Not HFD Rodents (A) Experimental procedure for pancreatic (basal insulin)-euglycemic clamp protocol. (B D) The glucose infusion rate (B), glucose production (C), and glucose uptake (D) during the pancreatic clamps of healthy rats that received an upper small intestinal infusion of saline (n = 1), PBS with bile salts (n = 7), oleic acid (n = 11), or linoleic acid (n = 7). **p <.1 versus saline and PBS with bile salts as determined by ANOVA with Tukey s post hoc test. (E) Glucose production during clamps of healthy rats that received an upper small intestinal infusion of MK-39 (n = 7), Exendin-9 (n = 5), MK-39 + oleic acid (n = 5), Exendin-9 + oleic acid (n = ), MK-39 + linoleic acid (n = 5), or Exendin-9 + linoleic acid (n = 5). **p <.1 versus all other groups as determined by ANOVA with Tukey s post hoc test. (F) Glucose production during clamps of healthy rats that received an upper small intestinal infusion of tetracaine (n = 5), tetracaine + oleic acid (n = 5), and tetracaine + linoleic acid (n = 5). (G) Glucose production during clamps in 3-day high-fat diet-fed rats that received an upper small intestinal infusion of saline (n = 5), oleic acid (n = 5), and linoleic acid (n = 5). Values are shown as mean + SEM. See also Tables S1, S, and Figure S. SRIF, somatostatin; basal, the average GP over the period from to 9 min during the pancreatic (basal insulin)-euglycemic clamp; clamp, the average GP from 18 to min; Ex-9, exendin-9. HFD-induced hyperphagia induces insulin and leptin resistance but does not increase body weight in rodents (Morgan et al., ; Scherer et al., 1; Thaler et al., 1). We found that upper small intestinal infusion of Intralipid, oleic acid, and linoleic acid failed to improve glucose tolerance (Figures 1H, 1I, 1J, S1F, S1G, and S1H) in HFD rats at comparable plasma insulin levels (Figures S1I, S1J, and S1K). Thus, upper small intestinal lipid (specifically oleic acid and linoleic acid) sensing, improves glucose tolerance in healthy but not HFD rodents. To determine whether upper small intestinal fatty acid-sensing regulates glucose tolerance via gut-mediated changes in GP or glucose uptake independent of changes in plasma insulin levels (Table S), oleic acid or linoleic acid were infused for 5 min into the upper small intestine and changes in glucose kinetics were evaluated via tracer dilution methodology under pancreatic basal insulin-euglycemic clamp conditions (Figure A; Table S). During the oleic acid and linoleic acid gut infusion, the exogenous glucose infusion rate increased by 5-fold relative to the vehicle infusion to prevent a drop in plasma glucose levels and to maintain euglycemia (Figure B; Table S). This glucoselowering effect was due to a reduction in GP (Figure C) rather than an increase in glucose uptake (Figure D). Of note, there was no change in plasma or portal free fatty acid levels at the end of the 5 min gut infusion relative to the pre-infused basal condition, as well as between treatment groups (Figure SA), ensuring the effects seen throughout the clamp and i.v.gtt (Figures 1 and S1) are due to pre-absorptive fatty acid sensing in healthy rats. In addition, the ability of oleic acid and linoleic acid to lower GP is conserved in healthy mice during the basal insulin pancreatic clamp (Figures SB SE). Together with the fact that upper small intestinal Intralipid infusion lowers GP (Wang et al., 8), our data collectively indicate that upper small intestinal fatty acid sensing increases glucose tolerance at least in part by lowering GP in rats and mice in vivo. 57 Cell Metabolism 7, , March, 18

5 Increased consumption of oleic acid and linoleic acid has beneficial glucoregulatory effects in rodents and humans (Riserus et al., 9); however, the mechanisms involved have been debated. Although Intralipid infusion into the upper small intestine activates a CCK-1 receptor-dependent pathway to lower GP (Cheung et al., 9), oleic acid stimulates the release of both CCK (Chang et al., ) and GLP-1 (Iakoubov et al., 7) in vitro. While CCK appears to mediate the satiety response to a duodenal infusion of oleic acid (Woltman and Reidelberger, 1995), the glucoregulatory effects of diets high in oleic acid have been attributed to GLP-1 action (Rocca et al., 1). Linoleic acid also stimulates the release of CCK (Shah et al., 1) and GLP-1 (Richards et al., 1) in vitro; however, intestinal linoleic acid infusion lowers food intake through a GLP-1-dependent mechanism (Dailey et al., 11). Thus, to evaluate whether a gut CCK- and/or GLP-1-dependent pathway is necessary for upper small intestinal fatty acid sensing, each fatty acid was coinfused with the CCK-1 receptor inhibitor MK-39 or the GLP-1 receptor (GLP-1R) inhibitor Exendin-9. Co-infusion of oleic acid with either MK-39 or Exendin-9 abolished the ability of an oleic acid 5 min infusion to increase the glucose infusion rate (Figure SF) and decrease GP (Figure E) with no change in glucose uptake (Figure SG) during the pancreatic clamp, suggesting (although warrants future investigation) that oleic acid-induced CCK-1 receptor and GLP-1R effect is additive in the current experimental context. In contrast, co-infusion of MK-39 with linoleic acid did not influence the ability of linoleic acid to decrease GP (Figures E, SF, and SG) compared with MK-39 alone (Figures E, SF, and SG). However, co-infusion of Exendin-9 with linoleic acid negated the ability of linoleic acid to regulate glucose metabolism compared with Exendin-9 alone (Figures E, SF, and SG). Therefore, oleic acid activates CCK-1- and GLP-1R-dependent pathways, while linoleic acid stimulates a CCK-1-independent and GLP-1R-dependent pathway in the upper small intestine to lower GP in healthy rats. The underlying mechanisms responsible for the oleic acid- versus linoleic acidinduced differential effect on the gut peptide-dependent glucoregulatory pathways warrant future investigation. To determine whether a downstream neuronal pathway is necessary for oleic acid and linoleic acid sensing, each fatty acid was co-infused into the upper small intestine with the local anesthetic, tetracaine. Infusion of tetracaine alone had no metabolic consequences but negated the ability of both fatty acids to increase the glucose infusion rate (Figure SH) and lower GP (Figure F) with no effect on glucose uptake (Figure SI), indicating that a gut-brain neuronal network is required for the suppressive effects of these fatty acids on GP. Taken together, oleic and linoleic acids trigger a common GLP-1R-dependent neuronal network to lower GP in healthy rats. To address the pathological relevance of these GP-lowering pathways, oleic acid or linoleic acid were infused into the upper small intestine of HFD rats. These HFD rats were hyperphagic relative to their RC counterparts (Figure SJ), but were not obese (Figure SK). During the pancreatic clamps, oleic acid and linoleic acid failed to increase the glucose infusion rate (Figure SL) and lower GP (Figure G) compared with RC-fed rats, while glucose uptake remained unaltered (Figure SM). Taken together with the i.v.gtt data, we have validated, via three different fatty acid treatments using two complementary tests, that an HFD disrupts specific upper small intestinal fatty acidsensing pathways, leading to a dysregulation of GP and glucose tolerance. Upper Small Intestinal Microbiota Transplantation from RC-Fed Donors Normalizes the Upper Small Intestinal Microbiota in HFD Recipient Rats A potential link between HFD and impaired upper small intestinal fatty acid sensing could be diet-induced changes in the gut microbiota. Indeed, the gut microbiota rapidly changes in response to an HFD (David et al., 1; Turnbaugh et al., 9), and increasing evidence suggests the gut microbiota plays an important role in glucose homeostasis (Kovatcheva-Datchary et al., 15; Plovier et al., 17; Shin et al., 1; Vrieze et al., 1; Wu et al., 17), as a recent study indicates metformin alters microbiota composition in the upper small intestine to impact a glucose-sensing mechanism that regulates GP as well (Bauer et al., 18). More importantly, conventionalization of germfree mice alters the expression of proteins involved in upper small intestinal lipid sensing, such as CD3, FABP, and ACSL (Derrien et al., 11; El Aidy et al., 13). Thus, we investigated whether an upper small intestinal microbiota transplant from RC-fed donors could normalize the upper small intestinal microbiota in HFD-recipient rats, and whether the change in upper small intestinal microbiota of transplanted HFD rats could rescue fatty acid-sensing mechanisms. We first characterized the microbiota composition in the upper small intestine of RC and HFD rats, as well as HFD rats receiving an upper small intestinal RC microbiota transplant. To ensure the microbiota transplants were targeted to the upper small intestine, upper small intestinal luminal contents of the donor rats were transplanted into recipient rats by slowly administering the transplanted material over the course of 3 s via the upper small intestinal cannula. RC, HFD, and HFD rats receiving an upper small intestinal RC microbiota transplant received a gut saline infusion and were sacrificed following the pancreatic clamp studies. The luminal contents were collected from the upper small intestine (between and 15 cm distal to the pyloric sphincter), and the variable region 3 (V3) of the bacterial 1S rrna gene was amplified by PCR and sequenced using an Illumina MiSeq platform (Table S3). Of note, the luminal contents used for bacterial transplantation and sequencing were from the same region of the small intestine. Principal coordinate analysis of weighted UniFrac distance between the upper small intestinal samples from each group showed a clear separation between RC and HFD communities (Figure 3A), while samples from HFD rats that received an upper small intestinal RC microbiota transplant clustered with RC samples and were well separated from HFD samples (Figure 3A). Analysis at the phylum level indicated that the upper small intestinal microbiota in all three groups was dominated by two phyla: Firmicutes and Proteobacteria (Figure 3B). The high abundance of Firmicutes in these groups (especially in the RC or the HFD rats transplanted with RC microbiota) is consistent with studies characterizing the upper small intestinal microbiota in RC mice (Gu et al., 13) and RC Wistar rats (Wirth et al., 1), as well as in the cecum and feces of RC Sprague-Dawley rats (Di Luccia et al., 15; Liu et al., 15). HFD increased the relative abundance of Proteobacteria and reduced relative abundance of Cell Metabolism 7, , March,

6 Figure 3. HFD-Induced Changes in the Upper Small Intestinal Microbiota Can Be Reversed with a Microbiota Transplant from RC Rats (A) Principal coordinate analysis of weighted UniFrac displays separation between samples from RC rats, HFD rats, and HFD rats that received an upper small intestinal RC microbiota transplant (HFDwRCM). 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 at the phylum level in the upper small intestinal community of RC, HFD, and HFDwRCM rats. Each column corresponds to one sample. (C) Relative abundance of families that are significantly altered by HFD feeding and restored with the upper small intestinal RC microbiota transplant (expressed as a percent of the total upper small intestinal community). **p <.1, ***p <.1 HFD versus all other groups, as assessed by ANOVA with Tukey s post hoc test. Values are shown as the mean + SEM. (D) Relative abundance at the genus level in the upper small intestinal community of RC, HFD, or HFDwRCM rats. Each column corresponds to one sample. (E) Heatmap of the relative abundance of species from the Lactobacillus genus. Each row corresponds to one sample. **p <.1 HFD versus all other groups, as assessed by ANOVA with Tukey s post hoc test. See also Table S3 and Figure S3. RC, regular chow (n = ); HFD, high-fat diet (n = ); HFDwRCM, high-fat diet with RC microbiota transplant (n = 5). Firmicutes compared with RC rats (Figure 3B and Table S3, as identified by LefSE and assessed by ANOVA). At the family level, HFD versus RC reduced Lactobacillaceae and modestly yet significantly increased Clostridiaceae (both Lactobacillaceace and Clostridiaceae are from the Firmicutes Phylum) (Figure 3C; Table S3). At the genus level, and consistent with previous literature (Almiron et al., 13), the upper small intestinal microbiota of RC rats was dominated by Lactobacillus (Figure 3D; Table S3). However, the abundance of Lactobacillus was reduced in response to HFD (Figure 3D; Table S3). Importantly, analysis (as identified by LefSE and assessed by ANOVA) of the upper small intestinal luminal contents of HFD rats transplanted with RC microbiota revealed that HFD-induced changes in the upper small intestinal microbiota at the phylum (Firmicutes and Proteobacteria) (Figure 3B; Table S3), family (Lactobacillaceae and Clostridiaceae) (Figure 3C; Table S3), and genus (Lactobacillus) (Figure 3D; Table S3) levels were all reversed to RC conditions with transplantation of upper small intestinal RC microbiota. We next performed a detailed characterization of the species within the Lactobacillus genus and found that HFD versus RC reduced the abundance of Lactobacillus (L.) gasseri and increased L. animalis, while the effect of HFD on L. gasseri and L. animalis were abolished in HFD rats transplanted with upper small intestinal RC microbiota (Figure 3E; Table S3). Of note, 57 Cell Metabolism 7, , March, 18

7 Figure. Transplantation of Upper Small Intestinal RC Microbiota Restores Fatty Acid-Sensing Pathways that Increase Glucose Tolerance in HFD Rodents (A) Experimental procedure of the microbiota transplant protocol. (B and C) Percent change in plasma glucose levels (inset: integrated AUC) (B) and absolute plasma insulin levels (C) during the i.v.gtt in HFD rats that received an RC upper small intestinal microbiota transplant and an upper small intestinal saline (n = ) or Intralipid (n = ) infusion and HFD rats that received an HFD upper small intestinal microbiota transplant and an upper small intestinal saline (n = ) or Intralipid (n = ) infusion. (D) Percent change in plasma glucose levels (inset: integrated AUC) during the i.v.gtt in HFD rats that received heat-shock (n = ) or filtered (n = 5) RC upper small intestinal microbiota and an upper small intestinal Intralipid infusion. (E) Percent change in plasma glucose levels (inset: integrated AUC) during the i.v.gtt in HFD rats that received a colonic RC microbiota transplant and an upper small intestinal saline (n = 5) or Intralipid (n = 5) infusion. *p <.5, **p <.1 HFD + RCM + Intralipid versus all other groups, as assessed by ANOVA with Tukey s post hoc test. Values are shown as mean ± SEM. See also Figure S. IVGTT, intravenous glucose tolerance test; RC, regular chow; HFD, high-fat diet. all changes in the microbiota among the three groups occurred in the context of comparable bacterial diversity (Figure S3). Thus, HFD-induced changes in upper small intestinal microbiota composition can be reversed in HFD rats with a direct upper small intestinal RC microbiota transplant. Upper Small Intestinal Microbiota Transplantation from RC-Fed Donors Restores Upper Small Intestinal Fatty Acid-Sensing Mechanisms in HFD-Recipient Rats Given that an HFD disrupts selective upper small intestinal fatty acid-sensing pathways and glucose homeostasis (Figures 1 and ) in parallel to changes in upper small intestinal microbiota composition (Figure 3), and that an upper small intestinal RC microbiota transplant is sufficient to normalize upper small intestinal microbiota in HFD rats (Figure 3), we next assessed the glucoregulatory relationship between the gut microbiota and fatty acid-sensing mechanisms in the transplanted rats (Figure A). We found that upper small intestinal infusion of Intralipid versus saline improved glucose tolerance in HFD rats that received an upper small intestinal RC microbiota transplant (Figure B), independent of changes in plasma insulin levels (Figure C), and this improvement in glucose tolerance recapitulated the gut lipidsensing effect seen in RC rats without a transplant (Figures 1B and S1A). To ensure the transplant procedure per se did not improve glucose tolerance, the microbiota of HFD rats was transferred to HFD recipients and we found that gut Intralipid versus saline failed to improve glucose tolerance in these rats (Figure B). These data demonstrate that RC microbiota restores lipid sensing to regulate glucose homeostasis in HFD rats. To ensure the detected changes in gut lipid sensing were due to the transfer of microbiota rather than metabolites found within the luminal contents, the upper small intestinal microbiota was either heat killed (Giannakis et al., 9; Kovatcheva-Datchary et al., 15) or removed via filtering (Gareau et al., 11) before the transplant. Transfer of heat-killed or filtered luminal contents of RC rats failed to restore gut lipid sensing in HFD-recipient rats (Figure D), illustrating that restoration of upper small intestinal lipid sensing in HFD rats transplanted with RC upper small intestinal luminal contents is dependent upon the transfer of live bacteria. Changes in the distal intestinal microbiota correlate with improved glucose regulation following microbiota manipulation with pre- and probiotics (Balakumar et al., 1; Cani et al., b; Ejtahed et al., 1). Therefore, to assess whether changes in the distal gut microbiota are responsible for the improvements in upper small intestinal glucoregulatory lipidsensing pathways following upper small intestinal RC microbiota transplantation, we transplanted the upper small intestinal microbiota of healthy RC rats directly into the colon of HFD rats that will receive upper small intestinal Intralipid infusion during the i.v.gtt the following day. Intralipid versus saline infusion failed to improve glucose tolerance in HFD rats receiving a colon RC microbiota transplant (Figure E), indicating the restoration of upper small intestinal lipid sensing in HFD rats transplanted with upper small intestinal RC microbiota is reliant on the normalization of the upper small intestinal microbiota, and not the microbiota in the lower intestinal tract. To further address whether HFD microbiota per se is sufficient to disrupt glucoregulatory lipid-sensing pathways, upper small intestinal microbiota obtained from 3-day HFD rats was transplanted into RC rats receiving gut saline or Intralipid infusion during the i.v.gtt (Figure SA). Intralipid failed to improve glucose tolerance relative to a saline infusion in these rats (Figure SB), independent of changes in plasma insulin levels (Figure SC), while upper small intestinal Intralipid versus saline infusion for 5 min improved glucose tolerance in RC rats transplanted Cell Metabolism 7, , March,

8 Figure 5. Upper Small Intestinal Microbiota Alters Fatty Acid-Sensing Pathways that Impact GP Regulation (A C) The glucose infusion rate (A), glucose production (B), and glucose uptake (C) during the clamps with an oleic acid upper small intestinal infusion in HFD rats that received an HFD microbiota transplant (n = ), HFD rats that received an RC microbiota transplant (n = ), RC rats that received an RC microbiota transplant (n = ), and RC rats that received an HFD microbiota transplant (n = ). (D F) The glucose infusion rate (D), glucose production (E), and glucose uptake (F) during the clamp with a linoleic acid upper small intestinal infusion in HFD rats that received an HFD microbiota transplant (n = ), HFD rats that received an RC microbiota transplant (n = ), RC rats that received an RC microbiota transplant (n = ), and RC rats that received an HFD microbiota transplant (n = ). **p <.1 versus RC + HFDM and HFD + HFDM rats, as determined by ANOVA with Tukey s post hoc test. Values are shown as mean + SEM. RC, regular chow; HFD, high-fat diet; basal, the average glucose production over the period of 9 min during the pancreatic (basal insulin)-euglycemic clamp; clamp, the average glucose production from 18 to min. with RC microbiota (Figure SB), and this elevation was identical to what was seen in RC rats without a transplant receiving an Intralipid infusion (Figures 1B and S1A). Heat-killed or filtered luminal contents of HFD rats failed to impair gut lipid sensing in RC-recipient rats (Figure SD). Taken together, changes in the upper small intestinal microbiota alter lipid-sensing-dependent pathways to impact glucose homeostasis. To next examine whether bidirectional changes in the upper small intestinal microbiota impact a selective glucoregulatory fatty acid-sensing-dependent pathway, microbiota transplant studies were repeated in rats that underwent the gut fatty acid infusion-pancreatic clamp studies the day after transplantation (Figure A). We discovered that RC rats receiving an HFD microbiota transplant exhibited impaired fatty acid sensing, as oleic acid and linoleic acid failed to increase the glucose infusion rate (Figures 5A and 5D) and lower GP (Figures 5B and 5E) relative to RC rats receiving an RC microbiota transplant. HFD rats receiving an RC microbiota transplant responded to oleic acid and linoleic acid infusion, leading to an increased glucose infusion rate (Figures 5A and 5D) and a suppression of GP (Figures 5B and 5E) compared with the disruption in fatty acid sensing seen in HFD rats receiving an HFD microbiota transplant. Glucose uptake was comparable among groups (Figures 5C and 5F). Thus, an RC microbiota transplant restores the microbiota to a healthy condition in HFD rats, leading to the restoration of an 578 Cell Metabolism 7, , March, 18

9 upper small intestinal glucoregulatory fatty acid-sensing-dependent pathway. Upper Small Intestinal Microbiota Alters ACSL3- Dependent Glucoregulatory Fatty Acid-Sensing Pathway The gut microbiota alters the expression of genes involved in lipid metabolism in the upper and lower gut (Derrien et al., 11; El Aidy et al., 13). For example, inoculation of germfree mice alters jejunal expression of ACSL (El Aidy et al., 13). Interestingly, HFD feeding lowers hepatic protein expression of the ACSL isoforms ACSL3 and ACSL (Bowman et al., 1); however, whether this effect occurs in the upper small intestine and whether it is dependent upon diet-induced changes in the upper small intestinal microbiota has not been investigated. Further, whether these postulated gut microbiotainduced changes in ACSL expression impact lipid sensing and subsequent glucose regulation requires consideration. There are five members of the ACSL gene family, numbered 1, 3,, 5, and (Soupene and Kuypers, 8). Studies exploring mrna expression of the genes encoding the five ACSLs in various rat tissues found that all ACSL isoforms are expressed in the small intestine (Bowman et al., 1; Mashek et al., ), with ACSL3 and ACSL5 being the most highly expressed isoforms (Bowman et al., 1). However, the protein expression for the five ACSL isoforms has not yet been examined in the upper small intestinal mucosa. Therefore, to begin evaluating whether HFD-induced changes in the upper small intestinal microbiota alter upper small intestinal fatty acid-sensing pathways via changes in ACSL expression, we first sought to determine which isoforms are expressed in the upper small intestinal mucosa and whether protein expression is altered in response to an HFD with or without an RC microbiota transplant. The upper small intestinal mucosa of RC, HFD, and HFD rats with RC microbiota transplant was collected following the clamp procedure. First, ACSL1,, 5, and were detected in the upper small intestine using brain tissue as a positive control (Figures S5A S5D). Expression of ACSL1,, 5, and was not significantly altered in HFD versus RC rats (Figures S5A S5D). In addition, the expression of these ACSL isoforms in HFD rats that received an upper small intestinal RC microbiota transplant was comparable with both RC and HFD rats (Figures S5A S5D). ACSL3 was also detected in the upper small intestinal mucosa using brain tissue as a positive control and heart as a negative control (Figure A). However, HFD versus RC feeding consistently and significantly reduced ACSL3 protein expression in the upper small intestinal mucosa (Figure A), and this reduction was restored with an upper small intestinal RC microbiota transplant in HFD rats (Figure A). To next investigate whether the upper small intestinal RC microbiota transplant restores lipid sensing via upregulation of ACSL3, we prevented ACSL3 upregulation via injection of lentivirus expressing either ACSL3 small hairpin RNA (shrna) (LV- ACSL3 shrna) or mismatch (LV-MM). LV-ACSL3 shrna or LV-MM was injected into the upper small intestine 3 days prior to the upper small intestinal RC microbiota transplant procedure. The recipient rats underwent the pancreatic clamp studies the following day. A comparable lentiviral shrna injection protocol has been demonstrated to knock down protein specifically in the upper small intestine (Cote et al., 15). We found that upper small intestinal lipid versus saline infusion in LV-MM-injected HFD rats with an RC upper small intestinal microbiota transplant increased the glucose infusion rate (Figure S5E) and lowered GP (Figure B), as reported previously for oleic acid and linoleic acid sensing (Figures 5A, 5B, 5D, and 5E). Importantly, LV-ACSL3 shrna-injected HFD rats that received an upper small intestinal RC microbiota transplant did not respond to upper small intestinal lipid infusion, as the glucose infusion rate (Figure S5E) and GP (Figure B) was unaltered when compared with saline-infused LV-ACSL3 shrna-injected transplanted rats, while glucose uptake was comparable among groups (Figure S5F). This inability of upper small intestinal lipid infusion to lower GP was associated with a significant 5% reduction in protein levels of upper small intestinal ACSL3 in LV-ACSL3 shrna-injected HFD transplanted rats versus LV-MM-injected HFD transplanted rats (Figure C). Since oleic acid- and linoleic acid-sensing pathways in the upper small intestine are dependent upon GLP-1R signaling in RC rats (Figure E), and that upper small intestinal infusion of Intralipid versus saline also failed to increase glucose infusion rate (Figure S5G) and lower GP (Figure S5H) in LV- ACSL3 shrna-injected RC rats, compared with the effects seen in LV-MM-injected RC rats, with no changes in glucose uptake (Figure S5I), we next investigated whether the restoration of upper small intestinal ACSL3-dependent Intralipid sensing is also GLP-1R dependent in HFD rats that received an upper small intestinal RC microbiota transplant. We found that, while infusion of Exendin-9 alone had no metabolic consequences in these transplanted rats (Figures D, S5J, and S5K), co-infusion of Intralipid with Exendin-9 completely abolished the ability of Intralipid to increase the glucose infusion rate (Figure S5J) and lower GP (Figure D) in HFD rats that received an upper small intestinal RC microbiota transplant, with no changes in glucose uptake (Figure S5K). Thus, transplantation of upper small intestinal RC microbiota is sufficient to restore an upper small intestinal ACSL3- and GLP-1R-dependent glucoregulatory lipid-sensing pathway in HFD rats. Of interest, given that inhibition of gut CCK-1 receptor is sufficient to abolish the GP-lowering effect of upper small intestinal Intralipid infusion as well in non-transplanted healthy rats (Cheung et al., 9), we put forward a working hypothesis (although this clearly warrants future investigation) that Intralipid-induced CCK-1 receptor and GLP-1R effect is additive in the current experimental context. Given that lipid sensing in HFD rats transplanted with RC microbiota is GLP-1R dependent, and that nutrients stimulate GLP-1 release via cell depolarization and the opening of voltage-gated Ca + channels (Diakogiannaki et al., 13; Gribble et al., 3; Kuhre et al., 15; Reimann et al., 8), we postulate that a 5% knock down of ACSL3 (Figure C) is sufficient to prevent the threshold for the opening of Ca + channels to be reached, resulting in an absence of GLP-1 release and an inability of lipid sensing to exert glucose control in these rats. This hypothesis is consistent with a study demonstrating that a % knock down of PKCz in the rat ileum inhibits oleic acidinduced GLP-1 release (Iakoubov et al., 11), although the working hypothesis warrants future investigation. Interestingly, upper small intestinal glucose infusion increases portal active Cell Metabolism 7, , March,

10 Figure. Upper Small Intestinal Microbiota Alters an ACSL3-Dependent Fatty Acid-Sensing Pathway to Impact GP Regulation (A) Protein expression of ACSL3 in the upper small intestinal mucosa of RC rats (n = 5), HFD rats (n = 5), and HFD rats transplanted with RC upper small intestinal microbiota (n = 5). Brain tissue was used as a positive control; heart was used as a negative control. b-actin and GAPDH were used as loading controls as b-actin is normally not detected in heart tissue. **p <.1 versus all other groups, as assessed by ANOVA with Tukey s post hoc test. (B) Glucoseproductionduring the clamp ofhfdratsinjected withmismatchoracsl3lentiviralshrnathatreceived anuppersmallintestinalrcmicrobiotatransplant and an upper small intestinal saline (n = 5, 5), or Intralipid (n = 5, 5) infusion. **p <.1 versus all other groups, as assessed by ANOVA with Tukey s post hoc test. (C) Protein expression of ACSL3 in the upper small intestinal mucosa of mismatch (n = ) or ACSL3 lentiviral shrna (n = 5)-injected HFD rats that received an upper small intestinal RC microbiota transplant. Brain tissue was used as a positive control; heart was used as a negative control. b-actin and GAPDH were used as loading controls as b-actin is normally not detected in heart tissue. *p <.5 versus mismatch as determined by t test. (D) Glucose production during the clamp of HFD rats that received an upper small intestinal RC microbiota transplant and a saline (n = ), Intralipid (n =), Exendin-9 (n = 5), or Exendin-9 + Intralipid (n = ) upper small intestinal infusion. **p <.1 versus all other groups, as assessed by ANOVA with Tukey s post hoc test. Values are shown as mean + SEM. See also Figure S5. RC, regular chow; HFD, high-fat diet; HFD + RCM, HFD with RC microbiota transplant; ACSL3, acyl coa synthetase 3; LV-ACSL3 shrna, lentiviral ACSL3 shrna; basal, the average glucose production over the period of 9 min during the pancreatic (basal insulin)-euglycemic clamp; clamp, the average glucose production from 18 to min. GLP-1 levels and lowers GP in RC, but not HFD, rats during the pancreatic clamp conditions, while co-infusion of glucose with Exendin-9 abolishes the glucoregulatory ability of the upper small intestinal glucose infusion in RC rats (Bauer et al., 18). Given that upper small intestinal fatty acid infusion similarly lowers GP through a GLP-1R-dependent pathway in RC rats, but failed to exert a GP-lowering effect in HFD rats (Figures E and G), it is likely that upper small intestinal fatty acid infusion, as conducted in the current study, also stimulates the release of GLP-1 in RC but not HFD rats. Our findings demonstrate that the restoration of upper small intestinal ACSL3 by transplantation of RC upper small intestinal microbiota in HFD rats is necessary to restore a glucoregulatory lipid-dependent pathway. Upper Small Intestinal L. gasseri Administration Restores Upper Small Intestinal ACSL3-Dependent Lipid Sensing in HFD Rats Upper small intestinal L. gasseri (from the Lactobacillus genus) levels were reduced in response to HFD and increased to normal 58 Cell Metabolism 7, , March, 18

11 Figure 7. Upper Small Intestinal Administration of L. gasseri Alters an ACSL3-Dependent Fatty Acid-Sensing Pathway via Inhibition of FXR to Impact GP Regulation (A) Schematic representation of the working hypothesis. (B) Glucose production during the clamp of HFD rats pre-administered with PBS that received an upper small intestinal saline or Intralipid infusion (n = 5, respectively) and HFD rats pre-administered with L. gasseri that received a saline or Intralipid (n =, respectively) upper small intestinal infusion. *p <.5 versus all other groups, as assessed by ANOVA with Tukey s post hoc test. (C) Protein expression of ACSL3 in the upper small intestinal mucosa of RC rats (n = 5), HFD rats administered with PBS (vehicle) (n = 5), HFD rats administered with L. gasseri (n = 5), and HFD rats administered with L. gasseri followed by GW (n = 5). Brain tissue was used as a positive control; heart was used as a negative control. b-actin and GAPDH were used as loading controls as b-actin is normally not detected in heart tissue. *p <.5, **p <.1 versus RC and HFD + LG, as assessed by ANOVA with Tukey s post hoc test. (D) Glucose production during the clamp of HFD rats administered with PBS vehicle with or without GW and infused with Intralipid (n =, respectively), HFD rats administered with L. gasseri that received 1% CMC and a saline or Intralipid infusion (n = 5, respectively), or HFD rats administered with L. gasseri that received GW and a saline or Intralipid infusion (n =, 5, respectively). *p <.5 versus all other groups, as assessed by ANOVA with Tukey s post hoc test. Values are shown as mean + SEM. See also Figure S. FXR, farnesoid X receptor; ACSL3, acyl CoA synthetase 3; LCFA-CoA, long-chain fatty acyl CoA; RC, regular chow; HFD, high-fat diet; LG, L. gasseri; basal, the average glucose production over the period of 9 min during the pancreatic (basal insulin)- euglycemic clamp; clamp, the average glucose production from 18 to min. levels in HFD rats that received an upper small intestinal RC microbiota transplant (Figure 3E; Table S3). This suggests that the increase in upper small intestinal L. gasseri could restore glucoregulatory fatty acid-sensing pathways and improve glucose control in these HFD transplanted rats. L. gasseri exhibits bile salt hydrolase activity (Rani et al., 17), and administration of L. gasseri probiotics alters the bile acid pool and promotes bile acid excretion in rats (Usman and Hosono, 1). In parallel, the bile acid sequestrant colesevelam improves glycemic control in diabetic humans (Beysen et al., 1; Marina et al., 1) and rodents (Prawitt et al., 11; Trabelsi et al., 15) and inhibits intestinal FXR in rodents (Prawitt et al., 11; Trabelsi et al., 15). Inhibition of intestinal FXR per se improves metabolic homeostasis (Jiang et al., 15; Li et al., 13; Prawitt et al., 11; Trabelsi et al., 15) in the presence of increased ACSL in the intestinal mucosa (Li et al., 13), while FXR activation downregulates nutrient-stimulated GLP-1 secretion (Trabelsi et al., 15). These collective and correlative findings raise the possibility that L. gasseri enhances lipid sensing via FXR inhibition and subsequent upregulation of ACSL3 expression in the upper small intestine (Figure 7A). Of note, L. animalis was also changed in our experimental context (Figure 3E; Table S3). However, given that the change in L. animalis was in the opposite direction as L. gasseri and that, to the best of our knowledge, studies to date have not documented a role for L. animalis in bile acid homeostasis, we here first tested whether L. gasseri is sufficient to restore upper small intestinal ACSL3-dependent glucoregulatory lipid-sensing pathways via FXR in HFD rats. HFD rats were administered with L. gasseri probiotics (1 9 colony-forming units) or PBS vehicle by slowly infusing the probiotic over the course of 3 s via the upper small intestinal cannula 1 day prior to the pancreatic clamp studies in the same way the upper small intestinal RC microbiota transplant was performed (Figure A). During the clamps, although upper small intestinal Intralipid infusion for 5 min had no metabolic impact relative to a saline infusion in non-probiotic-infused (or PBS-infused) Cell Metabolism 7, , March,

12 HFD rats, HFD rats administered with L. gasseri probiotics exhibited a restoration of upper small intestinal lipid sensing, as upper small intestinal Intralipid versus saline infusion increased the glucose infusion rate (Figure SA) and decreased GP (Figure 7B) in these rats, independent of changes in glucose uptake (Figure SB). To investigate whether this improvement in lipid sensing was associated with a rise in ACSL3 expression, the upper small intestinal mucosa of HFD rats administered with PBS vehicle or L. gasseri was collected immediately following the saline-infused clamp procedure and ACSL3 expression was compared with untreated RC upper small intestinal mucosal samples. HFD rats administered with PBS vehicle (non-probiotic-infused) exhibited significantly reduced ACSL3 protein expression in the upper small intestinal mucosa relative to RC samples (Figure 7C), which is consistent with ACSL3 protein expression in untreated HFD rats (Figure A). Importantly, this reduction in ACSL3 expression was prevented with L. gasseri probiotic administration in HFD rats (Figure 7C). Finally, to investigate whether L. gasseri restores upper small intestinal ACSL3-dependent lipid-sensing pathways via FXR inhibition, we repeated the probiotic L. gasseri experiments in the presence or absence of FXR activation. Probiotic- (or non-probiotic PBS)-infused HFD rats received GW (FXR agonist) or vehicle 1% carboxymethyl cellulose (CMC) 3 min after probiotic administration. GW or 1% CMC was slowly administered over the course of 3 s via the upper small intestinal cannula, and was administered again the following morning before commencing the pancreatic clamp studies. First, GW did not worsen upper small intestinal lipid-sensing mechanisms in non-probiotic-infused (i.e., PBS-infused) HFD rats compared with non-probiotic-infused and non-gw HFD rats (Figures 7D and SC). Second, an Intralipid versus saline 5 min infusion instead was able to increase the glucose infusion rate (Figure SC) and lower GP (Figure 7D) in probiotic-infused HFD rats but, importantly, this glucoregulatory effect was lost in probiotic-infused HFD rats administered with GW (Figures 7D and SC), while glucose uptake remained unchanged (Figure SD). Of note, we found that GW inhibited the stimulatory effect of L. gasseri on upper small intestinal ACSL3 expression in these HFD rats as well (Figure 7C). Taken together, L. gasseri enhances an upper small intestinal ACSL3-dependent glucoregulatory lipid-sensing pathway via FXR inhibition in HFD rodents. DISCUSSION We here first report that upper small intestinal oleic and linoleic acid sensing in healthy rodents pre-absorptively increase whole-body glucose tolerance independent of a rise in plasma insulin levels and lower GP when plasma insulin levels were maintained at basal with a pancreatic-euglycemic clamp. Both upper small intestinal fatty acid-sensing pathways fail to regulate glucose tolerance and GP in lard oil-enriched HFD-fed rodents, suggesting that HFD feeding disrupts upper small intestinal fatty acid sensing to lower GP, leading to a disruption of whole-body glucose homeostasis. Given that defective fatty acid sensing arises in response to a saturated fat lard oil-enriched diet, saturated palmitic and stearic acid sensing in the upper small intestine may not share the same metabolic effect as the monounsaturated oleic and polyunsaturated linoleic acids, as currently described, although such a working hypothesis warrants future investigation. HFD reduces upper small intestinal L. gasseri levels and ACSL3 expression, while both reductions were reversed in HFD rats that received an upper small intestinal RC microbiota transplant, together with a restoration of the glucoregulatory ACSL3-dependent lipid-sensing pathway. We further discovered that directly administering non-transplanted HFD rats with L. gasseri probiotics is sufficient to recapitulate the effect of the RC microbiota transplant, as upper small intestinal ACSL3 expression and the subsequent glucoregulatory function of upper small intestinal lipid sensing are also restored in these rats. These studies together highlight that L. gasseri directly impacts an ACSL3-dependent glucoregulatory lipid-sensing pathway in the upper small intestine. Given that probiotic supplementation with various species from the Lactobacillus genus improve glucose parameters in rodents and humans with diabetes (Asemi et al., 13; Ejtahed et al., 1; Yadav et al., 7), future studies are warranted to determine whether L. gasseri-induced restoration of upper small intestinal sensing mechanisms could rescue glucose homeostasis in long-term HFD-induced obese and/or diabetic rodents. How does L. gasseri increase ACSL3 expression in the upper small intestine? Although the mechanistic link remains largely unknown, our preliminary data suggest that inhibition of the upper small intestinal bile acid receptor, FXR, could represent a possible link. This is consistent with the fact that Lactobacillus probiotics inhibit intestinal FXR (Degirolamo et al., 1), and that FXR deficiency in rodents increases intestinal ACSL expression (Li et al., 13). Future gain- and loss-of-function genetic experiments targeting the upper small intestinal FXR nuclear receptor are necessary to assess the integrative role of FXR in microbiota-induced regulation of glucoregulatory intestinal lipid-sensing mechanisms. Another urgent question is which bile acid(s) are involved in L. gasseri-induced FXR inhibition? L. gasseri preferentially deconjugates glyco-conjugated bile acids rather than tauro-conjugated bile acids (Rani et al., 17), which would lead to a drop in glyco-conjugated bile acid with a relatively stable level of tauroconjugated bile acids. On one hand, deconjugation of glycoconjugated bile acids would result in increased excretion of these bile acids, as deconjugation increases the hydrophobicity of bile acids leading to increased excretion (Choi et al., 15; Schiff et al., 197). In fact, similarly to other Lactobacillus species (Degirolamo et al., 1; Jeun et al., 1; Kumar et al., 11; Lye et al., 17), administration of L. gasseri probiotics increases bile acid excretion in rodents (Usman and Hosono, 1). Consequently, Lactobacillus-induced reductions in intestinal bile acid uptake can reduce activity of the bile acid receptor FXR in the intestine (Degirolamo et al., 1). On the other hand, tauro-conjugated bile acids, such as tauro-b-muricholic acid, inhibit FXR and improve glucose homeostasis in obese rats (Li et al., 13; Sayin et al., 13). Thus the potential relative increase in tauro-conjugated bile acids could also contribute to a L. gasseri-induced suppression of FXR. Nonetheless, assessing bile acids as links between L. gasseri and ACSL3-dependent lipid-sensing pathways as well as how microbiota transplantation affects bile acid pool warrant future investigations. Microbiota changes in the large intestine and feces correlate with changes in host metabolism (Forslund et al., 15; Karlsson 58 Cell Metabolism 7, , March, 18

13 et al., 13; Le Chatelier et al., 13; Qin et al., 1; Ridaura et al., 13) and transplantation of large intestinal or fecal microbiota from HFD obese rodents impairs glucose homeostasis in recipient rodents (Kovatcheva-Datchary et al., 15; Parseus et al., 17; Perry et al., 1; Ridaura et al., 13). In parallel, administration of probiotics improves glucose homeostasis in diabetic rodents in association with changes in large intestinal and fecal microbiota composition (Balakumar et al., 1; Cani et al., b; Kovatcheva-Datchary et al., 15; Neyrinck et al., 1; Zhou et al., 8), together suggesting that changes in large intestinal microbiota affect glucose homeostasis. These studies clearly did not (and could not) rule out that upper small intestinal microbiota does (or does not) impact metabolic homeostasis. Instead, we here report that the upper small intestinal RC microbiota transplant rescues upper small intestinal lipidsensing pathways in HFD-recipient rats via specific changes in the upper small intestinal microbiota, rather than changes in the colonic tract. However, our studies are in no way implying that direct HFD-induced changes in colonic microbiota do not impact glucose homeostasis as well. Taken together, we propose a potential parallel role of the distal and upper small intestinal microbiota in glucose homeostasis. In fact, metformin has been documented to alter both distal intestinal (de la Cuesta-Zuluaga et al., 17; Forslund et al., 15; Shin et al., 1; Wu et al., 17) and upper small intestinal microbiota (Bauer et al., 18) in parallel to improvements in glucose homeostasis. While HFD feeding lowers upper small intestinal L. gasseri levels (Figure 3) (Bauer et al., 18), transplantation of metformin-treated microbiota into the upper small intestine of HFD-recipient rats does not alter L. gasseri levels (in contrast to what is currently described for the RC microbiota transplant in HFD rats (Figure 3)). Transplantation of metformin-treated microbiota instead increases Lactobacillus salivarius in parallel to a restoration of an upper small intestinal SGLT1-dependent glucoregulatory glucose-sensing pathway (Bauer et al., 18). Given that L. gasseri activates an ACSL3-dependent glucoregulatory lipid-sensing pathway via FXR inhibition in the upper small intestine (Figure 7), and that L. salivarius, like L. gasseri, exhibits bile salt hydrolase activity (Wang et al., 1; Xu et al., 1), which could impact the bile acid pool and subsequent FXR activity, we propose (which clearly warrants future investigation) that upper small intestinal FXR could be a common pathway that links members of the Lactobacillus genus, such as L. gasseri and L. salivarius, with nutrient-sensing mechanisms to exert glucoregulatory effects in obesity and diabetes. In summary, L. gasseri probiotic administration in HFD rats, or transplantation of RC upper small intestinal microbiota that increases upper small intestinal L. gasseri levels in HFD-recipient rats, enhances an upper small intestinal ACSL3-dependent glucoregulatory lipid-sensing pathway in rodents. These findings strengthen the claim on the causative glucoregulatory role of the gut microbiota and highlight L. gasseri as an important regulator of nutrient sensing mechanisms in the upper small intestine to exert metabolic benefits. Limitations of Study The first key limitation of our study is that the current working hypothesis has yet to be tested in chronic obese and/or diabetic animal models as well as in healthy, obese, and diabetic humans. A second key limitation is that the glucoregulatory role of ACSL isoforms other than ACSL3, as well as the definitive glucoregulatory role of the bile acids, has not been investigated in the current as well as chronic obese and/or diabetic models. Lastly, the glucoregulatory role of the members of the Lactobacillus genus other than L. gasseri has yet to be assessed. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d KEY RESOURCES TABLE d CONTACT FOR REAGENT AND RESOURCE SHARING d EXPERIMENTAL MODEL AND SUBJECT DETAILS B Animals d METHOD DETAILS B Surgical Procedures B Virus Injection B High-fat Diet Model B Intravenous Glucose Tolerance Test B Rat Pancreatic (Basal Insulin) Clamp Procedure B Mouse Pancreatic (Basal Insulin) Clamp Procedure B Fatty Acid Preparation for Upper Small Intestinal Infusions B Upper Small Intestinal Infusions B Microbiota Transplant B Microbiota Heat-Shock B Microbiota Filter B Lactobacillus Gasseri Administration B GW Administration B Genomic DNA Extraction and 1S rrna Gene Sequencing B Sequence Processing and Data Analysis B Tissue Collection and Western Blotting B Biochemical Analysis d QUANTIFICATION AND STATISTICAL ANALYSIS SUPPLEMENTAL INFORMATION Supplemental Information includes six figures and three tables and can be found with this article online at ACKNOWLEDGMENTS This work was supported by a Canadian Institutes of Health Research (CIHR) Foundation Grant to T.K.T.L. (FDN-13). P.V.B. is supported by an Ontario Graduate Scholarship and a Banting and Best Diabetes Center Graduate Studentship. F.A.D. was a Banting Fellow. T.M.Z.W. is supported by a Banting and Best Diabetes Center Post-Doctoral Fellowship. H.J.D. is supported by a CIHR and a Diabetes Canada post-doctoral fellowship. B.A.R. was a Vanier Scholar. T.K.T.L. holds the John Kitson McIvor ( ) Endowed Chair in Diabetes Research and the Canada Research Chair in Obesity at the Toronto General Hospital Research Institute and the University of Toronto. AUTHOR CONTRIBUTIONS P.V.B. conducted and designed the experiments, performed the data analyses, and wrote the manuscript. F.A.D., T.M.Z.W., and H.J.D. assisted with the experiments and edited the manuscript. B.A.R., A.P., M.R., and C.A.O. assisted with the experiments. T.K.T.L. supervised the project, designed the experiments, and edited the manuscript. Cell Metabolism 7, , March,

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18 STAR+METHODS KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies GAPDH antibody Cell Signaling Biotechnology Cat# 118S b-actin antibody Sigma Aldrich Cat# A1978 ACSL1 antibody Cell Signaling Technology Cat# 7 ACSL3 antibody Santa Cruz Biotechnology Cat# sc-137 ACSL antibody Abcam Cat# ab1558 ACSL5 antibody Novus Biologicals Cat# NPB1-595 ACSL antibody Abcam Cat# ab159 Bacterial and Virus Strains ACSL3 shrna (r) Lentiviral Particles Santa Cruz Cat# sc-79-v Control shrna Lentiviral Particles-A Santa Cruz Cat# sc-188 Lactobacillus gasseri Lauer and Kandler (ATCC 3333) American Type Culture Collection Cat # 3333 Chemicals, Peptides, and Recombinant Proteins D-(+)-Glucose solution Sigma Aldrich Cat# G879 [3-3H] Glucose Perkin Elmer Part# NECV5UC Oleic acid Sigma Aldrich Cat# O18 Linoleic acid Sigma Aldrich Cat# L137 L-a-phosphatidylcholine Sigma Aldrich Cat# P73 Sodium salt of taurcholic acid Sigma Aldrich Cat# 8339 Intralipid Sigma Aldrich Cat# I11 MK-39 Tocris Bioscience Cat# 3 Exendin-9 Tocris Bioscience Cat# 81 Tetracaine Sigma Aldrich Cat #T7383 Lactobacilli MRS Broth (ATCC Medium 1) Fischer Scientific Cat# DF8817 GW Sigma Aldrich Cat# G517 Sodium carboxymethyl cellulose Sigma Aldrich Cat# 1973 Mutaniolysin Sigma Aldrich Cat# M991 RNase A Qiagen Cat# 1911 Tris-HCl Roche Cat# EGTA Sigma Aldrich Cat# E3889 EDTA Sigma Aldrich Cat# E988 Nonidet P Roche Cat# Sodium orthovanadate Sigma Aldrich Cat# S58 Sodium fluoride Sigma Aldrich Cat# S79 Dithiotritol Sigma Aldrich Cat# D9779 Protease inhibitor cocktail Roche Cat# HR series NEFA-HR color reagent A Wako Diagnostics Cat# HR series NEFA-HR solvent A Wako Diagnostics Cat# HR series NEFA-HR color reagent B Wako Diagnostics Cat# HR series NEFA-HR solvent B Wako Diagnostics Cat# NEFA standard solution Wako Diagnostics Cat# Wako NEFA linearity set Wako Diagnostics Cat# Critical Commercial Assays Rat Insulin Radioimmunoassay EMD Millipore Cat# RI-13K Pierce nm Protein Assay Thermo Fischer Scientific Cat# DNA Clean and Concentrator Kit Zymo Research Cat# D3T (Continued on next page) e1 Cell Metabolism 7, e1 e, March, 18

19 Continued REAGENT or RESOURCE SOURCE IDENTIFIER Experimental Models: Organisms/Strains Sprague Dawley Rats Charles River Laboratories Strain# C57BL/J Mice Jackson Laboratories Strain# Software and Algorithms QIIME bioinformatics pipeline Open Source qiime.org R programming language Open Source RRID: SCR_195 GraphPad Prism 7 GraphPad Software CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Tony Lam (tony.lam@uhnres.utoronto.ca). EXPERIMENTAL MODEL AND SUBJECT DETAILS Animals Eight-week old adult male Sprague-Dawley rats (8-3g) were obtained from Charles River Laboratories (Montreal, Quebec, Canada). Eighteen-week-old adult male C57/BL/ mice (5-3g) were obtained from Jackson Laboratories (Bar Harbor, Maine, USA). Sex-differences were not examined in the current manuscript. Both rats and mice were housed individually and maintained on a standard light-dark cycle with ad libitum access to chow (Teklad Diet #7, Harlan Laboratories) (Table S1) and water. All animal protocols were reviewed and approved by the Toronto General and Western Animal Care Committee at UHN. METHOD DETAILS Surgical Procedures Animals were given at least days to acclimatize before surgeries were performed. Rat surgeries were performed days prior to intravenous glucose tolerance tests (ivgtt) and clamp experiments. An upper small intestinal catheter was placed cm distal to the pyloric sphincter to target the lower duodenum and upper jejunum (between and 1 cm distal to the pyloric sphincter) during a 5-min upper small intestinal infusion, while carotid artery and jugular vein cannulations were performed for infusion and sampling during the clamp. Two to three days before the mouse pancreatic clamp studies, a catheter was placed in the upper small intestine (1.5- cm distal to the pyloric sphincter) to target the lower duodenum and upper jejunum, while a catheter was placed in the jugular vein for infusion purposes and tail sampling was performed during the clamp studies. Following surgery, both rats and mice were housed individually and maintained on a standard light-dark cycle with ad libitum access to food and water. Food intake and body weight was monitored to ensure recovery from surgery, and rats that did not recover to 9% of their baseline body weight were excluded from the study. Rats were randomly designated into treatment groups prior to experiments and no blinding was done during the experimental procedures described below. Virus Injection A subset of rats received an upper small intestinal lentiviral injection prior to the insertion of the intestinal cannula as described (Cote et al., 15; Kokorovic et al., 11). Briefly, the upper small intestine was elevated from to 1 cm distal to the pyloric sphincter and was ligated with sutures at both ends to restrict outward flow of virus and inward flow of intestinal fluids. The elevated intestinal portion was first flushed with.-.5 ml of saline via a 3-gauge needle inserted right below the cm ligation, and then. ml of the lentivirus expressing mismatch or ACSL3 shrna (both at 1. x 1 IFU; ml was diluted into a total volume of. ml for injection) (Santa Cruz, CA, USA) was administered via the 3-gauge needle. After min, ligations sutures were removed and the intestine was flushed with saline. A catheter was then inserted in the site of the virus injection and vascular cannulations were performed as described above. High-fat Diet Model Rats were placed on a lard oil-enriched high fat diet (HFD; TestDiet #571R, Purina Mills, IN, USA) (Table S1) and allowed to overeat for three days before the ivgtt or pancreatic clamp procedure, which results in hyperphagia (Figures S1D and SJ) and upper small intestinal lipid sensing defects (Breen et al., 11; Cheung et al., 9; Rasmussen et al., 1) but not obesity (Figures S1E and SK). The same 3-day HFD-induced hyperphagic response has been documented by others to be sufficient to induce insulin and leptin resistance but insufficient to increase body weight (Morgan et al., ; Scherer et al., 1; Thaler et al., 1), thus representing an early onset HFD-induced obese model. Under rare circumstances when rats were not hyperphagic they were excluded from the study. Cell Metabolism 7, e1 e, March, 18 e

20 Intravenous Glucose Tolerance Test Experiments were performed in overnight-fasted rats (1-18 hr) days after surgery. Basal blood samples were obtained in conscious, unrestrained rats immediately before beginning the gut infusion (.1 ml/min), which began at t = -15 min and was maintained until the end of the experiment at t = 35 min. At t = min blood samples were obtained and an intravenous bolus of glucose (% glucose,.5 g/kg) was injected into the jugular vein and flushed with saline. Blood was collected via the carotid arte. ry catheter to measure plasma glucose and insulin levels for 35 min following the glucose injection, as previously described (Yue et al., 1). Rat Pancreatic (Basal Insulin) Clamp Procedure The clamp was performed as described previously (Cheung et al., 9; Wang et al., 8; Yue et al., 1). After a - hr fast, a primed continuous infusion of [3-3 H]-glucose ( mci bolus;. mci min 1 ; Perkin Elmer, Woodbridge, ON, Canada) was given from t = to t = min (start to end of the experiment) through an intravenous catheter to assess glucose kinetics based on the tracer dilution methodology. Starting at t = 9 min and continuing to the end of the experiment (t = ), a pancreatic clamp was conducted through infusion of insulin (1. mu kg 1 min 1 ) and somatostatin (3 mgkg 1 min 1 ) to inhibit endogenous insulin and glucagon secretion. Somatostatin-1 was used, which has been shown to have a very minor inhibitory effect on GLP-1 release when compared with somatostatin-8 (Brubaker, 1991; Chisholm and Greenberg, ; Hansen et al., ). Furthermore, upper small intestinal glucose infusion was shown to stimulate GLP-1 release during the pancreatic basal insulin clamp with the same dose of somatostatin-1 (Bauer et al., 18), suggesting this dose of somatostatin-1 does not inhibit GLP-1 release under these conditions. Between t = 1 and t =, blood samples were taken every 1 min to determine the rate of an exogenous 5% glucose infusion needed to maintain basal glucose levels (averaged from t = -9). At t = 15 min, the upper small intestinal infusion (.1 ml/min) was started and continued until the end of the experiment (t = ). To determine insulin levels and specific activity of [3-3 H]-glucose, plasma samples were taken every 1 min during the basal period (t = -9) and the gut infusion period (t = 15-). Upon completion of the experiment rats were anaesthetized and upper small intestinal microbiota luminal samples (between and 15 cm distal to the pyloric sphincter), as well as mucosal scrapings (between and 1 cm distal to the pyloric sphincter), were collected, snap-frozen in liquid nitrogen and stored at -8 C until use. Mouse Pancreatic (Basal Insulin) Clamp Procedure Mice were fasted for - hr prior to experimentation. Through an intravenous catheter, a primed-continuous intravenous infusion of [3-3 H]-glucose (1 mci bolus;.1 mci/min; Perkin Elmer, Woodbridge, ON, Canada) was given from t = to t = 17 min (start to end of the experiment) to assess glucose kinetics based on the tracer dilution methodology. Starting at t = and continuing to the end of the experiment (t = 17), a pancreatic clamp was conducted through infusion of insulin (1. mu/kg/min) and somatostatin (8.3 mg/kg/min). Blood samples were taken via tail sampling every 1 min to determine if an exogenous 1% glucose infusion was needed to maintain basal glucose levels (averaged from t = 5-) between t = to t = 17. Upper small intestinal infusions ( ml/min) of oleic acid or linoleic acid ( x 1-3 kcal/min) were performed from t = 1 to t = 17. To determine specific activity of [3-3 H]-glucose levels, plasma samples were taken every 1 minutes during the basal period (t = 5-) and the upper small intestinal infusion period (t = 1-17). Fatty Acid Preparation for Upper Small Intestinal Infusions Oleic acid (99% purity; Sigma, St. Louis, MO) and linoleic acid (99% purity; Sigma) were administered at kcal/ml to mimic the concentration of the Intralipid infusate, which has been shown to lower glucose production when infused directly into the upper small intestine at.1 ml/min (Wang et al., 8). In addition, this dose of oleic acid and linoleic acid (total of 1 kcal delivered) has been shown to inhibit food intake in rats when infused into the upper small intestine (Phifer and Berthoud, 1998; Woltman et al., 1995). Fatty acid solutions were prepared as previously described (Woltman et al., 1995). Briefly, fats were emulsified in phosphate buffered saline (PBS; 115 mm NaCl, 1.5 mm NaH PO,.75 mm Na HPO, and 5 mm KCl; ph.) containing.1% wt/vol (1. mmol/ml) lecithin (L-a-phosphatidylcholine, 99% pure; Sigma) and 1% wt/vol (17.5 mmol/ml) sodium taurocholate (sodium salt of taurcholic acid, 98% pure; Sigma). These bile acid concentrations are comparable to levels detected in the upper small intestine of humans in postprandial (9 min after a standardized meal) conditions [1..5 mmol/ml (lecithin) and 1 17 mmol/ml (sodium taurocholate)] (Clarysse et al., 9; Mansbach et al., 1975). Solutions were emulsified until homogenous with an Autotune Series High Intensity Ultrasonic Processor. Upper Small Intestinal Infusions The following substances were infused into the upper small intestine at a rate of.1 ml/min as described: (1) saline, () PBS + bile salts (PBS with lecithin and sodium taurocholate) (3) Intralipid ( kcal/ml) () Oleic acid ( kcal/ml; in PBS and bile salts) (5) Linoleic acid ( kcal/ml; in PBS and bile salts), (5) MK-39 (.8 mg/ml; Tocris Bioscience), () Exendin-9 (15 mg/ml; Tocris Bioscience) and () tetracaine (1 mg/ml; Sigma). The dose of Intralipid (1 kcal delivered over 5 min) was chosen to ensure the effects observed were in the pre-absorptive state (Greenberg et al., 1995; Wang et al., 8). The dose used for oleic acid and linoleic acid were chosen to mimic the Intralipid dose (1 kcal). In addition, this dose of oleic acid and linoleic acid (total of 1 kcal) has been shown to inhibit food intake in rats (Phifer and Berthoud, 1998; Woltman et al., 1995). The dose of MK-39 was previously shown to inhibit lipid sensing in the upper small intestine (Cheung et al., 9). The dose of Exendin-9 has been shown to inhibit the effects of ileal fatty acid sensing (Zadeh-Tahmasebi et al., 1). The dose of tetracaine was previously shown to inhibit the glucose production-lowering effects of an upper small intestinal Intralipid infusion (Wang et al., 8). e3 Cell Metabolism 7, e1 e, March, 18

21 Microbiota Transplant Donor rats were anaesthetized after a - hr fast and the upper small intestinal luminal contents were removed from the upper small intestine (between -15 cm distal to the pyloric sphincter to collect a sufficient amount of luminal contents for transplantation). The luminal contents were homogenized and diluted 1: in PBS and.5 ml of the luminal contents were slowly infused (over the course of 3 seconds) into - hr-fasted recipient rats via the upper small intestinal gut line to target the lower duodenum and upper jejunum. To validate that the transplant effectively altered the upper small intestinal microbiota composition, the upper small intestinal luminal contents of anaesthetized recipient rats obtained from -15 cm distal to the pyloric sphincter was collected immediately after the infusion experiments, snap frozen in liquid nitrogen and stored at -8 C for subsequent 1S rrna gene sequencing (see below). For the colonic microbiota transplant, the upper small intestinal contents were removed and prepared for infusion in the same way as the upper small intestinal microbiota transplant. The luminal contents were then transplanted into recipient rats via a colonic cannulation located cm distal to the cecum. Recipient rats were subjected to the ivgtt or the basal insulin euglycemic clamp the following day. Recipient rats undergoing the ivgtt were immediately fasted following the transplant procedure (1-18 hr fast). Recipient rats undergoing the clamps were given their original diet (the diet being consumed prior to transplantation) and fasted for - hr prior to the clamp procedure. Microbiota Heat-Shock Before transferring the upper small intestinal luminal contents to the recipient rat, bacteria was heat-killed by incubation at 95-1 C for two 5 min intervals. The heat-treated samples were cultured on LB agar plates at 37 C for 9 hr to prove that no viable organisms remained. Microbiota Filter To obtain bacteria-free samples, luminal contents were filtered through. mm (Millex) filters (Gareau et al., 11) before being transferred to recipient rats. The filtered samples were cultured on LB agar plates at 37 C for 9 hr to prove that no viable organisms remained. Lactobacillus Gasseri Administration Lactobacillus gasseri Lauer and Kandler (ATCC 3333) was freshly prepared for each administration. Briefly, the probiotic was grown overnight in Lactobacilli MRS Broth (ATCC Medium 1) under anaerobic conditions at 37 C the evening prior to administration. The following day, 1 9 colony-forming units (cfu) were dissolved in a total volume of.5 ml of PBS and slowly infused (over the course of 3 seconds) into - hr-fasted recipient rats via the upper small intestinal gut line to target the lower duodenum and upper jejunum. Control HFD-fed rats received.5 ml of vehicle PBS. Basal insulin euglycemic clamps were performed the following day after a - hr fast. The Lactobacillus gasseri probiotic dose (1 9 cfu) was shown to increase bile acid excretion in male Sprague Dawley rats (Usman and Hosono, 1). In addition, a comparable dose (5 x 1 9 cfu) of a probiotic mixture containing a variety of species from the Lactobacillus genus increased bile acid excretion and decreased small intestinal FXR expression in mice (Degirolamo et al., 1). GW Administration A subset of Lactobacillus gasseri-infused and control PBS-infused HFD rats were administered with the FXR agonist, GW (3 mg/kg; Sigma; dissolved in 1% carboxymethyl cellulose; administered via the gut line over the course of 3 seconds and followed by a saline gut line flush), 3 min following the Lactobacillus gasseri or PBS administration and again the next morning after a - hr fast (immediately prior to the basal insulin-euglycemic clamps). Control HFD-fed rats received 1% carboxymethyl cellulose. The dose of GW (3 mg/kg) was chosen based on previous work reporting an inhibition of glucose-stimulated GLP-1 release in response to GW (3 mg/kg) administered via gavage for 5 days (Trabelsi et al., 15). In addition, oral GW was shown to inhibit probiotic-induced increased bile acid excretion after two treatments at a comparable dose (5 mg/kg) (Degirolamo et al., 1). Lactobacillus gasseri-infused HFD rats were initially given a single dose of GW (3 mg/kg) 3 min following the probiotic administration. However, we found that a single GW treatment only partially blocked the restoration of upper small intestinal lipid sensing in Lactobacillus gasseri-infused HFD rats during the pancreatic clamps (Gut Lipid-infused, GP was 8.9 mg kg 1 min 1 (n = 1) vs. Gut saline-infused, GP was 11. mg kg 1 min 1 (n = 1)). Given that the effect of GW on glucose-stimulated GLP-1 release and bile acid excretion occurred following daily treatment for 5 and days, respectively (Degirolamo et al., 1; Trabelsi et al., 15), we next administered the same dose of GW (3 mg/kg) immediately following the Lactobacillus gasseri-infusion and again the next morning prior to the clamp. We found that two treatments with GW (3 mg/kg) blocked the restorative effect of Lactobacillus gasseri administration on upper small intestinal lipid sensing, with no metabolic impact in non-probiotic-infused (PBS-infused) HFD rats (Figure 7D). Genomic DNA Extraction and 1S rrna Gene Sequencing Upper small intestinal luminal contents obtained from between -15 cm distal to the pyloric sphincter were collected from rats immediately following upper small intestinal saline-infused basal insulin-euglycemic pancreatic clamps. Genomic DNA extraction and purification was performed as described previously (Whelan et al., 1). Intestinal luminal contents were added to a ml plastic screw top tube containing. g of.1 mm glass beads (MO BIO laboratories, Inc.). Subsequently, 8 ml of mm NaPO (ph 8) Cell Metabolism 7, e1 e, March, 18 e

22 and 1 ml of guanidine thiocyanate-edta-n-lauroyl sarcosine were added to the tube and homogenization was performed for 3 min with a bench-top bead-based homogenizer (Minilys personal homogenizer). For the first enzymatic lysis step, 5 ml of mutanolysin (Sigma-aldrich, 1 U/mL), 1 ml of RNase A (Qiagen, 1 mg/ml) and 1 ml of 5M NaCl were added to each sample. The samples were vortexed and incubated at 5 C (water bath) for 1 hr. After incubation, the tubes were spun at maximum speed for 5 min in a bench-top centrifuge and 9 ml of the supernatant was removed and transferred to a ml tube containing 9 ml of phenolchloroform-isoamyl alcohol (5::1). After vortexing the tubes for 1 seconds (shear DNA), they were again spun at maximum speed in the bench-top centrifuge for 1 min. After centrifugation, the top layer was carefully transferred to a 1.5 ml microfuge tube. DNA clean and concentrator-5 kit (Zymo Research) was used for purifying the DNA, where DNA was eluted from the column with 5 ml of water and the samples were diluted to a final concentration of ng/ml. DNA samples were quantified using Nanodrop spectrophotometer (NanoDrop c, Thermo Scientific) and isolated DNA was stored at - C. PCR amplification of the V3 region of the 1S rrna gene was performed as previously described (Bartram et al., 11) with the following modifications: a 5 ml reaction containing 1.5 mm MgCl,.5 mm of each dntp, 1 nm of each barcoded primer, and 1.5 U Taq polymerase. Amplifications were carried out in triplicate, where the amplified product was purified by separation with agarose gel electrophoresis and gel extraction. PCR conditions consisted of an initial denaturation at 9 C for min followed by 3 cycles at 9 C for 3 seconds, 5 C for 3 seconds and a final elongation at 7 C for 1 min. Purified PCR products were sequenced using the Illumina MiSeq platform at the McMaster Genome Facility (Hamilton, ON, Canada). Sequence Processing and Data Analysis PCR products were sequenced using Illumina MiSeq with paired-end reads. 1S rrna gene sequencing was completed as previously described (Whelan et al., 1). Custom Perl scripts were developed in-house to process the sequences. First, Cutadapt was used to trim these sequences to the V3 region, removing any sequences surpassing this region. Next, sequences were aligned with their pair using PANDAseq (Masella et al., 1). During this alignment any mismatches or ambiguous bases were culled. Operational taxonomic units (OTUs) were picked using AbundantOTU and as described previously (Ye, 11) with a clustering cut-off of 97%. Taxonomy of the resultant OTUs was assigned via comparison of a representative sequence of the unit to the GreenGenes reference database ( February 11 release) (DeSantis et al., ) using the Ribosomal Database Project (RDP) classifier (Wang et al., 7). Summaries of the relative abundances of taxonomies, as well as beta and alpha diversity measurements were calculated using Quantitative Insights Into Microbial Ecology (QIIME, v1.9.1) (Caporaso et al., 1). The rarefaction depth was 1, sequences/sample. Three-dimensional Principal Coordinate Analysis plots were visualized using Emperor (Vazquez- Baeza et al., 13). The significance of clustering was determined by passing the UniFrac distance matrices through QIIME using Adonis and ANOSIM non-parametric statistical methods. LefSe (Linear Discriminant Analysis with Effect Size) analyses were performed on the website (Segata et al., 11). The differential features were identified at the Phylum (p), Class (c), Order (o), Family (f), and Genus (g) levels. The alpha value for the factorial Kruskal-Wallis test among classes was <.5 and the threshold on the logarithmic LDA score for discriminative features was >.. For group comparisons, the one-way ANOVA test was used with a Tukey s multiple comparisons test. Statistical analyses were performed using GraphPad Prism. A p value <.5 was considered significant. Tissue Collection and Western Blotting A section of the upper small intestine -1 cm distal to the pyloric sphincter, containing both lower duodenum and upper jejunum, was removed from anaesthetized rats immediately following the infusion experiments. The upper small intestinal mucosa was separated from the smooth muscle and quickly placed in liquid nitrogen. The tissues were lysed on ice with a handheld homogenizer in a lysis buffer containing 5 mm Tris-HCl (ph 7.5), 1 mm EGTA, 1 mm EDTA, 1% (w/v) Nonidet P, 1 mm sodium orthovanadate, 5 mm sodium fluoride, 5 mm sodium pyrophosphate,.7 M sucrose, 1 mm Dithiotritolo (DTT) and protease inhibitor cocktail (Roche). The protein concentration of homogenized tissues was determined using the Pierce nm protein assay (Thermo Scientific). 5 mg of tissues lysates were subject to electrophoresis on 1% acrylamide gels and transferred to nitrocellulose membranes. The membranes were incubated for 1 hr with blocking buffer (5% bovine serum albumin in TBS-T), followed by incubation in primary antibody hr at C. b-actin (Sigma, #A1978) and GAPDH (Cell Signaling, #118S) were used as loading controls. The antibodies were prepared in 5% bovine serum albumin in TBS-T at the following dilutions: 1:1 for ACSL1 (#7, Cell Signaling Technology), 1:5 for ACSL3 (sc-137, Santa Cruz Biotechnology), 1:1 for ACSL (ab1558, Abcam), 1:1 for ACSL5 (NPB1-595, Novus Biologicals), and 1:1 for ACSL (ab159, Abcam). The blots were then washed three times in TBS-T and incubated with a horseradish peroxidase (HRP)-linked secondary antibody (1: dilution in 5% skim milk) for hr. Blots were washed 5 times in TBS-T and protein expression was detected using an enhanced chemiluminescence reagent (Clarity Western ECL Blotting Substrate, Bio-Rad). Immunoblots were imaged using a MicroChemi chemiluminescent imaging system (DNR Bio- Imaging Systems). Biochemical Analysis Plasma insulin concentrations were measured using a radioimmunoassay (Linco Research, St Charles, MO) and plasma free fatty acid concentrations were measured by an enzymatic assay (Wako Pure Chemical Industries, Osaka, Japan). e5 Cell Metabolism 7, e1 e, March, 18

23 QUANTIFICATION AND STATISTICAL ANALYSIS The sample size for each group was chosen based on study feasibility and previously published experiments. Data from all groups showed normal variance. Therefore, the results were analyzed using unpaired Student s t test when analyzing statistical difference between two groups. When comparisons were made across more than two groups, ANOVA was performed and if significant this was followed by Tukey s post hoc test. Measurements that were taken repeatedly over time were compared using repeated measures two-way ANOVA; if the time and treatment interaction between groups was found to be significant Tukey s multiple comparisons test was used to determine the statistical significance at specific time points between groups. p value <.5 was considered statistically significant. For clamp experiments, the time period of -9 min was averaged for the basal condition and the period of 18- min was averaged for the clamp condition. Cell Metabolism 7, e1 e, March, 18 e

24 Cell Metabolism, Volume 7 Supplemental Information Lactobacillus gasseri in the Upper Small Intestine Impacts an ACSL3-Dependent Fatty Acid-Sensing Pathway Regulating Whole-Body Glucose Homeostasis Paige V. Bauer, Frank A. Duca, T.M. Zaved Waise, Helen J. Dranse, Brittany A. Rasmussen, Akshita Puri, Mozhgan Rasti, Catherine A. O'Brien, and Tony K.T. Lam

25 Table S1 Diet content of the regular chow and the saturated fat-enriched high fat diet relating to Figures 1-. Calories Provided Regular Chow High-Fat Diet Carbohydrate (%) 9 Protein (%) 33 Fat (%) 18 3 Saturated Monounsaturated Polyunsaturated 1 Total Calorie provided (kcal/g)

26 Table S Plasma insulin and glucose concentrations of the groups receiving an upper small intestinal infusion of saline, PBS with bile salts, oleic acid or linoleic acid during the basal and clamp time periods relating to Figure. SAL (N = 8) PBS + BILE SALTS (N = 5) OLEIC ACID (N = ) LINOLEIC ACID (N = 5) Basal Insulin (ng/ml) Glucose (mm).9 ± ±.5.9 ±.1 7. ±. 1. ±.1 7. ±.3.9 ±.1. ±. Clamp Insulin (ng/ml) Glucose (mm).7 ± ±..9 ± ±..9 ± ±.3.8 ±.1 7. ±.1 Data are means ± SEM (Basal: -9 min; Clamp: 18-).

27 Table S3 Summary of the % relative abundance of the bacterial groups that are significantly different in HFD rats vs. RC rats and HFD rats that received an upper small intestinal RC microbiota transplant, as assessed by ANOVA and identified using LefSE, relating to Figure 3. Taxonomic level RC vs. HFD Adjusted P values Mean relative abundance RC vs. HFD vs. HFD HFDwRCM HFDwRCM RC HFD wrcm PHYLUM ORDER FAMILY GENUS SPECIES Root;p Proteobacteria % 3.%.1% Root;p_Firmicutes < % 7.% 9.% Root;p Firmicutes;c Bacilli;o Lactobacillales <.1.5 < %.1% 7.7% Root;p Firmicutes;c Bacilli;o Lactobacillales; f Lactobacillaceae <.1.3 < % 7.8% 75.% Root;p Firmicutes;c Clostridia;o Clostridiales; f Clostridiaceae % 5.9% 9.5% Root;p Firmicutes;c Bacilli;o Lactobacillales; f Lactobacillaceae;g Lactobacillus <.1.3 <.1 88.% 7.8% 75.% Root;p Firmicutes;c Bacilli;o Lactobacillales; f Lactobacillaceae;g Lactobacillus;s Lactobacillus gasseri <.1.89 < %.9% 85.1% Root;p Firmicutes;c Bacilli;o Lactobacillales; f Lactobacillaceeae;g Lactobacillus;s Lactobacillus animalis <.1.91 <.1 9.8% 57.% 5.%

28 A B C Glucose (mg/dl) F Glucose (mg/dl) Saline Intralipid * HFD Saline HFD Intralipid Glucose (mg/dl min) Glucose (mg/dl min) 1 8 Time (min) 1 8 Time (min) AUC Glucose 1 * * AUC Glucose 1 Glucose (mg/dl) G Glucose (mg/dl) PBS + bile salts Oleic acid Glucose (mg/dl min) 1 Time (min) HFD PBS + bile salts HFD Oleic acid Glucose (mg/dl min) Time (min) AUC Glucose 1 * * * AUC Glucose 1 Glucose (mg/dl) Time (min) H Glucose (mg/dl) PBS + bile salts Linoleic acid Glucose (mg/dl min) 1 8 * HFD PBS + bile salts HFD Linoleic acid Glucose (mg/dl min) AUC Glucose * * * AUC Glucose 1 Fold Change vs. RC (kcal) I D Insulin (ng/ml) ** ** HFD + Saline HFD + Intralipid Time (min) Time (min) Time (min) Time (min) ** J Insulin (ng/ml) * E Body weight (g) HFD Saline HFD Oleic acid K Insulin (ng/ml) HFD Saline HFD Linoleate Figure S1. Upper small intestinal fatty acid infusion improves absolute glucose tolerance in RC, but not hyperphagic HFD rats independent of changes in body weight or insulin levels relating to Figure 1. (A-C) Absolute plasma glucose levels (inset: integrated area under the curve (AUC)) during the intravenous glucose tolerance test (ivgtt) in regular chow (RC) rats that received an upper small intestinal infusion of saline (n=), Intralipid (n=), PBS with bile salts (n=), oleic acid (n=8), or linoleic acid (n=). Values shown as mean ± SEM. (D) Cumulative food intake for three days prior to overnight fast and subsequent ivgtt for high fat diet (HFD)-fed rats given a saline, Intralipid, oleic acid, or linoleic acid upper small intestinal infusion, expressed as a fold change from their RC counterparts. *p<.5, **p<.1 versus their RC counterparts, as assessed by t-test. Values are shown as the mean + SEM. (E) Body weight on the day of the ivgtt for RC and HFD rats receiving a saline, Intralipid, oleic acid, or linoleic acid upper small intestinal infusion. Values are shown as the mean + SEM. (F-H) Absolute plasma glucose levels (inset: integrated area under the curve (AUC)) and (I-K) absolute plasma insulin levels during the ivgtt in HFD rats that received upper small intestinal infusion of saline (n=), Intralipid (n=), PBS with bile salts (n=), oleic acid (n=8), or linoleic acid (n=). Values shown as mean ± SEM. See also Figure 1. RC, regular chow; HFD, high fat diet; PBS, phosphate buffered saline.

29 Plasma FFA (mm) A Basal Clamp Portal B Day 1 5 C D basal clamp E Cannulation surgery Clamp [3-3 H]-Glucose (.1µCi min- 1 ) SRIF (8.3 µg kg -1 min -1 ) Insulin (1. mu kg -1 min -1 ) Glucose (as needed) Intestinal infusion ( µl min -1 ) Glucose infusion rate ** * Glucose Production * ** Glucose uptake F Glucose infusion rate ** G Glucose uptake H Glucose infusion rate I Glucose uptake J Fold change vs. RC (kcal) ** ** ** K L N M O Body weight (g) Glucose infusion rate Glucose uptake Figure S. Preabsorptive upper small intestinal fatty acids lower hepatic glucose production in mice and in rats through a gut peptide-dependent neuronal network relating to Figure. (A) Plasma free fatty acid during basal and clamp periods and portal free fatty acids immediately following the clamp. (B) Experimental and clamp procedure in mice. (C-E) The glucose infusion rate (C), glucose production (D), and glucose uptake (E) during the clamps of healthy mice that received upper small intestinal saline (n=5), PBS + bile salts (n=), oleic acid (OA) (n=8), or linoleic acid (LA) (n=7). *p<.5, **p<.1 versus saline and PBS + bile salts, as assessed by ANOVA with Tukey s post hoc test. (F-G) The glucose infusion rate (F) and glucose uptake (G) during clamps of healthy rats that received upper small intestinal infusion of MK-39 (n=7), Ex-9 (n=5), MK-39 + OA (n=5), Ex-9 + OA (n=), MK-39 + LA (n=5), or Ex-9 + LA (n=5). **p<.1 versus all other groups, as determined by ANOVA with Tukey s post hoc test. (H-I) The glucose infusion rate (H) and glucose uptake (I) during clamps of rats that received upper small intestinal infusion of tetracaine (n=5), tetracaine + OA (n=5), and tetracaine + LA (n=5). (J-M) Cumulative food intake for 3 days prior to clamp (expressed as fold change from RC counterparts) (J), body weight (K), glucose infusion rate (L) and glucose uptake (M) in high fat-fed rats that received upper small intestinal infusion of saline (n=5), OA (n=5), and LA (n=5). **p<.1 versus RC counterparts, as assessed by t-test. Values are shown as mean + SEM. See also Figure. FFA, free fatty acid; OA, oleic acid; LA, linoleic acid; SRIF, somatostatin; Ex-9, exendin-9; Tet, tetracaine; RC, regular chow; HFD, high fat diet.

30 A B Figure S3. Bacterial diversity of microbial communities in regular chow, high fat diet and high fat diet rats given a regular chow upper small intestinal microbiota transplant relating to Figure 3. Bacterial diversity within a sample (i.e. alpha diversity) was measured by calculating Shannon s diversity index (A) and observed OTUs (B). Each point on the graph represents one sample with the line representing the mean for all samples. See also Figure 3. RC, regular chow; HFD, high fat diet; HFDwRCM, HFD with RC microbiota transplant.

31 A B Recipient Donor Infusion C D Recipient Donor Infusion Day 1 5 Cannulation surgery Upper small intestinal microbiota transplant: RC Microbiota (RCM) IVGTT or Clamp HFD Microbiota (HFDM) Percent change (%) RC RC + RCM + Saline RC Saline RC RC + RCM + Intralipid RC Intralipid RC RC + HFDM HFD + Saline Saline RC RC + HFDM HFD + Intralipid Intralipid ** AUC % Change ** ** * Recipient Donor Infusion Insulin (ng/ml) RC RC+ HFDM HFD + Saline Saline RC RC+ HFDM HFD + Intralipid Intralipid Percent change (%) RC+ HFDM + HFD Intralipid RC+ Heat-shock HFD (Heat-shock) HFDM + Intralipid RC+ Filtered HFD HFDM (Filtered) + Intralipid Intralipid AUC % Change 15 * 1 5 ** ** * Intralipid RC rat RC rat Figure S. Transplantation of upper small intestinal HFD microbiota impairs fatty acid sensing pathways to dysregulate glucose tolerance in regular chow-fed rats relating to Figure. (A) Experimental procedure of the microbiota transplant protocol. (B-C) Percent change in plasma glucose levels (inset: integrated area under the curve (AUC)) (B) and absolute plasma insulin levels (C) during the intravenous glucose tolerance test (ivgtt) in regular chow (RC) rats that received a RC upper small intestinal microbiota transplant and an upper small intestinal infusion of saline (n=) or Intralipid (n=) and RC rats that received a high fat diet (HFD) upper small intestinal microbiota transplant and an upper small intestinal saline (n=) or Intralipid (n=) infusion. *p<.5, **p<.1 RC+RCM+Intralipid versus all other groups, as assessed by ANOVA with Tukey s post hoc test. (D) Percent change in plasma glucose levels (inset: integrated AUC) during the intravenous glucose tolerance test in RC rats that received heat-shock (n=) or filtered (n=5) HFD upper small intestinal microbiota and an upper small intestinal Intralipid infusion. *p<.5, **p<.1 RC + HFDM + Intralipid versus all other groups, as assessed by ANOVA with Tukey s post hoc test. Values are shown as mean ± SEM. See also Figure. ivgtt, intravenous glucose tolerance test; RC, regular chow; HFD, high fat diet; RCM, regular chow microbiota transplant; HFDM, high fat diet microbiota transplant.

32 A E ACSL1 expression relative to RC ACSL1 Glucose infusion rate I Glucose uptake Actin RC HFD HFD + RCM Mismatch (+) HFD HFD + RCM + HFD HFD + HFD RCM + HFD Saline +RCM +RCM Saline +RCM +RCM Saline Intralipid Saline Intralipid Mismatch RC Saline ACSL expression relative to RC RC HFD HFD + RCM (+) ACSL5 expression relative to RC B C D Glucose uptake J Glucose infusion rate HFD +RCM Saline HFD +RCM Intralipid HFD +RCM Saline HFD +RCM Intralipid HFD + HFD + HFD + HFD + RCM + RCM + RCM + RCM + Saline Sal + IL - Ex-9- IL + -Ex- Intralipid Ex RC HFD HFD + RCM (+) ACSL expression relative to RC RC HFD HFD + RCM 7 kda ACSL5 ACSL 7 kda 7 kda ACSL 7 kda Actin Actin Actin LV-ACSL3 shrna F 18 Mismatch LV-ACSL3 shrna G Mismatch LV-ACSL3 shrna H 7 Mismatch LV-ACSL3 shrna ** 1 Figure S5. Upper small intestinal microbiota alters ACSL3-dependent fatty acid sensing pathway to impact glucose production regulation with no effect on expression of ACSL1,, 5 or relating to Figure. (A-D) Protein expression of ACSL1 (A), ACSL (B), ACSL5 (C), and ACSL (D) in the upper small intestinal mucosa of RC rats, HFD rats, and HFD rats transplanted with upper small intestinal RC microbiota (n=5). Brain tissue was used as a positive control. (E-F) The glucose infusion rate (E) and glucose uptake (F) during the clamps of HFD rats injected with mismatch or ACSL3 lentiviral shrna that received an upper small intestinal RC microbiota transplant and an upper small intestinal saline (n=5, 5 respectively) or Intralipid (n=5, 5 respectively) infusion. (G-I) The glucose infusion rate (G), glucose production (H) and glucose uptake (I) during the clamps of RC rats injected with mismatch or ACSL3 lentiviral shrna that received an upper small intestinal infusion of saline (n=5, respectively) or Intralipid (n=5, 5 respectively). (J-K) The glucose infusion rate (J) and glucose uptake (K) during the clamp of HFD rats that received an upper small intestinal RC microbiota transplant and a saline (n=), Intralipid (n=), Ex-9 (n=5) or Ex-9 + Intralipid (n=) upper small intestinal infusion. **p<.1, ***p<.1 versus all other groups, as assessed by ANOVA with Tukey s post hoc test. Values are shown as mean + SEM. See also Figure. RC, regular chow; HFD, high fat diet; HFD + RCM, high fat diet with regular chow microbiota transplant; ACSL, acyl coa synthetase; LV-ACSL3 shrna, lentiviral ACSL3 shrna; Ex-9, exendin-9. K Glucose infusion rate ** *** RC Intralipid LV-ACSL3 shrna RC Saline RC Intralipid *** Glucose uptake RC Saline RC Intralipid RC Saline RC Intralipid HFD + HFD + HFD + HFD + RCM + RCM + RCM + RCM + Saline Sal + IL - Ex-9- IL -+ Intralipid Ex-9 + Ex Glucose production RC Saline RC Intralipid RC Saline (+) RC Intralipid