Upregulation of Intestinal Glucose Transporters after Roux-en-Y Gastric Bypass to Prevent Carbohydrate Malabsorption

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1 Upregulation of Intestinal Glucose Transporters after Roux-en-Y Gastric Bypass to Prevent Carbohydrate Malabsorption Nam Q. Nguyen 1,2, Tamara L. Debreceni 1, Jenna E. Bambrick 1, Bridgette Chia 3, Adam M. Deane 2,4, Gary Wittert 2, Chris K. Rayner 1,2, Michael Horowitz 2 and Richard L. Young 2,3 Objective: To determine the effect of Roux-en-Y gastric bypass (RYGB) on the expression of intestinal sweet taste receptors (STRs), glucose transporters (GTs), glucose absorption, and glycemia. Methods: Intestinal biopsies were collected for mrna expression of STR (T1R2) and GTs (SGLT-1 and GLUT2) from 11 non-diabetic RYGB, 13 non-diabetic obese, and 11 healthy subjects, at baseline and following a 30 min small intestinal (SI) glucose infusion (30 g/150 ml water with 3 g 3-O-methyl-D-glucopyranose (3-OMG)). Blood glucose, plasma 3-OMG, and insulin were measured for 270 min. Results: In RYGB patients, expression of both GTs was 2-fold higher at baseline and after glucose infusion than those of morbidly obese or healthy subjects (P < 0.001). STR expressions were comparable amongst the groups. Peak plasma 3-OMG in both RYGB (r , P ) and obese (r , P ) correlated with baseline expression of SGLT-1, as was the case with peak blood glucose in RYGB subjects (r , P ). Conclusions: The upregulated intestinal GTs in RYGB patients are associated with increased glucose absorption when glucose is delivered at a physiological rate, suggesting a molecular adaptation to prevent carbohydrate malabsorption from rapid intestinal transit after RYGB. (2014) 22, doi: /oby Introduction Roux-en-Y gastric bypass (RYGB), arguably, is the most effective current surgical treatment for morbid obesity and results in a small gastric pouch (20 ml) and a short circuit for nutrient delivery from the stomach to the mid-jejunum through a Roux-en-Y limb (1,2). While the mechanisms underlying the effects of RYGB on body weight are incompletely understood, the anatomical alterations are thought to reduce oral intake via the restrictive gastric pouch, nutrient malabsorption and neurohormonal changes arising from the intestinal short circuit that favor weight loss, and improved glycemic control (3-5). It has been suggested that weight loss following RYGB is predominantly due to restricted food intake (79%) and malabsorption (11%), particularly malabsorption of fats (40% reduction) (6-8). Surprisingly, carbohydrate absorption is not significantly altered following RYGB in humans, despite accelerated emptying of the gastric pouch and accelerated intestinal transit (9), and the underlying reasons are unclear. Intestinal glucose is absorbed primarily by two glucose transporters (GTs) sodium dependent glucose co-transporter 1 (SGLT1) (10,11) and glucose transporter-2 (GLUT2) (12). Data derived from animal experiments indicate that intestinal glucose absorption is modulated, in large part, by intestinal sweet taste receptors (STRs), heterodimers of the G-protein-coupled receptors T1R2 and T1R3 (11-13). STRs, in the presence of intestinal sweet tastants, trigger increased expression of SGLT1 and GLUT2 to promote glucose absorption (14,15). There is no information about the impact of RYGB on the expression of intestinal STRs and GTs in humans, and their relationships to intestinal glucose absorption. Stearns et al. reported that glucose transport was reduced by up to 68% in the Roux limb after RYGB in rats, despite an increase in villus height and crypt depth compared with sham jejunum (16). More recent work, however, indicates that intestinal glucose metabolism in the Roux limb of RYGB rats is reprogrammed to meet increased bioenergetic demands after RYGB, and is characterized by upregulation of glucose transporter-1 (GLUT-1), enhanced basolateral glucose uptake, and augmented aerobic glycolysis. These changes direct glucose toward 1 Department of Gastroenterology and Hepatology, Royal Adelaide Hospital, Adelaide, South Australia 5000, Australia. Correspondence: Nam Q. Nguyen (quoc.nguyen@health.sa.gov.au) 2 Discipline of Medicine, University of Adelaide, Royal Adelaide Hospital, Adelaide, South Australia 5000, Australia 3 Discipline of Medicine, Nerve-Gut Research Laboratory, Level-1 Hanson Institute, Adelaide, South Australia 5000, Australia 4 Intensive Care Unit, Royal Adelaide Hospital, North Terrace, Adelaide, South Australia 5000, Australia Funding agencies: This study was conducted using a National Health and Medical Research Council (NHMRC) research grant. Disclosures: There are no conflicts of interest. Author contributions: NN, RY, CR, GW, AD and MH designed Study, NN, TD and JB carried out experiments, NN, BC and RY analysed data, NN wrote the paper. All authors had final approval of the submitted version. Additional Supporting Information may be found in the online version of this article. Received: 6 March 2014; Accepted: 13 June 2014; Published online 2 July doi: /oby VOLUME 22 NUMBER 10 OCTOBER

2 Figure 1 Outline of study protocol. metabolic pathways that support tissue growth and may contribute to the improvement in glycemic control after RYGB (17) We hypothesized that the absence of carbohydrate malabsorption after RYGB is related to compensatory upregulation of intestinal STRs and/ or GTs in humans. We, accordingly, evaluated the expression of intestinal STR and GTs and their relationship to glucose absorption in RYGB patients in comparison to morbidly obese and healthy subjects. Methods Subjects Eleven RYGB subjects who underwent surgery >12 months ago were studied between January 2012 and November The subjects were recruited from advertising flyers placed on hospital boards. The data were compared to 13 gender- and age-matched morbidly obese (BMI > 35 kg/m 2 ) subjects and 11 healthy volunteers. The age matched was within 3 years. The following were exclusion criteria: age less than 18 years, pregnancy, diabetes, contraindication to endoscopy, history of surgery on the stomach, duodenum, or small intestine (other than RYGB), use of medication known to alter platelet aggregation or thrombus formation, an International Normalized Ratio (INR) > 1.5 or platelet count < 50,000. The study was approved by the Human Research Ethics Committee of the Royal Adelaide Hospital, and informed, written, consent was obtained from all subjects. Protocol The study protocol is summarized in Figure 1. All subjects were studied at the same time of day (0900 h), after an overnight fast, to minimize the impact of a circadian variation in the expression of intestinal STR and GTs. An intravenous cannula was inserted for blood sampling, and following local anesthesia of the pharynx (Co-phenylcaine Forte, ENT Technologies, Hawthron East, Victoria, Australia), a small diameter (5.3 mm) video endoscope was inserted, via the mouth, into the distal duodenum in obese or healthy subjects, or the proximal Roux-limb in RYGB subjects (10-15 cm from the gastroenterotomy), using minimal insufflation of air. Three mucosal biopsies were collected at baseline and stored in RNAlater (Qiagen). From 0 to 30 min (T0-T30), a glucose solution containing 30 g glucose and 3 g 3- O-methyl-D-glucopyranose (3-OMG) (Sigma-Aldrich) in water to a total volume of 150 ml was infused directly into the intestine at a rate of 4 kcal/min; the endoscope was left in situ for the 30-min duration of intestinal glucose infusion. At the end of the glucose infusion, three further mucosal biopsies were taken and the endoscope then removed. The Roux limb was chosen as the site of glucose infusion and biopsy in RYGB patients as in rats marked changes in GTs after RYGB have been demonstrated at this site (17). Blood was sampled immediately prior to intra-intestinal glucose infusion (baseline), and at regular intervals over 240 min for measurements of blood glucose and plasma 3-OMG concentrations; glycated hemoglobin (HbA1c) was also measured on one sample. Samples were stored at 270 C until assayed. Absolute expression of SGLT-1, GLUT2, and T1R2 transcripts was quantified from duodenal mucosal biopsies by quantitative real time reverse transcriptase PCR (RT-PCR) (18). Primers for these targets were used as validated primer assays (QuantiTect, Qiagen; Supporting Information Table 1) or for absolute PCR standards, designed from target sequences obtained from the NCBI nucleotide database and product size was confirmed by gel electrophoresis (Supporting Information Table 2). RT-PCR was performed on a Chromo4 (MJ Research) real time instrument attached to a PTC- 200 Peltierthermal cycler (MJ Research) using a QuantiTect SYBR Green one-step RT-PCR kit (Qiagen) according to the manufacturer s specifications. Each assay was performed in triplicate and included internal no-template and no-rt controls. Minimum detectable levels for SGLT-1 and T1R2 transcripts were copies; when levels were not detected, this threshold value was substituted (18). Immunolabeling was performed on frozen tissues that were sectioned at 6-10 lm (Cryocut 1800, Leica Biosystems, Germany) and thawmounted onto gelatin-coated slides. Immuno-reactivity in human tissues was detected using a rabbit T1R2 primary (H90, 1:400, SC , Santa Cruz Biotechnology), a monoclonal GLUT-2 (1:800, AB85715, Abcam), and goat SGLT-1 (1:400, SC-47398, Santa Cruz) (18). All were visualized using species-specific secondary antibodies conjugated to Alexa Fluor dyes (1:200 in PBST) according to earlier methods. Antigen retrieval (S1700, Dako) was performed for T1R2 and SGLT-1 according to manufacturer s instruction. Plasma concentrations of 3-OMG were measured using high performance exchange chromatography with an assay sensitivity of VOLUME 22 NUMBER 10 OCTOBER

3 Gut Glucose Transporter Changes in RYGB Nguyen et al. TABLE 1 Demographic and clinical characteristics of healthy, morbidly obese, and RYGB subjects Lean healthy (n 5 11) Morbidly obese (n 5 13) RYGB subjects (n 5 11) Age (years) Male:female ratio 10:1 5:8 4:7 Glycated hemoglobin (Hb A1c,%) Body mass index (kg/m 2 ) * * HOMA-IR * * Data are mean 6 SD, * P < 0.01, vs. healthy subjects mmol/l (19). Peak and time to peak 3-OMG concentrations, as well as the area under the curve (AUC) over 270 min (AUC 0-270min ), were quantified. HbA1c was measured using cation exchange high performance liquid chromatography. Blood glucose was measured using a portable glucometer (Medisense Optimum, USA). Plasma insulin was measured by enzyme-linked immunosorbent assay (ELISA, Diagnostic Systems Laboratories, Inc., Webster, TX) with assay sensitivity of 0.26 mu/l and coefficient of variation was 2.6% within assay and 6.2% between assays (18). In each subject, the degree of insulin resistance was estimated at baseline by homeostasis model assessment (HOMA), according to the method described by Matthews et al. (20). Data analysis Data are presented as mean 6 SD. The primary endpoint was the differences in the expression of STR (T1R2) and TGs (SGLT1 and GLUT2). Based on our work (13,18,21) comparing duodenal RNA levels in type 2 diabetes, critically ill, and healthy subjects, the sample size provided 80% power to detect a difference in RNA expression between groups of 20%, accepting an a error of Differences in blood glucose, plasma 3-OMG, and insulin concentrations between the subject groups were assessed by repeated measures analysis of variance (ANOVA) with treatment and time as factors. In addition, the differences in copy number of intestinal SGLT- 1, T1R2, and GLUT2 transcripts were compared using Student s t- tests. Linear relationships were evaluated using Spearman s Rank Test. Analyses were performed using GraphPad Prism (version 6, GraphPad Software Inc., La Jolla, CA). Significance was accepted at P < 0.05; data is presented as mean 6 SD. Results RYGB subjects underwent surgery 4.5 (2-12) years prior to the study with a mean reduction of BMI of kg/m 2. In all subjects, the procedure was well tolerated and normal small intestinal mucosa was evident. Differences in demographics between the groups are summarized in Table 1. Expression of STR and GTs Baseline levels of SGLT-1 and GLUT2 transcript were higher in RYGB, than in either morbidly obese or healthy subjects (P < 0.001); T1R2 levels, in contrast, were comparable across study groups. In obese subjects, levels of SGLT-1 transcript were higher compared to healthy subjects at baseline (P ; Figure 2A). T1R2 levels were reduced following acute glucose infusion in RYGB patients (P ; Figure 2B), but were unchanged in morbidly obese and healthy subjects. In RYGB subjects, SGLT-1 and GLUT-2 transcript levels remained higher after glucose infusion when compared to morbidly obese and healthy subjects (P < 0.001; Figure 2B). Immunolabeling for T1R2 was evident in single cells dispersed throughout the duodenal epithelium in healthy, morbidly obese, and RYGB subjects (Figure 3A). These cells were largely open or flask shaped and found with equal frequency within villi or crypts; they showed label distribution throughout the cytoplasm, but a concentration at the brush border membrane. SGLT-1 labeling was detected in the brush border membrane of duodenal epithelial cells (Figure 3B), while GLUT2 was present in basolateral aspects of these cells (Figure 3C). Intestinal glucose absorption The rate of increase in plasma 3-OMG and peak concentrations was higher in RYGB and morbidly obese subjects than in healthy controls (Figure 4A). Both absolute (P < 0.001) and integrated AUC 0-270min plasma 3-OMG concentrations were higher in RYGB patients than in healthy subjects ( vs mmol/l min, P ; Figure 4B). In RYGB patients, there was also a trend for higher absolute (P ) and integrated AUC 0-270min plasma 3- OMG when compared to morbidly obese subjects ( vs mmol/l min, P ). Blood glucose and plasma insulin, GIP and GLP-1 Fasting (baseline) blood glucose concentrations were higher in morbidly obese ( mmol/l) when compared to healthy subjects ( mmol/l, P ) and RYGB patients ( mmol/l, P ; Figure 4B). Blood glucose concentrations were higher in morbidly obese and RYGB subjects between 45 and 90 min after commencing glucose infusion than in healthy subjects (subject 3 time interaction, P < 0.01; Figure 4C). During this time, blood glucose concentrations were also higher in RYGB than obese subjects (P < 0.05). Fasting plasma insulin was higher in obese ( mmol/l) subjects than RYGB ( mmol/l; P ) and healthy subjects ( mmol/l; P < 0.01). In all groups, plasma insulin increased markedly following intra-duodenal glucose infusion, and was highest in the obese subjects (Figure 4D). In the RYGB group, the rise in plasma insulin was greater than that in healthy subjects, but substantially less than that in obese subjects VOLUME 22 NUMBER 10 OCTOBER

4 Figure 2 Transcript levels of intestinal sweet taste receptor (T1R2) and glucose transporters (SGLT-1 and GLUT-2) at baseline and after small intestinal glucose exposure in RYGB, morbidly obese, and healthy subjects. Relationships between variables Although peak plasma 3-OMG concentrations correlated with peak blood glucose levels in all groups, the relationship was strongest in RYGB patients (r , P < , Figure 5A). Peak plasma 3- OMG correlated with the baseline expression of SGLT-1 in both RYGB (r , P ) and obese (r , P ), and peak blood glucose correlated with the baseline expression of SGLT-1 in RYGB subjects (r , P ; Figure 5B,C). No significant relationships between intestinal transcript levels of T1R2 or GLUT2 and plasma concentrations of blood glucose and 3-OMG Figure 3 Immunolabelling for T1R2, SGLT-1, and GLUT2 in human duodenum. (A) T1R2 immunolabel was present near the brush border membrane in sweet taste cells in transected villi. (B) SGLT-1 immunolabelling was evident in the brush border membrane of duodenal enterocytes, while (C) GLUT2 labeled within basolateral membranes. Intestinal lumen is to the top of B, C. Scale Bar (A) 5 20 mm, (B, C) 5 50 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] VOLUME 22 NUMBER 10 OCTOBER

5 Gut Glucose Transporter Changes in RYGB Nguyen et al. Figure 4 Intestinal glucose absorption (A, B), blood glucose (C), and plasma insulin (D) in RYGB, morbidly obese, and healthy groups. were evident. There were also no relationships between the expression of T1R2, either at baseline or after glucose exposure, and plasma concentrations of blood glucose or 3-OMG. Discussion This study is the first to demonstrate that expression of the intestinal GTs, SGLT-1 and GLUT2, but not the STR (T1R2), are upregulated substantially after RYGB in humans an increase that is preserved after small intestinal glucose exposure. Increased GT expression was associated with augmented intestinal glucose absorption in both obese subjects and RYGB patients in response to enteral delivery of glucose, but not in healthy controls. These findings support recent animal data (17) and provide important insights into the mechanisms underlying gut adaptation in humans after RYGB. Given the active gut adaptation to normalize glucose absorption after RYGB, ongoing attention to minimize carbohydrate intake may well be essential to optimize weight loss, prevent weight gain, and potentially, the development of type 2 diabetes, after surgery. The anatomical consequences of RYGB surgery dictate that the Roux-limb is usually rapidly exposed to ingested nutrient following a meal. The loss of absorptive surface for glucose uptake, but increased GT expression and gain in absorptive function evident in RYGB patients in the current study, indicate that the Roux-limb is remodeled, likely as a result of increased absorptive capacity of 2168 VOLUME 22 NUMBER 10 OCTOBER

6 Figure 5 Relationships between glucose absorption, glycemia, and expressional activity of intestinal glucose transporter SGLT-1 in healthy, obese, and RYGB subjects (NS 5 non-significant; SGLT-1 5 sodium dependent glucose transporter 1). existing enterocytes and/or recruitment of additional enterocytes to participate in glucose absorption (22). Whilst increased levels of SGLT-1 transcript in the current study were shown to be linked directly to both peak glucose absorption and blood glucose, it remains uncertain whether increased GLUT-2 expression may have additional consequences. In general, GLUT2 is immunolocalized to the basolateral membrane of enterocytes in healthy humans (18), although there is recent evidence that it may also be present at the apical membrane in obese humans (23). It remains uncertain whether GLUT2 participates in augmented luminal glucose flux, in concert with SGLT1, as well as serve as a bi-directional efflux transporter for increased intestinal glucose absorption and utilization in RYGB patients, as shown for GLUT1 in RYGB rats (17). The relative contribution of these GTs to intestinal glucose metabolism VOLUME 22 NUMBER 10 OCTOBER

7 Gut Glucose Transporter Changes in RYGB Nguyen et al. in humans warrants further investigation by the combined use of selective inhibitors, such as phloridzin (SGLT-1) and phloretin (GLUT-2), and assessments of glucose disposal. Our study did not reveal differences in baseline levels of intestinal T1R2 transcript amongst the patient groups, in contrast to Bueter et al., who reported a reduction in T1R2 expression and protein at 60 days after RYGB in rats (24). The specific decrease in T1R2 evident after acute glucose infusion in RYGB patients, however, indicates that this receptor may exhibit an increased sensitivity to tachyphylaxis in this patient group, although the overlying mechanisms involved are unknown. Together, these findings suggest that intestinal STRs are not tightly linked to GTs in human RYGB, and that disordered GT regulation per se may be the major contributor to augmented glucose absorption. Direct and physiological infusion of glucose into the proximal intestine, in the current study, excluded the potential confounding effects of differences in the rate of gastric emptying on assessments of intestinal glucose absorption, other studies which have assessed intestinal glucose absorptive capacity after an oral stimulus failed to quantify gastric emptying (9,25,26). The rate of intestinal glucose infusion (4 kcal/min) was chosen to mimic the physiological rate of nutrient delivery to the small intestinal (27), and the duration of 30- min was selected given that it has the capacity to induce changes in STR and GTs in humans (18,21). Our observations of increased SGLT1 expression, accelerated glucose absorption, and positive associations between SGLT-1 and glucose absorption in morbidly obese and RYGB subjects suggest an intrinsic dysregulation of intestinal GT expression and absorption in obesity, which is magnified by RYGB. This hypothesis warrants further evaluation, probably in a prospective longitudinal study design. The increased glucose absorption (3-OMG) and blood glucose AUC in RYGB patients in the current study reflected accelerated uptake and sustained exposure to intestinal glucose (i.e., 30 min infusion). After RYGB, pouch emptying of a meal is usually very rapid, leading to a marked reduction in the duration of the exposure of the gut to nutrients. Although pouch emptying of solids and mixed meal is variably rapid, it is less rapid than liquid pouch emptying (28,29). Despite the rapid pouch emptying, the overall absorption of glucose has been reported to be largely unaffected after RYGB at 1 year, and comparable with normal subjects (9). These observations attest to the importance of the restrictive component of RYGB in triggering and maintaining weight loss (6-8). It is intriguing that the glucose excursion curve in the RYGB subjects was higher than that of obese subjects, and is likely to be related to the nature of glucose stimulation, which consisted of a 4 kcal/min intra-duodenal glucose infusion over 30 min. This is in contrast to the responses to an oral glucose drink after RYGB, characterized by a higher early peak level and earlier return to baseline due to the presumed rapid GI transit (30,31). In the current study, the higher early peak was more sustained in response to the constant intra-duodenal glucose infusion. Whilst the underlying reason remains unclear, we speculate that it relates to the combination of substantially lower rise in plasma insulin and insulin resistance (as reflected by the high HOMA-IR score). The greater glucose absorption in the RYGB subjects, as compared to the obese and healthy subjects, may also contribute to the higher blood glucose between 60 to 90 min in these subjects. It is also possible the accelerated glucose absorption in these patients increases the release of gut hormones, such as GIP and glucagon, favoring hyperglycemia (32). Although the current age-, gender-matched cross-sectional study can be improved by a prospective longitudinal study design and a larger sample size, it should be recognized that this type of study is technically demanding. Whilst a screening oral glucose tolerance test was not performed in all subjects, type 2 diabetes was effectively excluded by HbA1c of less than 6.5%, and fasting blood glucose less than 7 mmol/l, without differences between the groups. Importantly, the observed changes were major and consistent. As we quantified SGLT1, GLUT2, and T1R2 transcripts in the current study it is not known whether transcript expression reflects the activity of mucosal STRs and GT proteins, although the observed relationship between SGLT1 transcript and peak plasma 3-OMG provides strong support. While we cannot exclude the possibility that observed differences in STR/GT expression relate to differences in the biopsy collection site (healthy, obese 5 duodenum, RYGB 5 jejunum), this appears most unlikely given that we have reported previously that the expression of STRs and GTs in humans is not significantly different between the duodenum and proximal jejunum (i.e., Roux limb) (13). Although there was a gender difference between the obese/rygb subjects and healthy controls, data from studies using an oral glucose tolerance test suggest that gender does not impact on glucose absorption (33,34). Furthermore, our earlier studies have shown that gender does not influence the expression of intestinal STRs or GTs (18,21). In conclusion, intestinal GTs are upregulated after RYGB in association with increased glucose absorption when nutrient is delivered directly to the proximal intestine. 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