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Obesity Research Center (B.L., H.T., J.R.E., A.C., B.K., B.B., K.Y., B.O.) and Bariatric Division (J.T., J.M., N.K., H.L.), St. Luke’s/Roosevelt Hospital Center, Columbia University College of Physicians and Surgeons, New York, New York 10025
Address all correspondence and requests for reprints to: Dr. Blandine Laferrère, St. Lukes Roosevelt Hospital, Obesity Research Center, 1111 Amsterdam Avenue, Room 1020 Babcock, New York, New York 10025. E-mail: BBL14{at}columbia.edu.
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Abstract |
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Objective: Our objective was to determine whether the magnitude of the change of the incretin levels and effect is greater after GBP compared with a low caloric diet, after equivalent weight loss.
Design and Methods: Obese women with T2DM studied before and 1 month after GBP (n = 9), or after a diet-induced equivalent weight loss (n = 10), were included in the study. Patients from both groups were matched for age, body weight, body mass index, diabetes duration and control, and amount of weight loss.
Setting: This outpatient study was conducted at the General Clinical Research Center.
Main Outcome Measures: Glucose, insulin, proinsulin, glucagon, gastric inhibitory peptide (GIP), and glucagon-like peptide (GLP)-1 levels were measured after 50-g oral glucose. The incretin effect was measured as the difference in insulin levels in response to oral and to an isoglycemic iv glucose load.
Results: At baseline, none of the outcome variables (fasting and stimulated values) were different between the GBP and diet groups. Total GLP-1 levels after oral glucose markedly increased six times (peak:17 ± 6 to 112 ± 54 pmol/liter; P < 0.001), and the incretin effect increased five times (9.4 ± 27.5 to 44.8 ± 12.7%; P < 0.001) after GBP, but not after diet. Postprandial glucose levels (P = 0.001) decreased more after GBP.
Conclusions: These data suggest that early after GBP, the greater GLP-1 and GIP release and improvement of incretin effect are related not to weight loss but rather to the surgical procedure. This could be responsible for better diabetes outcome after GBP.
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Introduction |
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The two main incretins, GIP and GLP-1 (11), are secreted by the endocrine cells of the intestinal mucosa (12) in response to food and are responsible for 50–60% of insulin secretion after meals (12, 13). The incretin effect is impaired in patients with T2DM (14). GLP-1 levels are blunted (15), but the effect of administered GLP-1 on insulin secretion persists (16). GIP levels are usually normal in patients with T2DM, but the effect of administered GIP on insulin secretion is blunted (17), although can be restored under normal glycemic conditions (18). GIP and GLP-1 are rapidly degraded by the enzyme dipeptidyl peptidase-IV (DPPIV) (19). GLP-1 and GIP analogs and DPPIV inhibitors are in use or currently being developed as antidiabetic agents (20).
The markedly increased incretin levels and effect observed after GBP could be one of the key mediators of the antidiabetic effects of the surgery. However, weight loss occurs very rapidly after GBP, and it is unclear whether the early changes in incretin levels and effect are a result of the surgery or could be attributed to weight loss per se. The blunted postprandial response of GLP-1 observed in severely obese individuals (21) has improved after diet-induced weight loss (22). Other studies suggest that it is not the weight loss, but the surgical procedure, that is responsible for the improvement of glucose tolerance. Ileal transposition, which increases GLP-1 levels, results in improved glucose control (23) independently of weight loss in rodent models (24) as well as in humans (25).
The goal of this study was to determine whether the magnitude of the change of the incretin levels and effect is greater after GBP compared with a low caloric diet, under conditions of short-term equivalent weight loss, in morbidly obese patients with T2DM. Specifically, we measured the changes in GLP-1 and GIP levels after oral glucose stimulation, and their incretin effect on insulin, in obese patients with T2DM before and 1 month after GBP, or after an equivalent diet-induced weight loss. A second goal was to determine whether an equivalent weight loss, achieved by GBP or by diet, would result in the same improvement of blood glucose levels in patients with T2DM.
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Subjects and Methods |
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Obese patients with body mass index (BMI) above or equal to 35 kg/m2, eligible candidates for GBP, younger than 60 yr of age, of both genders and all ethnic groups, with T2DM diagnosed for less than 5 yr, not on insulin, thiazolidinedione, exenatide, or DPPIV inhibitors, with an glycosylated hemoglobin (HbA1c) less than 8%, were invited to participate in the study. All participants signed an informed consent, approved by our institution, before enrolling in the study. One group of patients was studied before and 1 month after GBP (surgical group). A second group of patients, fulfilling the same recruitment criteria, was studied before and after a 10-kg diet-induced weight loss (diet group). In addition, patients in the diet group were matched for age, weight, BMI, T2DM duration and control (HbA1c) to patients from the surgical group.
Diet-induced weight loss and diabetes treatment
The diet consisted of a meal replacement plan of 1000 kcal/d. A 1-wk supply of meal replacement products (Robard Corp., Mt. Laurel, NJ), including high-protein shakes, bars, fruit drinks, and soups, was given to each patient during an individual weekly visit at the General Clinical Research Center. Fresh fruits and vegetables were allowed. Body weight was measured weekly and the diet adjusted when necessary. If no weight loss, or if weight gain occurred at two consecutive weekly visits, the patients were excluded from the study. Patients were kept on the 1000-kcal diet and in negative energy balance (active weight loss) while retested for incretin levels and effect after a 10-kg weight loss. Although there was no time limit, the expectation was that patients would lose 10 kg in 4–8 wk. During the weight loss, patients were asked to monitor blood glucose levels by finger stick and keep logs. Diabetes medications were adjusted by a nurse educator or a diabetologist to avoid hypoglycemia and to fulfill the American Diabetes Association standard of treatment, based on fasting and postprandial glucose levels. In most cases, patients on sulfonylureas had their medication decreased or discontinued to avoid hypoglycemia.
Roux-en-Y gastric bypass (GBP) protocol
All patients underwent a laparoscopic GBP. In brief, the jejunum was divided 30 cm from the ligament of Treitz and anastomosed to a 30-ml proximal gastric pouch. The jejunum was reanastomosed 150-cm distal to the gastrojejunostomy. All mesenteric defects were closed. The post-GBP diet recommendations included a daily intake of 600–800 kcal, 70 g protein, and 1.8 liter fluid. This was achieved, on an individual basis, with multiple small meals and snacks with various commercial protein supplements. The diet after GBP was monitored by food records but not directly supervised. The diet in the few days preceding the testing in surgical or diet patients before weight loss was not controlled for.
Incretin effect: insulin secretion after oral and isoglycemic iv glucose load (IsoG IVGT)
Subjects were studied for the oral glucose tolerance test (OGTT) and IsoG IVGT in the morning after a 12-h overnight fast, on 2 different days, separated by less than 5 d.
Three-hour OGTT
All patients underwent first a 3-h OGTT with 50 g glucose (noncarbonated, in a total volume of 200 ml). After iv insertion, at 0800 h, subjects received orally 50 g glucose. Blood samples, collected on chilled EDTA tubes with added aprotinin (500 kallikrein inhibitory U/ml blood) and DPPIV inhibitor (LINCO Research, Inc., St. Charles, MO) (10 µl/ml blood), were centrifuged at 4 C before storage at –70 C.
IsoG IVGT
The goal of the IsoG IVGT was to expose the pancreas to blood glucose levels matched to the ones obtained during the OGTT in the same subject. Glucose (sterile 20% dextrose solution in water) was infused iv over 3 h using a Gemini pump (Gemini, Inc., St. Louis, MI). A sample of blood was collected every 5 min, using a contralateral antecubital iv catheter, then transferred in a microcentrifuge tube without any additive and centrifuged at bedside for immediate measure of glucose levels. The glucose infusion rate was adjusted to match the glucose concentrations obtained for the same patient during the OGTT at each time point for 3 h. For insulin levels, blood samples were collected every 15 min for the first 90 min, then every 30 min until 180'. During the OGTT and IsoG IVGT, the arm used for blood sampling was kept warm with a heating pad.
Incretin effect
The difference in β-cell responses (insulin total area under the curve or INS AUC (0–180') to the oral and isoglycemic iv glucose stimuli represents the action of the incretin factor expressed as the percentage of the physiological response to oral glucose, which is taken as the denominator (100%) (26). The formula is:
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Assays
Total GLP-1, an indicator of GLP secretion, was measured by RIA (LINCO Research) after plasma ethanol extraction. The intraassay and interassay coefficients of variation (CVs) were 3–6.5% and 4.7–8.8%, respectively. This assay cross-reacts 100% with GLP-17–36, GLP-19–36, and GLP-17–37 but does not cross-react with glucagon (0.2%), GLP-2 (<0.01%), or exendin (<0.01%). Active GLP-1, an indicator of GLP potential action, was measured by ELISA (LINCO Research). The intraassay and interassay CVs were 3–7% and 7–8%, respectively. The assay cross-reacts 100% with GLP-17–36 and GLP-17–37 but does not cross-react with GLP-19–36, glucagon, or GLP-2. Total GIP was measured by ELISA. The assay cross-reacts 100% with GIP 1–42 and GIP 3–42 but does not cross-react with GLP-1, GLP-2, oxyntomodulin, or glucagon. The intraassay and interassay CVs were 3.0–8.8% and 1.8–6.1%, respectively. Plasma insulin, C peptide, proinsulin, and glucagon concentrations were measured by RIA (LINCO Research) with an intraassay CV of 3–8% and interassay CV of 5.5–9%. The glucagon assay cross-reacts 100% with glucagon but cross-reacts less than 0.1% with oxyntomodulin. Glucose concentration was measured at the bedside by the glucose oxidase method (Beckman glucose analyzer; Beckman Coulter, Inc., Fullerton, CA). All hormonal and metabolites assays were performed at the Hormone and Metabolite Core Laboratory of the New York Obesity Research Center.
Statistical analysis
Outcome variables were plasma glucose and plasma insulin, C peptide, glucagon, proinsulin, GLP-1, and GIP concentrations. Total AUCs 0–180' for outcome variables were calculated using the trapezoidal method. ANOVA with repeated measures was used to detect glucose and hormonal changes over time during the OGTT within each condition, and for comparison before and after GBP and before and after diet, or between diet and surgical groups with T2DM. Paired t tests were used to compare data between before and after GBP or diet. Data are expressed as the mean ± SD, except in the figures where SEMs are used. Statistical significance was set at P < 0.05. Statistical analyses were performed with SPSS 14.0 (SPSS, Inc., Chicago, IL).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Subject characteristics are shown in Table 1
. Obese women with T2DM and normal liver enzymes, thyroid function tests, and blood pressure were studied before and 1 month after GBP (n = 9), and before and after an equivalent diet-induced weight loss (n = 10). Of the 12 women recruited in the diet group, two did not complete the weight loss due to pregnancy or breast cancer, and data from 10 diet completers are presented. Diabetes medications, sulfonylureas and/or metformin, were discontinued 3 d before being studied at baseline in all patients and were adjusted during the diet-induced weight loss to avoid hypoglycemia. Patients from the diet and surgical group were matched for age, body weight, BMI, diabetes duration and control (HbA1c) (Table 1). Before weight loss, neither fasting glucose (P = 0.874), proinsulin (P = 0.797), insulin (P = 0.629), C peptide (P = 0.589), glucagon (P = 0.363), GLP-1 (P = 0.832) and GIP (P = 0.414) and incretin effect (P = 0.245), nor stimulated variables during the OGTT were significantly different between the diet and GBP groups.
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Although 50 g glucose drink was used rather than 75 g to minimize the risk of dumping syndrome after GBP, four patients experienced stomach cramping and discomfort, nausea, sweating, flushing, and palpitations 5–20 min into the OGTT. No severe adverse effects were observed. There was no adverse effect from the diet.
Effect of weight loss
All patients in the surgical group discontinued their diabetes medications the day of the surgery. In the diet group, diabetes medications were either discontinued (n = 2), or the dosage was decreased, with patients taking only low doses of metformin (n = 8) at the completion of the weight loss. The duration of weight loss was shorter for the GBP group (32.3 ± 13.1 d) compared with the diet group (55.0 ± 9.9 d; P = 0.001). Body weight, BMI, fasting glucose, insulin, C peptide, proinsulin, proinsulin to insulin ratio, and homeostasis model of assessment of insulin resistance decreased significantly and equally in the surgical and diet groups (Table 1
). Fasting incretins did not change with either weight loss treatment.
Glucose AUC and glucose levels at 120' were significantly lower after GBP compared with diet (P = 0.014 and P = 0.001, respectively) (Table 1). Although the changes of insulin with weight loss (fasting, AUC, peak response) were not different between GBP and diet, the pattern of secretion of insulin changed considerably after GBP (P = 0.001), with recovery of the early phase with a peak at 30 min and a return to baseline after 180 min (Fig. 1).
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and Fig. 1). On the contrary, after diet, GLP-1 levels (total and active) tended to decrease (P, Not significant), and GIP did not change significantly. There was no change in GLP-1 or GIP levels during the IsoG IVGT (data not shown). A significant linear relationship was found between total GLP-1 release and insulin release during the OGTT, only in patients after GBP (r = 0.762; P = 0.028).
The glucose concentrations were well matched between the IsoG IVGT and OGTT in the surgical and diet groups before and after the weight loss intervention (Table 2). The insulin response was not greater after oral than iv glucose before GBP or before diet, with a resulting blunted incretin effect. The incretin effect increased significantly by a factor of 3.8 after GBP (+35.4 ± 22.7%; P = 0.009), but only minimally after diet (+7.15 ± 18.13%; P = 0.244).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The data from this study suggest that the effect of GBP on the incretins is likely not weight loss related. However, the mechanism by which the incretins increase after GBP remains unclear. Whether it is the rapid and direct stimulation of the L cells of the distal ileum, referred to as the hindgut mechanism, or the bypass of the duodenum (the foregut hypothesis) is still unclear. Elegant studies in rodents support the foregut hypothesis. In these studies in Goto-Kakizaki type 2 diabetic rats, the improvement of glucose tolerance after surgical exclusion of the duodenum, but not after gastrojejunostomy, is independent of calorie restriction or weight loss (33). The hindgut hypothesis is based on the results of experimental ileal transposition, a surgical procedure that improves diabetes in rodents (23) independently of weight loss (24). The foregut/hindgut hypothesis has not been tested in humans. Recent data demonstrate that ileal transposition with sleeve gastrectomy can improve diabetes, even after minimal weight loss, in patient with BMI less than 35 kg/m2 (34). Our experiment was designed to address the effect of weight loss on incretins and did not allow us to separate the effect of duodenal bypass vs. rapid stimulation of the distal gut on incretin stimulation after GBP. Gastric emptying (GE) and intestinal transit time have increased after GBP (27, 35), but not after diet (22). The release of GIP and GLP-1 is related to the rate of carbohydrate entry into the small intestine (36). Faster small intestinal glucose delivery increases plasma GIP and GLP-1 levels (37). The rapid delivery of nutrient after GBP could represent a mechanism by which incretins are markedly released after the surgery. We did not measure GE or intestinal transit time in our study. Therefore, we cannot exclude the possibility that, in the diet group, the glucose solution was absorbed entirely in the duodenum and did not reach the lower part of the ileum to exert its stimulating effect directly on the L cells to release GLP-1. However, recent data showed that the enteroendocrine K and L cells, which secrete GIP and GLP-1, respectively, are distributed all along the gut (38). Therefore, it is unlikely that the L cells would have had no contact with the glucose solution in the diet group.
We cannot exclude that gut adaptation played a role in the increased incretin response after GBP because hyperplasia of intestinal endocrine cells have been described 3 months after JIB (39). We have shown persistent increased GLP-1 levels 1 yr after GBP (32), and others have shown increased incretin levels 20 yr after JIB (31). Adaptative changes in gut motility have also been shown after a period of energy restriction and weight loss (40). We cannot exclude a role of calorie restriction per se, independently of weight loss, in the improvement of incretins after GBP. The daily calorie intake of patients after GBP was 600–800 kcal (data not shown) compared with 1000 kcal in the diet group. Although the calorie restriction was not matched between the two groups from day to day, the overall calorie deficit and weight loss were identical.
Diet-induced (41, 42, 43, 44, 45, 46) or surgical weight loss (3, 47) improves T2DM control. In this study the effect of an equivalent weight loss on diabetes control was greater after GBP than after diet. Patients in the surgical group had a better clinical outcome and did not require diabetes medications after the weight loss. In addition, postprandial glucose levels were lower after GBP compared with diet. In the fed state, GE (36, 37), glucose absorption (48), and the release of incretins (12) are key determinants of postprandial glucose levels. In patients with T2DM, many components of the gut physiology are impaired, such as GE (40, 49), and incretin release and effect (14), resulting in postprandial hyperglycemia, a predictor of cardiovascular complications and mortality (50). Our data show that a diet-equivalent weight loss does not lower postprandial glucose levels to the same extent as GBP in patients with T2DM. This may indicate that the marked increase of incretins associated with GBP, and not with the diet, could be responsible for the better postprandial glucose control. The administration of the synthetic exendin-4, a compound that binds to the GLP-1 receptor and exerts similar effects as the native GLP-1 (51), or of vildagliptin, an inhibitor of DPPIV, the enzyme that rapidly inactivates the endogenous incretins (52, 53), has been shown to reduce postprandial glucose levels and improve diabetes control.
Patients with T2DM have typically hyperglucagonemia (54), which contributes to the postprandial hyperglycemia. It is puzzling to see that the decrease of postprandial glucose levels after GBP is associated with a paradoxical increase of glucagon levels during the OGTT. The increase in glucagon is seen despite a marked increase in GLP-1, a gut hormone that inhibits glucagon release (55). This increase of glucagon levels was previously shown after GBP (56) and ileal transposition in dogs (56). The source of this increase in glucagon is unclear. Although the commercial assay used in this study is specific for pancreatic glucagon, cross-reactivity with enteroglucagon or oxyntomodulin cannot be entirely excluded. Our study group was small and limited to women. Future studies will need to address gender differences in incretin levels and effect after bariatric surgery.
In summary, our data suggest that it is the surgical procedure per se, rather than weight loss, that stimulates incretin release and effect after GBP. The rapid and marked increase of GLP-1 levels after GBP plays an important role in insulin secretion, and could be a key determinant in the decrease of postprandial glycemia and the resolution of T2DM after GBP.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: B.L. received grant support through the Merck Investigator Initiated Studies Program in 2007. J.T., J.M., H.T., J.R.E., A.C., B.K., B.B., N.K., H.L., K.Y., and B.O. have nothing to declare.
First Published Online April 22, 2008
Abbreviations: AUC, Area under the curve; BMI, body mass index; CV, coefficient of variation; DPPIV, dipeptidyl peptidase-IV; GBP, Gastric bypass surgery; GE, gastric emptying; GIP, gastric inhibitory peptide; GLP, glucagon-like peptide; HbA1c, glycosylated hemoglobin; IsoG, isoglycemic; IVGT, iv glucose test; JIB, jejunoileal bypass; OGTT, oral glucose tolerance test; T2DM, type 2 diabetes.
Received December 28, 2007.
Accepted April 10, 2008.
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