| Abstract |
Alterations in gut hormones following laparoscopic sleeve gastrectomy
INTRODUCTION
Sleeve gastrectomy (SG) was initially described as a first-step procedure followed by either biliopancreatic diversion with duodenal switch or Roux-en-Y gastric bypass in severely obese patients or in high-risk patients. Adjacently, laparoscopic SG (LSG) was introduced as a multi-purpose bariatric operation. More recently, SG has been indicated as a definitive treatment in patients with a BMI΄&γτ40 kg/m2 or BMI΄&γτ35 kg/m2 associated with co-morbidities, and it has also been proposed for patients with moderate obesity (BMI=30-35 kg/m2
The coordination of body weight and energy reserves is regulated by multiple interactions between the gastrointestinal tract, adipose tissue and the central nervous system. A number of peptides released from the gastrointestinal tract have been shown to regulate appetite and food intake, effecting both orexigenic and anorexic outcomes through actions on the hypothalamic arcuate nucleus. The aim of this prospective study was to investigate and analyze the potential effect of LSG on the entero-hypothalamic axis by examining fasting and meal-stimulated release of the gut hormones ghrelin, pancreatic polypeptide (PP), peptide-YY (PYY), glucagon-like peptide-1 (GLP-1) and amylin as well as of the adipocytokine leptin. ) -and metabolic syndrome. Although classified as a restrictive procedure, SG appears to be more than just a mechanical barrier to food consumption because the removal of the gastric fundus and part of the body of the stomach results in a significant reduction of serum ghrelin levels and alterations in gastric emptying.
METHODS
Patients
The study was performed according to the principles of the Declaration of Helsinki and was approved by the research and ethics committee of our hospital. Fifteen
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consecutive morbidly obese (MO) patients of Caucasian descent were prospectively enrolled in our study. All patients were evaluated preoperatively by a multidisciplinary team. The selection criteria for bariatric surgery were a BMI΄&γε40 kg/m
2 or BMI΄&γε35 kg/m2
A thorough discussion of the benefits of the operation, in conjunction with the individual characteristics of every patient, the postoperative quality of life, the expected weight loss, and the possible complications was performed in every case. Written, informed consent was obtained from each participant in the study. The LSG technique included removing the fundus and greater curvature portion of the stomach. The dissection of the stomach began with a linear stapler 5-6 cm proximal to the pylorus, alongside a calibration 34 Fr bougie up to the angle of Hiss. After the gastric transection, the excision line was reinforced with a running 2-0 polyglyconate monofilament suture. All LSGs were performed by the same surgical team. in the presence of significant co-morbidities that are ameliorated by weight loss. All patients had been obese for at least 5 years and had failed to achieve weight loss on an adequate weight control program. Exclusion criteria included psychiatric illness, substance abuse, and previous gastrointestinal surgery. As study controls, we evaluated 15 healthy, lean volunteers, without a dysmetabolic profile matched for age and sex.
. Body weight in kilograms (kg) and height in meters (m) were measured with a Detecto scale and a stadiometer and the BMI was calculated in kg/m2
Several parameters were evaluated and recorded preoperatively and at 6 and 12 months after LSG in the study group, before and after the consumption of a standard meal. The same parameters were recorded pre and postprandially in the lean group once. Venus blood samples were collected for the determination of glucose (GL), insulin (I), ghrelin, . The percentage of excess BMI loss (%EBL) was calculated using the formula %EBL = [(preoperative BMI-current BMI) / (preoperative BMI-25)] x 100.
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PP, PYY, GLP-1, amylin and leptin levels. All samples were collected at 3 different time points: after a 12 hour overnight fast (t
0) and 60 (t1) and 120 min (t2) after the consumption of a standard 447 kcal test meal (60.0% carbohydrate, 21.7% protein, and 18.3% fat). All blood tests were performed using serum samples assayed blindly under code. Venus blood was also collected at t0
Hormone Assays for measurement of glycosylated hemoglobin (HbA1c).
Blood was collected into tubes containing a protease inhibitor for active amylin (Sigma’s Protease inhibitor cocktail – P2714), DPPIV inhibitor for active GLP-1 (LINCO DPP4-010) and serine protease inhibitor for active ghrelin (Roche Pefabloc SC/ AEBSF – A154.2). Samples were allowed to clot for at least 30 minutes. After centrifugation at 4°C, serum samples were stored at -70°C until analysis. All hormones were measured in duplicate using a commercially available multiplex ELISA Kit (Miltiplex map Kit, Human Gut Hormone panel HGT-68K; Millipore Corp., St. Charles, Missouri 63304 U.S.A.) The panel measures all the hormones simultaneously. The intra- and inter-assay variability remained below 11% and 19%, respectively. The lower limit of detection was 1.8 pg/mL for active ghrelin, 8.4 pg/mL for total PYY, 5.2 pg/mL for active GLP-1, 2.4 pg/mL for PP, 31.5 pg/mL for active amylin and 157.2 pg/mL for leptin. All samples were measured at the same time using the same assay in order to reduce sources of variation. Serum insulin was assessed through a commercial chemiluminescence immunoassay (architect i2000, Abbott Diagnostics, USA). The HOMA index, which quantifies insulin resistance and β-cell function, was calculated according to the type: HOMA = [I0 (μUI/mL) x GL0
Statistical Analysis (mmol/L)] / 22.5.
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The statistical analysis of the data was performed using the SPSS 13.0 program (SPSS Inc., Chicago, IL, USA). The data are expressed as median and range. The data were compared using non-parametric tests. The Mann-Whitney U-test was used for comparisons between different groups (obese vs. controls) and the Wilcoxon paired test for comparisons between obese patients, before and after bariatric surgery. Differences at p΄&λτ0.05 were considered statistically significant. All p-values are two-tailed.
RESULTS
All patients tolerated the operation well and recovered within a short period of time. There were no conversions to open surgery. A significant reduction in the body weight of obese patients [130 kg (range 97-201 kg)] was evident at 6 [93 kg (range 75-180 kg)] and 12 [90 kg (range 69-167 kg)] months, postoperatively (p΄&λτ0.01). The reduction in BMI from 46.8 kg/m2 (range 37.6-58.7 kg/m2) to 34.6 kg/m2 (range 29.1-52.6 kg/m2) at 6 months and to 32.9 kg/m2 (range 26.1-48.8 kg/m2) at 12 months following LSG, was also significant (p΄&λτ0.01). Significant reduction in BMI between 6th and 12th
Lean vs MO group before and after weight loss. postoperative month was also observed (p΄&λτ0.01). This represents a median of 54.8 % EBL (range 18.0-73.0%) and a median 68.9 % EBL (range 19.0-94.0%) at 6 and 12 months after surgery, respectively (p=0.02 for the comparison between 6 and 12 months). No patient regained weight during the study period.
Although none of the patients was a known diabetic, when MO patients were compared to the lean controls before LSG, statistically significant differences in fasting glucose, insulin levels and HOMA index were detected.
Preoperatively, fasting ghrelin, GLP-1, PP, and PYY levels did not show significant differences between the MO patients and lean subjects. After the test meal, no differences between the 2 groups were observed for GLP1, PP, PYY levels. Yet, higher
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ghrelin levels in MO patients were found at 60 minutes postprandially. Before weight-loss fasting and postprandial levels of serum amylin and leptin in the MO patients were significantly higher than in the lean controls.
Postoperatively, significant difference between lean individuals and MO patients following weight loss, at 6 and 12 months was observed only in leptin levels (p΄&λτ0.01 for all the comparisons before and after the test-meal), which remained higher in the MO group. No statistically significant difference was noticed on fasting and postprandial levels of ghrelin, PP, PYY, GLP-1 and amylin between lean controls and MO patients both at 6 and 12 months following LSG.
Changes after LSG in the MO group
LSG resulted not only in significant body weight loss but also in an overall improvement of the glycemic profile of MO patients (table 2). The HbA1c decreased from 5.1% (range 4.0-6.4%) preoperatively to 4.8% (range 4.0-5.9%) at 6 months (p=0.02) and to 4.2% (range 4.0-5.4%) at 12 months (p=0.01), postoperatively. The HOMA index was also reduced significantly from 1.98 (range 0.51-5.71) to 1.26 (range 0.54-3.15) at 6 months (p=0.013) and to 1.12 (range 0.35-1.61) at 12 months (p=0.004), postoperatively. Fasting insulin levels were decreased, though not significantly, at 6 and 12 months following LSG. However, surgery induced a significant decrease in postprandial insulin secretion both at 6 (p=0.003 at 60 min and p=0.028 at 120 min) and 12 months (p=0.04 at 60 min and p=0.028 at 120 min), postoperatively.
Postoperatively, markedly decreased fasting ghrelin levels at 6 months (p=0.036) and 12 months (p=0.011) were noted in comparison to the preoperative values. Additionally, significantly decreased postprandial ghrelin levels were observed after LSG with even lower levels both at 6 and 12 months postoperatively (p=0.012 and p=0.008 at
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60 min and p=0.017 and p=0.028 at 120 min, for the comparisons with the preoperative values, respectively).
Interestingly, while no statistically significant changes were observed postoperatively in fasting PP levels, significantly increased levels were noted at 60 min after the test meal, both at 6 (p=0.012) and 12 months (p=0.028) after LSG.
No significant difference was noticed between fasting pre- and postoperative PYY levels. However, a considerable postprandial response resulted in increased levels of PYY both at 6 months (p=0.018 at 60 min and p=0.025 at 120 min) and 12 months (p=0.008 at 60 min and p=0.011 at 120 min), postoperatively.
Fasting GLP-1 levels did not significantly change after the operation. However, MO patients had an exaggerated postprandial GLP-1 response at 6 months (p=0.018 at 60 min and p=0.049 at 120 min), which further increased at 12 months (p=0.008 at 60 min and 120 min), postoperatively.
At 6 and 12 months after the operation, a remarkable reduction in fasting amylin levels was observed (p=0.018 and p=0.028, respectively). Postoperatively, meal-stimulated concentrations of amylin were also significantly reduced (p=0.012 and p=0.028 at 60 min and p=0.012 and p=0.038 at 120 min) at 6 and 12 months, respectively.
A significant reduction in fasting leptin levels was also noticed at 6 (p=0.012) and at 12 months (p=0.012), postoperatively. Similar changes were observed in postprandial leptin levels at 6 months (p=0.012 at 60 min and 120 min) and at 12 months (p=0.008 at 60 min and at 120 min), postoperatively.
DISCUSSION
Our main objective was to examine changes in the circulating levels of gastrointestinal hormones after LSG and their role in the underlying mechanism of weight loss.
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Ghrelin is a potent orexigenic polypeptide hormone that is produced predominantly from endocrine cells in the oxyntic glands of the gastric fundus. A number of cross-sectional and prospective studies have demonstrated that basal ghrelin levels in obese subjects are lower than in lean subjects. In our study, no difference was observed between lean and obese subjects at baseline, preoperatively. However, significantly increased levels of ghrelin were noted in the MO group compared to the lean group 60 min after the test meal. This result is consistent with previous findings that obese individuals demonstrate reduced postprandial ghrelin suppression compared to normal-weight individuals, suggesting that ghrelin is involved in the pathophysiology of obesity. LSG led to significantly decreased levels of serum ghrelin, a potent orexigenic hormone, at up to 12 months postoperatively. Meal-induced ghrelin release was also diminished postoperatively. In this series of LSG procedures, the greater curvature and the gastric fundus were completely resected. The gastric fundus is the major food storage compartment and the upper part of the body of the stomach, including the “gastric pacemaker” as well as the main locus of ghrelin production. The reduced ghrelin response may be one of the mechanisms contributing to the overall effectiveness and negative energy balance observed after LSG. Thus, LSG may be understood as a food-limiting procedure augmented by the reduction of ghrelin-producing tissue. On the contrary, studies on the effect of Roux-en-Y gastric bypass, an operation where the gastric fundus is not resected, on ghrelin levels have been conflicting.
PP is synthesized and released mainly by the pancreatic polypeptide cells of the pancreatic islets of Langerhans and has been associated with decreased food intake in humans. The circulating levels of PP rises postprandially in proportion to the ingested calorie load. Differences in circulating levels of PP between lean and obese individuals have been variable. In our study, while no difference was observed in fasting PP levels
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postoperatively, markedly increased levels were noticed at 60 min postprandially both at 6 and 12 months after the operation, suggesting that LSG may lead to changes in the levels of PP secreted by the pancreas. Previous reports have shown that gastric emptying for solids occurs faster at 6 months following SG, and this was found to persist 2 years thereafter. This accelerated gastric emptying after LSG, which peaks at approximately 60 min postprandially, may partially influence the increased pancreatic secretion of PP. The increased early postprandial PP response may have an effect on food intake and energy metabolism after LSG.
PYY is a satiety hormone secreted predominantly from the distal gut, particularly the ileum, colon and rectum. The enteroendocrine L-cells of the intestine release PYY in proportion to the amount of calories ingested at a meal. Studies of circulating levels of PYY in obese and lean people have been conflicting. No difference between fasting levels of total PYY was noted postoperatively in our study. After the test-meal stimulation, an exaggerated postprandial response was observed, resulting in increased levels of total PYY both at 6 and 12 months after the operation. It is known that the secretion of PYY is not altered by gastric distension and does not depend upon intact vagal input. Meal-stimulated PYY release in humans occurs primarily in response to incompletely digested nutrients, particularly fats, reaching the ileum. Thus, in the case of LSG, we could hypothesize that accelerated gastric emptying, which promotes the quicker passage of nutrients to the small intestine, in combination with other neurohormonal pathways modulated by other gut peptides may trigger such a response. The increased postprandial secretion of PYY may contribute to reduced food intake and to the ability of an individual to maintain weight loss after LSG.
GLP-1 is released from the lower-intestinal endocrine L-cells in response to ingested nutrients along with PYY. These peptides are believed to act synergistically with
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other postprandial gastrointestinal signals and cause satiation, leading to meal termination. Moreover, GLP-1 has a potent incretin effect by stimulating insulin secretion in a glucose-dependent manner. Clinical and preclinical studies have shown that GLP-1 promotes satiety and weight loss. Previous reports have shown that obese subjects have a blunted postprandial response compared with lean subjects. In our study, no difference was observed between lean and obese subjects, preoperatively. Fasting GLP-1 levels did not change significantly after the operation. However, an exaggerated postprandial GLP-1 response was observed postoperatively. The presence of indigested nutrients in the intestinal lumen, particularly carbohydrates and fat, seem to be the primary stimulus for GLP-1 secretion, which is dependent on a minimal caloric threshold. After nutrient ingestion, GLP-1 is rapidly secreted, a response that seems paradoxical, -given the distal location of the majority of L-cells. Therefore, after LSG, in addition to the neuroendocrine mechanisms that are proposed to be involved in postprandial GLP-1 secretion, this type of response may be enhanced by the rapid arrival of the food bolus at the level of the small intestine. The postprandial amplified response of GLP-1 may be associated not only with the weight loss but also with the improvement in glucose metabolism noted after LSG.
Amylin is co-secreted from pancreatic β-cells in equimolar quantities with insulin. Although the physiological basis of the relationship remains to be elucidated, previous reports are generally consistent with a role for amylin signaling in the sequence of peripherally and centrally mediated events that determine meal size and termination. In addition to its role as a satiation factor, amylin meets the definition of an adiposity signal. Similar to leptin and insulin, the basal circulating levels of amylin correlates positively with adipose mass. Basal circulating levels of amylin are higher in obese than in lean subjects. In healthy subjects, plasma amylin concentrations rise rapidly and by several
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fold in response to meals, whereas in obese individuals with impaired glucose tolerance, the postprandial increase of amylin is additively higher. The exact mechanism of amylin-mediated weight loss remains undefined. In rodents and in humans, the exogenous administration of an amylin agonist has been shown to dose-dependently reduce food intake and body weight by affecting the size and duration of meals. In our study, obese subjects had fasting preoperative amylin levels significantly higher than the lean subjects. Postoperatively, a significant reduction was observed in fasting and postprandial amylin levels. The pattern of levels of insulin and amylin observed in response to test-meal stimulation changed similarly after LSG, with increased early-phase and decreased late-phase secretion. Amylin, co-secreted with insulin, not only reflects β-cell function but may also exert an effect on insulin secretion and glucose metabolism.
There is a growing body of evidence supporting the existence of amylin and leptin interactions in the regulation of energy homeostasis. Ghrelin has also been shown to antagonize leptin action through the activation of a hypothalamic receptor pathway. Moreover, an interaction between leptin and the enteroinsular axis has been reported. Leptin, which is produced almost exclusively by adipocytes, inhibits food intake and positively correlates with BMI and body fat content. Human obesity is characterized by high plasma leptin levels because of the increase in adipose tissue mass. Several reports, though, have suggested that leptin resistance, rather than leptin production, may contribute to the development of obesity. In our study, obese subjects had preoperatively more than 1500% higher fasting leptin levels than the lean group. Remarkably, decreased fasting leptin levels were observed at 6 and 12 months after the operation. No significant changes in leptin levels were observed postprandially, pre- or postoperatively, a finding which is consistent with previous studies.
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Our study demonstrates improvement in glucose metabolism and significant changes in ghrelin, PP, PYY, GLP-1, amylin and leptin levels after LSG. These findings are consistent with recent studies suggesting that LSG is not just a restrictive procedure. Mounting evidence suggests that the significant improvement in insulin resistance, also noted after LSG by our study group, is a result not only of the reduction in fat mass and weight loss but also of gut hormonal changes induced by SG.
Obesity develops despite a complex and seemingly well-orchestrated network of signals that control eating, energy expenditure and, ultimately, body weight. Many of the involved signals derive from the gastrointestinal tract. The mechanisms of weight loss and improvement in glucose metabolism observed after SG are related not only to gastric restriction but also to neurohormonal changes related to gastric resection and altered gastric emptying. Our data, in addition to those demonstrating analogous effects on gut peptides involved in satiety responses, demonstrate a potential but as yet unproven link between altered motor function of the gut and appetite regulation after LSG. After LSG, nutrients pass from the stomach to the intestine more rapidly and a number of gastrointestinal signals are released. Recent studies indicate that the upper intestine plays a critical role in the regulation of food intake and glucose homeostasis by the activation of an intestine-brain-liver axis. Furthermore, novel data have demonstrated that intestinal gluconeogenesis and its hepatoportal sensor pathway may constitute a key mechanistic link in the central effects of satiety induced by food protein and may also have a key regulatory role in glucose homeostasis. These signals, in coordination with gut hormones and adipocytokines, act to optimize the digestive process, and some also function as short-term satiety signals and possibly, long-term regulators of body weight. Thus, besides its well-described effects on gastric motility and the secretion of ghrelin, LSG may have direct metabolic effects on other tissues and organs, such as the intestine and
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adipose tissue. Therefore, LSG may be considered not only as a food-limiting operation but also as a metabolic bariatric procedure.
Potential limitations of the present study are the relatively small number of subjects and the fact that food intake and satiety in obese subjects are triggered by multiple factors; therefore, more than one mechanism may account for the clustering outcome. Still, we attempted to explore the effects of LSG in islet- and gut-derived hormones as well as in adipocyte-derived long-term adiposity signals. In addition, we cannot definitely exclude the possibility that the observed changes in gut hormones could be also due to weight loss and not only due to anatomical and physiological gastrointestinal changes related to the procedure. Further investigations, particularly on a cohort of patients who regain weight after LSG, will be most insightful in order to clarify if the observed changes are more likely to be based on the anatomy and physiology of the operation than the physiology of weight loss alone.
CONCLUSION
LSG generates multiple hormonal actions that may have several beneficial effects on the underlying mechanism of weight loss. Longer follow-up is required to evaluate if changes in the enterohormonal profile of MO after LSG persist and therefore, to assess the lasting beneficial effect of surgical intervention on these hormonal alterations. Further studies are required to determine the complex mechanisms through which the gut neurohormonal signals interrelate after LSG.
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