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Probing the mechanisms of the metabolic effects of weight loss surgery in humans using a novel mouse model system
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Available online at www.sciencedirect.com
journal homepage: www.JournalofSurgicalResearch.com
Probing the mechanisms of the metabolic effects of weight loss surgery in humans using a novel mouse model system John Kucharczyk a, Eirini Nestoridi a, Stephanie Kvas a, Robert Andrews b, Nicholas Stylopoulos a,* a
Center for Basic and Translational Obesity Research, Division of Endocrinology, Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts b Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts
article info
abstract
Article history:
Background: Gastrointestinal weight loss surgery, especially Roux-en-Y gastric bypass
Received 10 November 2011
(RYGB), is the most effective treatment for severe obesity. RYGB is associated with
Received in revised form
a remarkable decrease in the rate of death from obesity-related complications, such as
17 January 2012
diabetes mellitus, coronary artery disease, and cancer. Dissecting the mechanisms of RYGB
Accepted 17 February 2012
effects could augment our understanding about the pathogenesis of obesity and its
Available online 10 March 2012
complications. Objectives and methods: In this study, we describe in detail a mouse model of RYGB that
Keywords:
closely reproduces the surgical steps of the human procedure.
Gastric bypass
Results: We show that RYGB in mice has the same effects as in human patients, proving the
Metabolism
high translational validity of this model system. We present an intraoperative video to
Animal models
facilitate the widespread use of this complex and difficult method.
Rodents
Conclusions: The study of the mechanisms of RYGB using this model system can greatly
Obesity
facilitate our understanding about the effects of RYGB in human patients. The reverse
RYGB
engineering of the physiological mechanisms of RYGB could lead to discovery of new, effective, and less invasive treatments. ª 2013 Elsevier Inc. All rights reserved.
1.
Introduction
Although we have made great strides in reducing important health problems, such as smoking and hypertension, we have been unable to find effective solutions for obesity. This trend is particularly disturbing because obesity promotes the development of more than 50 chronic disorders, costs more than $50 billion in lost worker productivity every year, accounts for approximately 9% of all U.S. health care
expenditures (more than $125 billion annually), and can shorten life expectancy by more than 10 y [1e3]. Although many treatment options have been proposed, few have demonstrated substantial long-term effectiveness. For severe and medically complicated obesity, gastrointestinal weight loss surgery (GIWLS), especially Roux-en-Y gastric bypass (RYGB), is currently the most effective treatment available, leading to profound weight loss, improvement or resolution of diabetes mellitus, amelioration of other comorbidities, and
* Corresponding author. Center for Basic and Translational Obesity Research, Division of Endocrinology, Children’s Hospital Boston, Harvard Medical School, Boston, MA, USA. Tel.: þ1 617 919 2714; fax: +1 617 730 0856. E-mail address: [email protected] (N. Stylopoulos). 0022-4804/$ e see front matter ª 2013 Elsevier Inc. All rights reserved. doi:10.1016/j.jss.2012.02.036
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prolonged survival [4e9]. Despite its obvious benefits, however, GIWLS is associated with appreciable morbidity and mortality that limits its overall application. Understanding the mechanisms by which RYGB works could strongly facilitate the development of alternate, less invasive means of achieving the same beneficial effects, which could therefore be used in a much broader subset of individuals with obesity. The mechanisms by which RYGB induces its therapeutic effects remain largely unknown. Nonetheless, recent clinical observations and the exploitation of surgery in animal models have begun to provide important clues. Recently, we and others have shown that RYGB in humans and rats induces weight loss by altering physiologic regulation of metabolic function [10e16]. More specifically, RYGB alters ingestive behavior, increases energy expenditure, changes neuroendocrine activity, improves glucose and lipid metabolism, and prolongs survival [9,11e14,17e21]. The growing need and interest in defining the mechanisms underlying these effects would be aided by the development and standardization of experimental model systems for sophisticated experimentation and analysis. Because GIWLS induces weight loss by altering regulation of metabolic function, examination of the effects of weight loss surgery in genetically engineered animals could help dissect the mechanisms underlying the physiologic effects of these procedures. Recognizing the need for a valid and reproducible model system of RYGB and the potential of its applications, we sought to develop a mouse model for RYGB. In this study, we present the technical aspects of the mouse RYGB procedure in sufficient detail to facilitate reproduction and use of this model by a broad range of obesity investigators. To this end, we also provide an intraoperative video of the whole procedure in the Supplementary Data section. In addition, we discuss the long-term effects of RYGB on body weight, food intake, body composition, and glucose and lipid metabolism, demonstrating that this model of RYGB closely reproduces the effects of RYGB observed in humans and is associated with long-term health and survival, normal behavior, substantial and sustained weight loss, and improved glucose metabolism.
2.
Methods
2.1.
Animals
All experiments were performed in compliance with and were approved by the Institutional Animal Care and Use Committee of Children’s Hospital Boston. Animals were individually housed and were maintained on 12-h lightedark cycle (lights on at 07:00 h) in a facility with an ambient temperature of 19 Ce22 C and 40%e60% humidity.
2.1.1.
C57BL/6 mice
We used C57BL/6 mice (C57BL/6Ntac, Taconic Farms Inc., Germantown, NY) for all studies. Obesity was induced in these mice by feeding the animals ad libitum with a high fat diet (HFD) that provides 60% of total energy as fat, 20% as carbohydrate, and 20% as protein (D12492 diet, Research Diets Inc., New Brunswick, NJ) for 20 wk from weaning.
2.2.
Surgical procedures
Animals were fasted overnight. They were maintained on inhalation anesthesia throughout the preparation and the operation, using a scavenged mask circuit of isoflurane 1%e4% to effect (Viking Medical, Medford Lakes, NJ). Standard sterile procedures were followed in all operations. The operating field was covered with a fenestrated drape, and the shaved abdomen was prepared with an alcohol-based solution. Sterile surgical gloves and gowns were used. On the day of the operation, mice were kept without food and water. Food was reintroduced 48e72 h after the operation, depending on the observed health and behavior of each animal. A liquid diet was provided first, and the animals were switched to solid food (the same HFD consumed before the operation) beginning on postoperative day 7. Mice tolerated the postoperative protocol well with no sign of distress. For postoperative analgesia, we used buprenorphine (0.1 mg/kg subcutaneously). The first dose was given as soon as the animal was anesthetized, and additional doses were given every 8 h for the first three postoperative days and then as needed thereafter.
2.2.1.
Sleeve gastrectomy
A midline abdominal incision was made extending about twothirds the length of the abdomen to the xiphoid cartilage and a self-retaining retractor was placed. The liver was gently retracted cranially, using 3-inch cotton tip applicators. A sleeve was created along the lesser curvature carefully preserving the gastroesophageal junction and the pylorus, by transecting the stomach. The sleeve was hand sewn, using an 8e0 continuous suture. The surgical incision was closed with 5e0 silk sutures in two layers.
2.2.2.
Gastric plication
The procedure was very similar to sleeve gastrectomy, and the same steps were followed. The only difference was that the sleeve was constructed by plicating the greater curvature of the stomach with interrupted 5e0 silk sutures.
2.2.3.
Surgical supplies and instruments
Surgical instruments used for the procedures included the following: a mouse retractor, a microsurgery needle holder, fine forceps, and microsurgery scissors. An operating microscope was required; we used a surgical microscope with a foot pedal to allow adjustment without compromising sterility.
2.2.4.
Sham operation
In all experiments, mice undergoing a sham operation were used as controls. The sham operation consisted of laparotomy, jejunal transection, and reanastomosis and was performed on groups of mice that were carefully age and weight matched with mice undergoing RYGB.
2.3.
Metabolic assessments
All metabolic assessments were performed 8e10 wk after surgery. All experiments were performed after at least 2 wk of acclimation to each of the procedures used.
2.3.1.
Body composition
For body composition studies, mice were anesthetized by intraperitoneal (i.p.) injection of a solution containing
j o u r n a l o f s u r g i c a l r e s e a r c h 1 7 9 ( 2 0 1 3 ) e 9 1 ee 9 8
ketamine (100 mg/kg) and acepromazine (2.5 mg/kg). Body composition was assessed by dual-energy X-ray absorptiometry (DEXA) using a DEXA Scanner (Lunar/GE Medical Systems, Madison, WI). After the scanner was calibrated for bone mineral density and percent fat content using the “phantom mouse,” mice were placed ventral side down on disposable plastic trays. DEXA scans were analyzed using the PIXImus2TM software with the head region excluded from the analysis.
2.3.2.
Body weight and food intake
Body weight was measured weekly using a bench-top laboratory scale of the appropriate size and dimensions for the animals. For the assessment of food intake, mice were individually housed in cages with elevated racks without bedding. Food intake was determined each day by weighing the amount of food remaining from the previous day’s allowance. Food intake also was measured during the energy expenditure assessment in the metabolic system.
2.3.3.
Histology
Tissues were immersed in 10% formalin. After 24 h of fixation, the excised tissue was inserted into cassettes, processed to paraffin blocks, microtome sectioned to 6 microns and stained with hematoxylin and eosin (H&E) or Oil Red O staining for microscopic examination. Lipid area was calculated using ImageJ software (http://rsbweb.nih.gov/ij/).
2.3.4.
Glucose metabolism
For the glucose tolerance testing (GTT), animals were fasted for 16 h and blood was obtained by tail stick with a lancet. Blood glucose level was determined using a hand-held glucometer (Lifescan, Milpitas, CA). GTT was performed after administration of 1 g/kg body weight D50 glucose solution by i.p. injection. Glucose tolerance was assessed by calculating the area under glucose excursion curves (AUC), using the trapezoidal rule. Animals were considered acclimated to the procedure if there was a minimal change in glucose levels immediately and 20 min after i.p. injection with saline. For the measurement of fasting and glucose-stimulated insulin levels, we used an ELISA kit (Alpco, Salem, NH). Homeostasis Model Assessment (HOMA) for insulin resistance (IR) was calculated based on the following equation: HOMA IR ¼
Fasting glucose Fasting insulin 135
Fasting insulin is given in pmol/L, and fasting glucose is given in mmol/L. Insulin is initially measured in ng/mL. To convert to pmol/L, we divided the ng/mL by 0.0058. Glucose was initially measured in mg/dL. To convert to mmol/L, the mg/dL values were divided by 18.
2.4.
Statistical analysis
Analysis was performed using PASW Statistics 18.0 (IBM, Armonk, NY) and Prism 5 software. All values are presented as mean standard error of the mean (GraphPad Software Inc., La Jolla, CA). The Mann-Whitney U test of PASW Statistics 18.0 was used to analyze the differences between sham-operated and RYGB-treated groups. The PASW Statistics 18.0 repeated measures analysis of variance was applied to analyze the
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progression of body weight over time, among the different study groups. A P < 0.05 was used for statistical significance.
3.
Results
3.1.
Description of the mouse RYGB model
The mouse RYGB model was based on the rat RYGB procedure [12], with significant modification to accommodate the smaller size of anatomy of the mouse GI tract (Fig. 1). A step-by-step description of the surgical steps can be found in the intraoperative video presented in the Supplementary Data section. After the animal was anesthetized, a 1.5e2 cm midline abdominal incision was made using fine scissors and a selfretaining retractor was placed. We then determined the approximate length of the Roux and biliopancreatic limbs. The operation in human patients usually includes a rearrangement of 20%e30% of the small intestine (i.e., Roux limb [RL] and biliopancreatic limb each about 10%e15% of total intestinal length). The total length of the small intestine was measured, the ligament of Treitz identified, and the jejunum divided at the appropriate distance downstream of this ligament. The RL was brought up to the level of gastric fundus. A 2e3 mm incision was made along the antimesenteric intestinal wall at the appropriate distance downstream of the jejunal transection. An endto-side jejunojejunostomy was created with an 8e0 prolene or nylon running suture. Using a cotton tip applicator, the liver was gently retracted cranially and the stomach was pulled caudally. Mice have a proximal rumen (nonglandular portion of the stomach) that is separated from the glandular portion by a white ridge. The greater and lesser curvatures of the stomach were dissected free, cutting their attachments with cotton tip applicators and fine scissors or forceps. A 2e3 mm incision was made in the gastric wall at the most lateral part of the forestomach, and a gastrojejunostomy was created with an 8e0 prolene or nylon running suture. The forestomach was then restricted, and a pouch was created with transverse placement of a surgical clip (titanium clips, product# LT400, Ethicon Inc., Cincinnati, OH) using a surgical clip applier (Fig. 1B). To secure the clip in place, two 4e0 silk sutures were placed around the clip and through the anterior and the posterior wall of the stomach. The laparotomy was closed with a 7e0 silk suture in two layers. The gastric pouch (GP) can also be created by transecting the stomach and closing the distal and proximal end with an 8e0 nylon running suture (Fig. 1E). Based on our experience, the major advantages of the surgical clip technique in comparison with hand-sewn technique in constructing the GP are the following: (1) less operative time (the approximate operative time for the surgical clip and hand-sewn technique is 45 and 60 min, respectively), (2) easier technique, and (3) reduced number of complications leading to better mortality rates (the mortality rate for the surgical clip and hand-sewn technique is 10% and 30%, respectively). The major complication of the hand-sewn technique is injury of the gastric artery leading to intraoperative hemorrhage. However, the surgical clip technique is associated with the disadvantage that the constructed GP is proportionally larger (approximately 30% of the total gastric volume) in comparison with the GP that is constructed in the human operation (approximately 5% of the
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Fig. 1 e Intraoperative snapshots of the mouse RYGB model. (A) Intraoperative photograph showing the operative field before the closure of the abdomen and after returning the stomach and the intestine to their normal intra-abdominal location. (B) Using a clip applicator, a surgical clip is placed above the junction of the forestomach with the glandular stomach and the GP is created (left). The operating field after clip application through surgical microscope (right). (C) Intraoperative photograph after completion of the operation showing the gastrojejunostomy, jejunojejunostomy, RL, and biliopancreatic limb. (D) Upper GI contrast study using gastrograffin after RYGB. (E) The GP can also be constructed by transecting the stomach and closing the distal and proximal end with an 8e0 nylon running suture. DS [ distal stomach; C [ clip separating GP from DS. total gastric volume). The hand-sewn technique offers the advantage that the GP can be designed and constructed to be, proportionally, as small as the human pouch.
3.2. The mouse RYGB model exhibits substantial weight loss To determine whether our operation replicates the significant weight loss observed in humans, we performed the operation on 10 diet-induced obese C57BL/6 mice (body weight 45 1 g) and compared this outcome with that of 10 age- and weightmatched mice that underwent a sham operation. As shown in Fig. 2A, RYGB in these mice induced a substantial and sustained weight loss. In the immediate postoperative period (2 wk), RYGB-treated and sham-operated mice lost 34 2.1% and 24 3.3% of their preoperative weight, respectively. This initial weight loss reflects both the catabolic effect of surgery and the postoperative feeding protocol used. After this period, the operated mice started to regain weight, suggesting that they had recovered well. Weight curves were a highly sensitive sign of health: in the initial stages of the development of the model, animals that developed complications, such as abscesses or partial obstruction failed to regain weight. RYGB-treated mice weighed 37% less than shamoperated controls (32.8 0.7 versus 52.2 0.4 g; P < 0.001, Fig. 2A) 8 wk after surgery. None of the mice after RYGB became cachectic as all mice weighed at least 28 g, which is the average,
age-adjusted weight for chow-fed lean C57BL/6 mice. Thus, despite being on an HFD after surgery, RYGB normalized the body weight of these mice. This normalization of body weight suggests that the observed weight loss is unlikely to be an adverse effect of the operation but is rather a physiologic effect of surgery. The difference in the volume of the GP did not affect the body weight data. The weight loss observed was similar in mice that had undergone RYGB with the surgical clip technique of constructing the GP to those with the hand-sewn technique. Similar to the effects of RYGB on humans, RYGB was the most effective operation in mice, inducing greater weight loss than gastric volume reduction procedures, such as sleeve gastrectomy and gastric plication (Fig. 2B), where a tube-like gastric canal is constructed, by either removing (sleeve gastrectomy) or plicating (invaginating) the greater curvature part of the stomach.
3.3. The mouse RYGB model exhibits selective reduction of fat mass To determine body composition after RYGB, we performed DEXA in mice that underwent an RYGB or a sham operation. RYGB-treated mice had a substantially lower fat mass compared with sham-operated controls (%body fat: 20.1 1.7 versus 48.5 1.5%; P < 0.001) (Fig. 2C). There was no difference in fat-free mass between the two groups of mice. Histologic assessment of tissues derived from RYGB-treated mice showed changes similar to the ones observed in human
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Fig. 2 e The mouse RYGB model exhibits substantial weight loss. (A) Weight changes after RYGB and sham operation. (B) RYGB was the most effective operation in mice. RYGB-treated animals lost more weight than animals that underwent sleeve gastrectomy (SGx) and gastric plication (GP). (C) Comparison of fat and fat-free mass after RYGB and sham operation. RYGB was associated with substantially less fat, without change in the fat-free mass (left panel). Screenshot (right) captured from the Lunar Piximus densitometer showing a mouse 6 mo after a sham operation (upper panel) or RYGB (lower panel). The radiopaque surgical clip that separates the proximal GP from the distal stomach (DS) in the RYGB-treated mouse is visible.
patients, after RYGB, such as significant reduction in subcutaneous fat depots and significant decrease in the accumulation of fat in the liver (Fig. 3).
3.4. The mouse model exhibits improved glucose metabolism and lipid profile Obesity in humans and in C57BL/6 mice is complicated with type 2 diabetes mellitus and dyslipidemia characterized by elevated triglycerides and low-density lipoprotein cholesterol and total cholesterol levels. To determine whether our RYGB mouse model reproduces the effects on glucose and lipid metabolism observed in human patients after RYGB, we measured several indicators of metabolic function (Fig. 4AeD). Mean fasting glucose decreased by 45% after RYGB (87 3.9 versus 158 8.7 mg/dL; P < 0.001) (Fig. 4A). RYGB-treated mice also exhibited substantial improvement in GTT. In a 120-min i.p. GTT, plasma glucose levels at each of the sampling time points were significantly lower in the RYGB group than in controls (Fig. 4B). The AUC was 45% lower in the RYGB group than in
sham-operated animals (1191 111 versus 2147 231; P < 0.001) (Fig. 4B). Insulin sensitivity also improved as evident by the improved response to insulin administration on insulin tolerance test (Fig. 4C). This also was confirmed by calculating the HOMA-IR, which was 5.8 times better in RYGB-treated animals (Fig. 4C inset). A well-documented effect of RYGB on humans is the increase in glucagon-like peptide 1 (GLP-1). This also was observed in our mouse model (Fig. 4D). The lipid profile also improved substantially after RYGB, and the total cholesterol, low-density lipoprotein cholesterol, and triglyceride levels were 42% (5.2 0.3 versus 3.1 0.2 mmol/L), 40% (3 0.19 versus 1.8 0.3 mmol/L), and 30% (0.7 0.03 versus 0.5 0.07 nmol/L) lower than the respective levels in sham-operated controls.
4.
Discussion
In this study, we present a novel, feasible, and valid translational model of RYGB that replicates the steps of the
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Fig. 3 e The mouse RYGB model exhibits decreased fat content in liver and subcutaneous adipose tissue. Histologic studies of the liver and subcutaneous adipose tissue in RYGB-treated and sham-operated mice. Despite consuming an HFD, RYGB-treated mice exhibited a reduction in fat accumulation. (A) H&E staining of liver sections. A decreased accumulation of fat was seen after RYGB. (B) The decreased fat content was confirmed by Oil Red O staining of liver sections (the lipid droplets are orange colored; please refer to the online version for colors). (C) H&E staining of subcutaneous adipose tissue sections. A reduction of fat stores was seen after RYGB.
operation performed in humans and also reproduces the effects of RYGB observed in humans. Postoperatively, the animals live normal, healthy lives, exhibiting a substantial weight loss and a similar metabolic profile to human patients that have undergone the procedure, with significant improvement in glucose and lipid metabolism.
This novel animal model could allow investigators to exploit the advantages of mouse systems to determine the mechanisms of action of RYGB. Mouse models have played an essential role in biological research, and the use of genetically manipulated mice has been invaluable for understanding the molecular basis of numerous human conditions, including obesity and diabetes mellitus. To date, however, the mouse has not been widely used as a model for studying the effects of GIWLS on weight regulation. Several investigators have examined the effects of gastrectomy, vagotomy, and short bowel syndrome in mice, but more complex GI weight loss procedures have rarely been studied using mouse models [22e30]. The limited use of mouse models of GIWLS likely results from the technical challenges associated with the performance of those complex procedures in a small-sized animal. Another hindrance to the widespread use of these models may be the traditional inference that weight loss procedures, including RYGB, induce their effects by direct mechanical means (i.e., malabsorption and/or gastric restriction). Recent clinical observations and physiological studies, however, have shown that the effects of RYGB cannot be explained by simple mechanical restriction and/or malabsorption. Rather, RYGB appears to alter the basic physiological mechanisms of weight regulation, leading to (1) diminished hunger, (2) increased or accelerated experience of satiety after meals, (3) altered release of various GI hormones that regulate food intake (e.g., GLP-1, peptide YY, gastric inhibitory polypeptide, and ghrelin), (4) increased energy expenditure, and (5) improved glucose tolerance by both weight loss-dependent and independent mechanisms [4,5,17,20,31e37]. The observation in this study that the body weight loss was similar in mice that have a GP that is 5% of the total gastric volume (the mice in which the hand-sewn technique was used to create the GP) or 30% of the total gastric volume (the mice in which the surgical clip was used) further suggests that RYGB does not work through mechanical means and that the size of the GP may not be a significant contributor to the observed RYGBinduced weight loss. This is further supported by studies showing that operations, in which the total volume of the stomach remains intact and no GP is created (such as the duodenojejunal bypass), induce significant weight loss in rodents. Understanding the mechanisms by which surgery works to reduce body weight and improve metabolic function will have profound effects on our understanding of obesity and diabetes mellitus and the development of new effective therapies for these diseases. If surgery worked primarily by mechanical mechanisms, its effects could be reproduced only by mechanical means. Because the effects of GIWLS appear to be primarily physiological, knowledge of the underlying physiological mechanisms will allow the development of alternative approaches that mimic the physiology of surgery. These approaches could include less invasive surgical or endoscopic techniques, devices, drugs, and nutritional manipulation. To systematically explore the mechanisms underlying the effects of surgery, we need a reliable, reproducible, and highly manipulable experimental model system. The laboratory mouse meets these criteria and allows exploitation of the genetic manipulation of these novel surgical models. Using the mouse as the experimental
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Fig. 4 e The mouse RYGB model exhibits improved glucose metabolism. (A) Fasting blood glucose levels after RYGB or sham operation. (B) Glucose excursion curves after i.p. injection of glucose after RYGB or sham operation. The inset shows the AUC for the i.p. GTT. AUC was calculated using the trapezoidal rule. (C) Insulin tolerance test performed after i.p. injection of insulin. HOMA-IR (inset) confirmed that RYGB-treated mice exhibited higher insulin sensitivity. (D) GLP-1 levels in RYGB and sham-operated animals. * denotes a statistically significant difference.
platform for surgical manipulation provides a unique opportunity for a detailed examination of the molecular effects and mechanisms of surgery because we can take advantage of the widely available genetic technologies to overexpress, silence, and/or reactivate a gene, globally or locally. Such studies could facilitate the identification of critical molecular components required for surgery to exert its effects. These molecular components would likely be attractive targets for the development of new, less invasive treatments for obesity, diabetes mellitus, and other metabolic conditions that are corrected by surgery. In summary, we have shown that our mouse RYGB model exhibits healthy weight loss without adverse behavioral effects or cachexia, and with improvements of glucose and lipid metabolism. The similar effects of RYGB in humans, rats, and mice demonstrate that the effects of RYGB are conserved across species, further suggesting that RYGB affects fundamental physiological mechanisms of weight regulation and metabolism. The ability to combine surgical with genetic and/ or pharmacological manipulation should accelerate efforts to understand the physiological mechanisms of action of RYGB and facilitate the development of therapies that exploit these mechanisms.
Acknowledgments Conflict of interest: None.
Appendix. Supplementary material Supplementary data related to this article can be found online at doi:10.1016/j.jss.2012.02.036.
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[22] Chandarana K, Gelegen C, Karra E, et al. Diet and gastrointestinal bypass-induced weight loss: the roles of ghrelin and peptide YY. Diabetes 2011;60:810. [23] Troy S, Soty M, Ribeiro L, et al. Intestinal gluconeogenesis is a key factor for early metabolic changes after gastric bypass but not after gastric lap-band in mice. Cell Metab 2008;8:201. [24] Stenstrom B, Loseth K, Bevanger L, et al. Gastric bypass surgery does not increase susceptibility to Helicobacter pylori infection in the stomach of rat or mouse. Inflammopharmacology 2005;13:229. [25] Dornonville de la Cour C, Lindqvist A, Egecioglu E, et al. Ghrelin treatment reverses the reduction in weight gain and body fat in gastrectomised mice. Gut 2005;54:907. [26] Erlanson-Albertsson C, Lindqvist A. Vagotomy and accompanying pyloroplasty down-regulates ghrelin mRNA but does not affect ghrelin secretion. Regul Pept 2008;151:14. [27] Bray GA. Hypothalamic and genetic obesity: An appraisal of the autonomic hypothesis and the endocrine hypothesis. Int J Obes 1984;8:119. [28] Schlager A, Khalaileh A, Mintz Y, et al. A mouse model for sleeve gastrectomy: Applications for diabetes research. Microsurgery 2011;31:66. [29] Zhang H, Wang Y, Zhang J, et al. Bariatric surgery reduces visceral adipose inflammation and improves endothelial function in type 2 diabetic mice. Arterioscler Thromb Vasc Biol 2011;31:2063. [30] Yin DP, Gao Q, Ma LL, et al. Assessment of different bariatric surgeries in the treatment of obesity and insulin resistance in mice. Ann Surg 2011;254:73. [31] Korner J, Bessler M, Cirilo LJ, et al. Effects of Roux-en-Y gastric bypass surgery on fasting and postprandial concentrations of plasma ghrelin, peptide YY, and insulin. J Clin Endocrinol Metab 2005;90:359. [32] Laferrere B, Heshka S, Wang K, et al. Incretin levels and effect are markedly enhanced 1 month after Roux-en-Y gastric bypass surgery in obese patients with type 2 diabetes. Diabetes Care 2007;30:1709. [33] le Roux CW, Aylwin SJ, Batterham RL, et al. Gut hormone profiles following bariatric surgery favor an anorectic state, facilitate weight loss, and improve metabolic parameters. Ann Surg 2006;243:108. [34] Adami GF, Campostano A, Gandolfo P, et al. Body composition and energy expenditure in obese patients prior to and following biliopancreatic diversion for obesity. Eur Surg Res 1996;28:295. [35] Carrasco F, Papapietro K, Csendes A, et al. Changes in resting energy expenditure and body composition after weight loss following Roux-en-Y gastric bypass. Obes Surg 2007;17:608. [36] Rubino F, Forgione A, Cummings DE, et al. The mechanism of diabetes control after gastrointestinal bypass surgery reveals a role of the proximal small intestine in the pathophysiology of type 2 diabetes. Ann Surg 2006;244:741. [37] Rubino F, Gagner M, Gentileschi P, et al. The early effect of the Roux-en-Y gastric bypass on hormones involved in body weight regulation and glucose metabolism. Ann Surg 2004; 240:236.
Available online at www.sciencedirect.com
journal homepage: www.JournalofSurgicalResearch.com
Probing the mechanisms of the metabolic effects of weight loss surgery in humans using a novel mouse model system John Kucharczyk a, Eirini Nestoridi a, Stephanie Kvas a, Robert Andrews b, Nicholas Stylopoulos a,* a
Center for Basic and Translational Obesity Research, Division of Endocrinology, Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts b Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts
article info
abstract
Article history:
Background: Gastrointestinal weight loss surgery, especially Roux-en-Y gastric bypass
Received 10 November 2011
(RYGB), is the most effective treatment for severe obesity. RYGB is associated with
Received in revised form
a remarkable decrease in the rate of death from obesity-related complications, such as
17 January 2012
diabetes mellitus, coronary artery disease, and cancer. Dissecting the mechanisms of RYGB
Accepted 17 February 2012
effects could augment our understanding about the pathogenesis of obesity and its
Available online 10 March 2012
complications. Objectives and methods: In this study, we describe in detail a mouse model of RYGB that
Keywords:
closely reproduces the surgical steps of the human procedure.
Gastric bypass
Results: We show that RYGB in mice has the same effects as in human patients, proving the
Metabolism
high translational validity of this model system. We present an intraoperative video to
Animal models
facilitate the widespread use of this complex and difficult method.
Rodents
Conclusions: The study of the mechanisms of RYGB using this model system can greatly
Obesity
facilitate our understanding about the effects of RYGB in human patients. The reverse
RYGB
engineering of the physiological mechanisms of RYGB could lead to discovery of new, effective, and less invasive treatments. ª 2013 Elsevier Inc. All rights reserved.
1.
Introduction
Although we have made great strides in reducing important health problems, such as smoking and hypertension, we have been unable to find effective solutions for obesity. This trend is particularly disturbing because obesity promotes the development of more than 50 chronic disorders, costs more than $50 billion in lost worker productivity every year, accounts for approximately 9% of all U.S. health care
expenditures (more than $125 billion annually), and can shorten life expectancy by more than 10 y [1e3]. Although many treatment options have been proposed, few have demonstrated substantial long-term effectiveness. For severe and medically complicated obesity, gastrointestinal weight loss surgery (GIWLS), especially Roux-en-Y gastric bypass (RYGB), is currently the most effective treatment available, leading to profound weight loss, improvement or resolution of diabetes mellitus, amelioration of other comorbidities, and
* Corresponding author. Center for Basic and Translational Obesity Research, Division of Endocrinology, Children’s Hospital Boston, Harvard Medical School, Boston, MA, USA. Tel.: þ1 617 919 2714; fax: +1 617 730 0856. E-mail address: [email protected] (N. Stylopoulos). 0022-4804/$ e see front matter ª 2013 Elsevier Inc. All rights reserved. doi:10.1016/j.jss.2012.02.036
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prolonged survival [4e9]. Despite its obvious benefits, however, GIWLS is associated with appreciable morbidity and mortality that limits its overall application. Understanding the mechanisms by which RYGB works could strongly facilitate the development of alternate, less invasive means of achieving the same beneficial effects, which could therefore be used in a much broader subset of individuals with obesity. The mechanisms by which RYGB induces its therapeutic effects remain largely unknown. Nonetheless, recent clinical observations and the exploitation of surgery in animal models have begun to provide important clues. Recently, we and others have shown that RYGB in humans and rats induces weight loss by altering physiologic regulation of metabolic function [10e16]. More specifically, RYGB alters ingestive behavior, increases energy expenditure, changes neuroendocrine activity, improves glucose and lipid metabolism, and prolongs survival [9,11e14,17e21]. The growing need and interest in defining the mechanisms underlying these effects would be aided by the development and standardization of experimental model systems for sophisticated experimentation and analysis. Because GIWLS induces weight loss by altering regulation of metabolic function, examination of the effects of weight loss surgery in genetically engineered animals could help dissect the mechanisms underlying the physiologic effects of these procedures. Recognizing the need for a valid and reproducible model system of RYGB and the potential of its applications, we sought to develop a mouse model for RYGB. In this study, we present the technical aspects of the mouse RYGB procedure in sufficient detail to facilitate reproduction and use of this model by a broad range of obesity investigators. To this end, we also provide an intraoperative video of the whole procedure in the Supplementary Data section. In addition, we discuss the long-term effects of RYGB on body weight, food intake, body composition, and glucose and lipid metabolism, demonstrating that this model of RYGB closely reproduces the effects of RYGB observed in humans and is associated with long-term health and survival, normal behavior, substantial and sustained weight loss, and improved glucose metabolism.
2.
Methods
2.1.
Animals
All experiments were performed in compliance with and were approved by the Institutional Animal Care and Use Committee of Children’s Hospital Boston. Animals were individually housed and were maintained on 12-h lightedark cycle (lights on at 07:00 h) in a facility with an ambient temperature of 19 Ce22 C and 40%e60% humidity.
2.1.1.
C57BL/6 mice
We used C57BL/6 mice (C57BL/6Ntac, Taconic Farms Inc., Germantown, NY) for all studies. Obesity was induced in these mice by feeding the animals ad libitum with a high fat diet (HFD) that provides 60% of total energy as fat, 20% as carbohydrate, and 20% as protein (D12492 diet, Research Diets Inc., New Brunswick, NJ) for 20 wk from weaning.
2.2.
Surgical procedures
Animals were fasted overnight. They were maintained on inhalation anesthesia throughout the preparation and the operation, using a scavenged mask circuit of isoflurane 1%e4% to effect (Viking Medical, Medford Lakes, NJ). Standard sterile procedures were followed in all operations. The operating field was covered with a fenestrated drape, and the shaved abdomen was prepared with an alcohol-based solution. Sterile surgical gloves and gowns were used. On the day of the operation, mice were kept without food and water. Food was reintroduced 48e72 h after the operation, depending on the observed health and behavior of each animal. A liquid diet was provided first, and the animals were switched to solid food (the same HFD consumed before the operation) beginning on postoperative day 7. Mice tolerated the postoperative protocol well with no sign of distress. For postoperative analgesia, we used buprenorphine (0.1 mg/kg subcutaneously). The first dose was given as soon as the animal was anesthetized, and additional doses were given every 8 h for the first three postoperative days and then as needed thereafter.
2.2.1.
Sleeve gastrectomy
A midline abdominal incision was made extending about twothirds the length of the abdomen to the xiphoid cartilage and a self-retaining retractor was placed. The liver was gently retracted cranially, using 3-inch cotton tip applicators. A sleeve was created along the lesser curvature carefully preserving the gastroesophageal junction and the pylorus, by transecting the stomach. The sleeve was hand sewn, using an 8e0 continuous suture. The surgical incision was closed with 5e0 silk sutures in two layers.
2.2.2.
Gastric plication
The procedure was very similar to sleeve gastrectomy, and the same steps were followed. The only difference was that the sleeve was constructed by plicating the greater curvature of the stomach with interrupted 5e0 silk sutures.
2.2.3.
Surgical supplies and instruments
Surgical instruments used for the procedures included the following: a mouse retractor, a microsurgery needle holder, fine forceps, and microsurgery scissors. An operating microscope was required; we used a surgical microscope with a foot pedal to allow adjustment without compromising sterility.
2.2.4.
Sham operation
In all experiments, mice undergoing a sham operation were used as controls. The sham operation consisted of laparotomy, jejunal transection, and reanastomosis and was performed on groups of mice that were carefully age and weight matched with mice undergoing RYGB.
2.3.
Metabolic assessments
All metabolic assessments were performed 8e10 wk after surgery. All experiments were performed after at least 2 wk of acclimation to each of the procedures used.
2.3.1.
Body composition
For body composition studies, mice were anesthetized by intraperitoneal (i.p.) injection of a solution containing
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ketamine (100 mg/kg) and acepromazine (2.5 mg/kg). Body composition was assessed by dual-energy X-ray absorptiometry (DEXA) using a DEXA Scanner (Lunar/GE Medical Systems, Madison, WI). After the scanner was calibrated for bone mineral density and percent fat content using the “phantom mouse,” mice were placed ventral side down on disposable plastic trays. DEXA scans were analyzed using the PIXImus2TM software with the head region excluded from the analysis.
2.3.2.
Body weight and food intake
Body weight was measured weekly using a bench-top laboratory scale of the appropriate size and dimensions for the animals. For the assessment of food intake, mice were individually housed in cages with elevated racks without bedding. Food intake was determined each day by weighing the amount of food remaining from the previous day’s allowance. Food intake also was measured during the energy expenditure assessment in the metabolic system.
2.3.3.
Histology
Tissues were immersed in 10% formalin. After 24 h of fixation, the excised tissue was inserted into cassettes, processed to paraffin blocks, microtome sectioned to 6 microns and stained with hematoxylin and eosin (H&E) or Oil Red O staining for microscopic examination. Lipid area was calculated using ImageJ software (http://rsbweb.nih.gov/ij/).
2.3.4.
Glucose metabolism
For the glucose tolerance testing (GTT), animals were fasted for 16 h and blood was obtained by tail stick with a lancet. Blood glucose level was determined using a hand-held glucometer (Lifescan, Milpitas, CA). GTT was performed after administration of 1 g/kg body weight D50 glucose solution by i.p. injection. Glucose tolerance was assessed by calculating the area under glucose excursion curves (AUC), using the trapezoidal rule. Animals were considered acclimated to the procedure if there was a minimal change in glucose levels immediately and 20 min after i.p. injection with saline. For the measurement of fasting and glucose-stimulated insulin levels, we used an ELISA kit (Alpco, Salem, NH). Homeostasis Model Assessment (HOMA) for insulin resistance (IR) was calculated based on the following equation: HOMA IR ¼
Fasting glucose Fasting insulin 135
Fasting insulin is given in pmol/L, and fasting glucose is given in mmol/L. Insulin is initially measured in ng/mL. To convert to pmol/L, we divided the ng/mL by 0.0058. Glucose was initially measured in mg/dL. To convert to mmol/L, the mg/dL values were divided by 18.
2.4.
Statistical analysis
Analysis was performed using PASW Statistics 18.0 (IBM, Armonk, NY) and Prism 5 software. All values are presented as mean standard error of the mean (GraphPad Software Inc., La Jolla, CA). The Mann-Whitney U test of PASW Statistics 18.0 was used to analyze the differences between sham-operated and RYGB-treated groups. The PASW Statistics 18.0 repeated measures analysis of variance was applied to analyze the
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progression of body weight over time, among the different study groups. A P < 0.05 was used for statistical significance.
3.
Results
3.1.
Description of the mouse RYGB model
The mouse RYGB model was based on the rat RYGB procedure [12], with significant modification to accommodate the smaller size of anatomy of the mouse GI tract (Fig. 1). A step-by-step description of the surgical steps can be found in the intraoperative video presented in the Supplementary Data section. After the animal was anesthetized, a 1.5e2 cm midline abdominal incision was made using fine scissors and a selfretaining retractor was placed. We then determined the approximate length of the Roux and biliopancreatic limbs. The operation in human patients usually includes a rearrangement of 20%e30% of the small intestine (i.e., Roux limb [RL] and biliopancreatic limb each about 10%e15% of total intestinal length). The total length of the small intestine was measured, the ligament of Treitz identified, and the jejunum divided at the appropriate distance downstream of this ligament. The RL was brought up to the level of gastric fundus. A 2e3 mm incision was made along the antimesenteric intestinal wall at the appropriate distance downstream of the jejunal transection. An endto-side jejunojejunostomy was created with an 8e0 prolene or nylon running suture. Using a cotton tip applicator, the liver was gently retracted cranially and the stomach was pulled caudally. Mice have a proximal rumen (nonglandular portion of the stomach) that is separated from the glandular portion by a white ridge. The greater and lesser curvatures of the stomach were dissected free, cutting their attachments with cotton tip applicators and fine scissors or forceps. A 2e3 mm incision was made in the gastric wall at the most lateral part of the forestomach, and a gastrojejunostomy was created with an 8e0 prolene or nylon running suture. The forestomach was then restricted, and a pouch was created with transverse placement of a surgical clip (titanium clips, product# LT400, Ethicon Inc., Cincinnati, OH) using a surgical clip applier (Fig. 1B). To secure the clip in place, two 4e0 silk sutures were placed around the clip and through the anterior and the posterior wall of the stomach. The laparotomy was closed with a 7e0 silk suture in two layers. The gastric pouch (GP) can also be created by transecting the stomach and closing the distal and proximal end with an 8e0 nylon running suture (Fig. 1E). Based on our experience, the major advantages of the surgical clip technique in comparison with hand-sewn technique in constructing the GP are the following: (1) less operative time (the approximate operative time for the surgical clip and hand-sewn technique is 45 and 60 min, respectively), (2) easier technique, and (3) reduced number of complications leading to better mortality rates (the mortality rate for the surgical clip and hand-sewn technique is 10% and 30%, respectively). The major complication of the hand-sewn technique is injury of the gastric artery leading to intraoperative hemorrhage. However, the surgical clip technique is associated with the disadvantage that the constructed GP is proportionally larger (approximately 30% of the total gastric volume) in comparison with the GP that is constructed in the human operation (approximately 5% of the
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Fig. 1 e Intraoperative snapshots of the mouse RYGB model. (A) Intraoperative photograph showing the operative field before the closure of the abdomen and after returning the stomach and the intestine to their normal intra-abdominal location. (B) Using a clip applicator, a surgical clip is placed above the junction of the forestomach with the glandular stomach and the GP is created (left). The operating field after clip application through surgical microscope (right). (C) Intraoperative photograph after completion of the operation showing the gastrojejunostomy, jejunojejunostomy, RL, and biliopancreatic limb. (D) Upper GI contrast study using gastrograffin after RYGB. (E) The GP can also be constructed by transecting the stomach and closing the distal and proximal end with an 8e0 nylon running suture. DS [ distal stomach; C [ clip separating GP from DS. total gastric volume). The hand-sewn technique offers the advantage that the GP can be designed and constructed to be, proportionally, as small as the human pouch.
3.2. The mouse RYGB model exhibits substantial weight loss To determine whether our operation replicates the significant weight loss observed in humans, we performed the operation on 10 diet-induced obese C57BL/6 mice (body weight 45 1 g) and compared this outcome with that of 10 age- and weightmatched mice that underwent a sham operation. As shown in Fig. 2A, RYGB in these mice induced a substantial and sustained weight loss. In the immediate postoperative period (2 wk), RYGB-treated and sham-operated mice lost 34 2.1% and 24 3.3% of their preoperative weight, respectively. This initial weight loss reflects both the catabolic effect of surgery and the postoperative feeding protocol used. After this period, the operated mice started to regain weight, suggesting that they had recovered well. Weight curves were a highly sensitive sign of health: in the initial stages of the development of the model, animals that developed complications, such as abscesses or partial obstruction failed to regain weight. RYGB-treated mice weighed 37% less than shamoperated controls (32.8 0.7 versus 52.2 0.4 g; P < 0.001, Fig. 2A) 8 wk after surgery. None of the mice after RYGB became cachectic as all mice weighed at least 28 g, which is the average,
age-adjusted weight for chow-fed lean C57BL/6 mice. Thus, despite being on an HFD after surgery, RYGB normalized the body weight of these mice. This normalization of body weight suggests that the observed weight loss is unlikely to be an adverse effect of the operation but is rather a physiologic effect of surgery. The difference in the volume of the GP did not affect the body weight data. The weight loss observed was similar in mice that had undergone RYGB with the surgical clip technique of constructing the GP to those with the hand-sewn technique. Similar to the effects of RYGB on humans, RYGB was the most effective operation in mice, inducing greater weight loss than gastric volume reduction procedures, such as sleeve gastrectomy and gastric plication (Fig. 2B), where a tube-like gastric canal is constructed, by either removing (sleeve gastrectomy) or plicating (invaginating) the greater curvature part of the stomach.
3.3. The mouse RYGB model exhibits selective reduction of fat mass To determine body composition after RYGB, we performed DEXA in mice that underwent an RYGB or a sham operation. RYGB-treated mice had a substantially lower fat mass compared with sham-operated controls (%body fat: 20.1 1.7 versus 48.5 1.5%; P < 0.001) (Fig. 2C). There was no difference in fat-free mass between the two groups of mice. Histologic assessment of tissues derived from RYGB-treated mice showed changes similar to the ones observed in human
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Fig. 2 e The mouse RYGB model exhibits substantial weight loss. (A) Weight changes after RYGB and sham operation. (B) RYGB was the most effective operation in mice. RYGB-treated animals lost more weight than animals that underwent sleeve gastrectomy (SGx) and gastric plication (GP). (C) Comparison of fat and fat-free mass after RYGB and sham operation. RYGB was associated with substantially less fat, without change in the fat-free mass (left panel). Screenshot (right) captured from the Lunar Piximus densitometer showing a mouse 6 mo after a sham operation (upper panel) or RYGB (lower panel). The radiopaque surgical clip that separates the proximal GP from the distal stomach (DS) in the RYGB-treated mouse is visible.
patients, after RYGB, such as significant reduction in subcutaneous fat depots and significant decrease in the accumulation of fat in the liver (Fig. 3).
3.4. The mouse model exhibits improved glucose metabolism and lipid profile Obesity in humans and in C57BL/6 mice is complicated with type 2 diabetes mellitus and dyslipidemia characterized by elevated triglycerides and low-density lipoprotein cholesterol and total cholesterol levels. To determine whether our RYGB mouse model reproduces the effects on glucose and lipid metabolism observed in human patients after RYGB, we measured several indicators of metabolic function (Fig. 4AeD). Mean fasting glucose decreased by 45% after RYGB (87 3.9 versus 158 8.7 mg/dL; P < 0.001) (Fig. 4A). RYGB-treated mice also exhibited substantial improvement in GTT. In a 120-min i.p. GTT, plasma glucose levels at each of the sampling time points were significantly lower in the RYGB group than in controls (Fig. 4B). The AUC was 45% lower in the RYGB group than in
sham-operated animals (1191 111 versus 2147 231; P < 0.001) (Fig. 4B). Insulin sensitivity also improved as evident by the improved response to insulin administration on insulin tolerance test (Fig. 4C). This also was confirmed by calculating the HOMA-IR, which was 5.8 times better in RYGB-treated animals (Fig. 4C inset). A well-documented effect of RYGB on humans is the increase in glucagon-like peptide 1 (GLP-1). This also was observed in our mouse model (Fig. 4D). The lipid profile also improved substantially after RYGB, and the total cholesterol, low-density lipoprotein cholesterol, and triglyceride levels were 42% (5.2 0.3 versus 3.1 0.2 mmol/L), 40% (3 0.19 versus 1.8 0.3 mmol/L), and 30% (0.7 0.03 versus 0.5 0.07 nmol/L) lower than the respective levels in sham-operated controls.
4.
Discussion
In this study, we present a novel, feasible, and valid translational model of RYGB that replicates the steps of the
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Fig. 3 e The mouse RYGB model exhibits decreased fat content in liver and subcutaneous adipose tissue. Histologic studies of the liver and subcutaneous adipose tissue in RYGB-treated and sham-operated mice. Despite consuming an HFD, RYGB-treated mice exhibited a reduction in fat accumulation. (A) H&E staining of liver sections. A decreased accumulation of fat was seen after RYGB. (B) The decreased fat content was confirmed by Oil Red O staining of liver sections (the lipid droplets are orange colored; please refer to the online version for colors). (C) H&E staining of subcutaneous adipose tissue sections. A reduction of fat stores was seen after RYGB.
operation performed in humans and also reproduces the effects of RYGB observed in humans. Postoperatively, the animals live normal, healthy lives, exhibiting a substantial weight loss and a similar metabolic profile to human patients that have undergone the procedure, with significant improvement in glucose and lipid metabolism.
This novel animal model could allow investigators to exploit the advantages of mouse systems to determine the mechanisms of action of RYGB. Mouse models have played an essential role in biological research, and the use of genetically manipulated mice has been invaluable for understanding the molecular basis of numerous human conditions, including obesity and diabetes mellitus. To date, however, the mouse has not been widely used as a model for studying the effects of GIWLS on weight regulation. Several investigators have examined the effects of gastrectomy, vagotomy, and short bowel syndrome in mice, but more complex GI weight loss procedures have rarely been studied using mouse models [22e30]. The limited use of mouse models of GIWLS likely results from the technical challenges associated with the performance of those complex procedures in a small-sized animal. Another hindrance to the widespread use of these models may be the traditional inference that weight loss procedures, including RYGB, induce their effects by direct mechanical means (i.e., malabsorption and/or gastric restriction). Recent clinical observations and physiological studies, however, have shown that the effects of RYGB cannot be explained by simple mechanical restriction and/or malabsorption. Rather, RYGB appears to alter the basic physiological mechanisms of weight regulation, leading to (1) diminished hunger, (2) increased or accelerated experience of satiety after meals, (3) altered release of various GI hormones that regulate food intake (e.g., GLP-1, peptide YY, gastric inhibitory polypeptide, and ghrelin), (4) increased energy expenditure, and (5) improved glucose tolerance by both weight loss-dependent and independent mechanisms [4,5,17,20,31e37]. The observation in this study that the body weight loss was similar in mice that have a GP that is 5% of the total gastric volume (the mice in which the hand-sewn technique was used to create the GP) or 30% of the total gastric volume (the mice in which the surgical clip was used) further suggests that RYGB does not work through mechanical means and that the size of the GP may not be a significant contributor to the observed RYGBinduced weight loss. This is further supported by studies showing that operations, in which the total volume of the stomach remains intact and no GP is created (such as the duodenojejunal bypass), induce significant weight loss in rodents. Understanding the mechanisms by which surgery works to reduce body weight and improve metabolic function will have profound effects on our understanding of obesity and diabetes mellitus and the development of new effective therapies for these diseases. If surgery worked primarily by mechanical mechanisms, its effects could be reproduced only by mechanical means. Because the effects of GIWLS appear to be primarily physiological, knowledge of the underlying physiological mechanisms will allow the development of alternative approaches that mimic the physiology of surgery. These approaches could include less invasive surgical or endoscopic techniques, devices, drugs, and nutritional manipulation. To systematically explore the mechanisms underlying the effects of surgery, we need a reliable, reproducible, and highly manipulable experimental model system. The laboratory mouse meets these criteria and allows exploitation of the genetic manipulation of these novel surgical models. Using the mouse as the experimental
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Fig. 4 e The mouse RYGB model exhibits improved glucose metabolism. (A) Fasting blood glucose levels after RYGB or sham operation. (B) Glucose excursion curves after i.p. injection of glucose after RYGB or sham operation. The inset shows the AUC for the i.p. GTT. AUC was calculated using the trapezoidal rule. (C) Insulin tolerance test performed after i.p. injection of insulin. HOMA-IR (inset) confirmed that RYGB-treated mice exhibited higher insulin sensitivity. (D) GLP-1 levels in RYGB and sham-operated animals. * denotes a statistically significant difference.
platform for surgical manipulation provides a unique opportunity for a detailed examination of the molecular effects and mechanisms of surgery because we can take advantage of the widely available genetic technologies to overexpress, silence, and/or reactivate a gene, globally or locally. Such studies could facilitate the identification of critical molecular components required for surgery to exert its effects. These molecular components would likely be attractive targets for the development of new, less invasive treatments for obesity, diabetes mellitus, and other metabolic conditions that are corrected by surgery. In summary, we have shown that our mouse RYGB model exhibits healthy weight loss without adverse behavioral effects or cachexia, and with improvements of glucose and lipid metabolism. The similar effects of RYGB in humans, rats, and mice demonstrate that the effects of RYGB are conserved across species, further suggesting that RYGB affects fundamental physiological mechanisms of weight regulation and metabolism. The ability to combine surgical with genetic and/ or pharmacological manipulation should accelerate efforts to understand the physiological mechanisms of action of RYGB and facilitate the development of therapies that exploit these mechanisms.
Acknowledgments Conflict of interest: None.
Appendix. Supplementary material Supplementary data related to this article can be found online at doi:10.1016/j.jss.2012.02.036.
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