BACKGROUND:The gold standard for treatment of obesity remains bariatric surgery. The mechanisms underlying the effectiveness of these operations remain incompletely understood. Rodent models of these surgeries have proven essential to efforts to elucidate these mechanisms. The most commonly used such model is the mouse vertical sleeve gastrectomy (VSG). OBJECTIVE:This study aimed to evaluate technical approaches and complications of mouse VSG. SETTING:Academic research laboratory, United States. METHODS:Wild-type male mice were subjected to variations of VSG. Important metabolic outcomes, including body weight, body composition, and glucose tolerance, were assessed at relevant time points postoperation. A necropsy was performed on all mice at 2 months postsurgery to evaluate the gastric sleeve for any complications. RESULTS:Chronic, contained gastric leak is a relatively high-frequency complication after mouse VSG. A scoring system was developed to quantify and sort mice with leaks. Data from mouse cohorts performed by different methods demonstrate that no surgical approach used in the study could prevent the risk of leak. It shows that significant leaks impact commonly measured metabolic parameters after VSG, likely secondary to the effects of chronic local and systemic inflammation. CONCLUSIONS:The presence of chronic gastric leak is common after mouse VSG. The effects of these leaks are likely to confound data analysis when using this surgical model. The use of the scoring and sorting approach developed and described in this study provides an important tool for quality control of murine VSG studies. Careful elimination of mice with this complication enables the effective and reliable use of this surgical model.

Regenerating islet-derived protein 3 gamma (Reg3g), a gut peptide has been implicated in host defense and various physiological functions including metabolic regulation. Emerging evidence has demonstrated that peripheral administration of Reg3g results in improved glucose regulation as a gut hormone. In this study, we explored the therapeutic potential of Reg3g through intraduodenal infusion in mouse models of metabolic disorders. The objective of this study was to test the hypothesis that administered Reg3g into the intestinal lumen contributes to metabolic improvements by enhancing gut barrier function. Our mouse studies revealed that duodenal infusion of Reg3g reduces gut permeability and systemic endotoxemia. Studies with intestinal organoids supported the role of Reg3g in preserving cellular integrity and antioxidant gene expression under fructose-induced stress. Although Reg3g treatment results in little change to body weight, food intake, or glucose tolerance, Reg3g-treated mice exhibited reduced hepatic lipid accumulation along with the downregulation of lipogenic pathway genes. These data point toward the positive impact of Reg3g administration through intraduodenal infusion to regulate the intricate cross talk between gut barrier function and hepatic steatosis with the gut-liver axis. NEW & NOTEWORTHY This study shows that intraduodenal administration of the gut peptide, regenerating islet-derived protein 3 g (Reg3g), reduces hepatic lipid accumulation, improves gut barrier function, and lowers systemic endotoxemia in mouse models of metabolic disorders. These findings elucidate the therapeutic benefits of Reg3g administration into the gut.

Obesity promotes triple-negative breast cancer (TNBC), and effective interventions are urgently needed to break the obesity-TNBC link. Epidemiologic studies indicate that bariatric surgery reduces TNBC risk, while evidence is limited or conflicted for weight loss via low-fat diet (LFD) or calorie restriction (CR). Using a murine model of obesity-driven TNBC, we compared the antitumor effects of vertical sleeve gastrectomy (VSG) with LFD, chronic CR, and intermittent CR. Each intervention generated weight and fat loss and suppressed tumor growth relative to obese mice (greatest suppression with CR). VSG and CR regimens exerted both similar and unique effects, as assessed using multiomics approaches, in reversing obesity-associated transcript, epigenetics, secretome, and microbiota changes and restoring antitumor immunity. Thus, in a murine model of TNBC, bariatric surgery and CR each reverse obesity-driven tumor growth via shared and distinct antitumor mechanisms, and CR is superior to VSG in reversing obesity's procancer effects.

Vertical sleeve gastrectomy (VSG), the most utilized bariatric procedure in clinical practice, greatly reduces body weight and improves a variety of metabolic disorders. However, one of its long-term complications is bone loss and increased risk of fracture. Elevated circulating sclerostin (SOST) and granulocyte-colony stimulating factor (G-CSF) concentrations have been considered as potential contributors to VSG-associated bone loss. To test these possibilities, we administrated antibodies to SOST or G-CSF receptor and investigated alterations to bone and marrow niche following VSG. Neutralizing either SOST or G-CSF receptor did not alter beneficial effects of VSG on adiposity and hepatic steatosis, and anti-SOST treatment provided a further improvement to glucose tolerance. SOST antibodies partially reduced trabecular and cortical bone loss following VSG by increasing bone formation, whereas G-CSF receptor antibodies had no effects on bone mass. The expansion in myeloid cellularity and reductions in bone marrow adiposity seen with VSG were partially eliminated by treatment with Anti-G-CSF receptor. Taken together, these experiments demonstrate that antibodies to SOST or G-CSF receptor may act through independent mechanisms to partially block effects of VSG on bone loss or marrow niche cells, respectively.

DNA methylation is dynamically regulated in metabolic diseases, but it remains unclear whether the changes are causal or consequential. Therefore, we used a longitudinal approach to refine the onset of metabolic and DNA methylation changes at high temporal resolution. Male C57BL/6N mice were fed with 60 % high-fat diet (HFD) for up to 12 weeks and metabolically characterized weekly. Liver was collected after 1, 2, 4, 5, 6, 7, 8, and 12 weeks and hepatic DNA methylation and gene expression were analyzed. A subset of obese mice underwent vertical sleeve gastrectomy (VSG) or metformin treatment and livers were studied. Distinct hepatic gene expression patterns developed upon feeding HFD, with genes from the fatty acid metabolism pathway being predominantly altered. When comparing metabolic data with gene expression and DNA methylation, in particular Fgf21 DNA methylation decreased before the onset of increased Fgf21 expression and metabolic changes. Neither weight loss induced by VSG nor improved glucose tolerance by metformin treatment could revert hepatic Fgf21 DNA methylation or expression. Our data emphasize the dynamic induction of DNA methylation upon metabolic stimuli. Reduced Fgf21 DNA methylation established before massive overexpression of Fgf21, which is likely an adaptive effort of the liver to maintain glucose homeostasis despite the developing insulin resistance and steatosis. Fgf21 DNA methylation resisted reversion by intervention strategies, illustrating the long-term effects of unhealthy lifestyle. Our data provide a temporal roadmap to the development of hepatic insulin resistance, comprehensively linking DNA methylation with gene expression and metabolic data.

Abstract Obesity is an established risk and prognostic factor for multiple breast cancer (BC) subtypes. Epidemiologic studies suggest that weight loss via bariatric surgery results in metabolic reprogramming that reverses systemic aberrations as well as the protumorigenic effects of obesity; however, data on the effects of nonsurgical weight loss interventions in obese women remain inconclusive. We previously established that diet-induced obesity (DIO) increases tumor progression in murine models of basal-like BC. The purpose of this study was to determine if surgical and dietary weight loss interventions could effectively reduce obesity-associated tumor progression. Our first experiment directly compared mammary tumor growth following weight loss in DIO mice. Female C57BL/6 mice were randomized to a low-fat control (CON; 10 kcal% fat) diet or DIO regimen (60 kcal% fat) for 15 weeks. DIO mice were then randomized to either remain on DIO diet (DIO-DIO) or initiate weight loss either via vertical sleeve gastrectomy (DIO-SURG, with concurrent switch to control diet) or switch to control diet without surgery (DIO-DIET). CON mice remained on the 10 kcal% fat diet throughout the study. Eight weeks later, all mice were orthotopically injected with E0771 basal-like mammary tumor cells and were euthanized when tumors from the DIO-DIO group reached 1.5cm3. Body weights were not significantly different between DIO-SURG and DIO-DIET mice but both groups weighed significantly less than DIO-DIO mice. Compared with CON mice, tumor burden was not different in DIO-SURG mice but was significantly greater in DIO-DIET mice, suggesting that surgical weight loss more effectively reverses the protumorigenic effects of obesity than a low-fat diet. We next sought to determine how the anticancer effects of calorie restricted diets compared with bariatric surgery. Another group of female C57BL/6 received DIO diet for 16 weeks, then randomized to the same CON or DIO-SURG interventions as above, or to receive one of two calorie restriction (CR) diets—chronic CR (DIO-CCR; 30% daily calorie reduction) or intermittent CR (DIO-ICR; 14% calorie reduction 5 days per week, 70% calorie reduction on 2 nonconsecutive days per week). At time of tumor collection, DIO-SURG, DIO-CCR, and DIO-ICR mice weighed significantly less than DIO-DIO mice; however, only DIO-CCR and DIO-ICR mice achieved body weights significantly lower than CON mice. Tumor weights were significantly reduced in DIO-CCR and DIO-ICR fed mice, relative to CON mice, while tumor weights of DIO-SURG mice were intermediate. Moreover, multiple linear regression indicated weight loss, irrespective of intervention, was the strongest predictor of tumor mass. Our results in a murine model of basal-like BC suggest that weight loss via CR regimens (CCR, ICR) and, to a lesser extent, bariatric surgery, mitigates the pro-tumorigenic effects of obesity. Citation Format: Kristina K. Camp, Emily Rossi, Tori L. McFarlane, Steven Doerstling, Subreen Khatib, Elaine Glenny, Michael Coleman, Laura Bowers, Erika Rezeli, Randy J. Seeley, Alfor G. Lewis, Joel Parker, Anthony Fodor, Farnaz Fouladi, Stephen Hursting. Calorie restriction reverses the tumorigenic effects of obesity to a greater extent than bariatric surgery in a murine model of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2576.

Bariatric surgery is still the most effective long-term weight-loss therapy. Recent data indicate that surgical outcomes may be affected by diurnal food intake patterns. In this study, we aimed to investigate how surgery-induced metabolic adaptations (i.e. weight loss) interact with circadian clock function. For that reason, vertical sleeve gastrectomy (VSG) was performed in obese mice and rhythms in behavior, tissue rhythmicity, and white adipose tissue transcriptome were evaluated. VSG under constant darkness conditions led to a maximum weight loss of 18% compared to a loss of 3% after sham surgery. Post-surgical weight development was characterized by two distinct intervals of catabolic and subsequent anabolic metabolic state. Locomotor activity was not affected. However, VSG significantly increased active phase meal frequency in the anabolic state. No significant effects on clock gene rhythmicity were detected in adrenal and white adipose tissue (WAT) explant cultures. Transcriptome rhythm analyses of subcutaneous WAT revealed a reduction of cycling genes after VSG (sham: 2493 vs VSG: 1013) independent of sustained rhythms in core clock gene expression. This may be a consequence of weight loss-induced morphological reconstruction of WAT that overwrites the direct influence of the local clock machinery on the transcriptome. However, VSG altered rhythmic transcriptional regulation of WAT lipid metabolism pathways. Thus, our data suggest a reorganization of diurnal metabolic rhythms after VSG downstream of the molecular clock machinery.

Objective: Post-bariatric surgery hypoglycemia (PBH) is defined as the presence of neuroglycopenic symptoms accompanied by postprandial hypoglycemia in bariatric surgery patients. Recent clinical studies using continuous glucose monitoring (CGM) technology revealed that PBH is more frequently observed in vertical sleeve gastrectomy (VSG) patients than previously recognized. PBH cannot be alleviated by current medication. Therefore, a model system to investigate the mechanism and treatment is required. Methods: We used CGM in a rat model of VSG and monitored the occurrence of glycemic variability and hypoglycemia in various meal conditions for 4 weeks after surgery. Another cohort of VSG rats with CGM was used to investigate whether the blockade of glucagon-like peptide-1 receptor (GLP-1R) signaling alleviates these symptoms. A mouse VSG model was used to investigate whether the impaired glucose counterregulatory system causes postprandial hypoglycemia. Results: Like in humans, rats have increased glycemic variability and hypoglycemia after VSG. Postprandial hypoglycemia was specifically detected after liquid versus solid meals. Further, the blockade of GLP-1R signaling raises the glucose nadir but does not affect glycemic variability. Conclusions: Rat bariatric surgery duplicates many features of human post-bariatric surgery hypoglycemia including postprandial hypoglycemia and glycemic variability, while blockade of GLP-1R signaling prevents hypoglycemia but not the variability. (C) 2020 The Authors. Published by Elsevier GmbH.

Objective: To study the relationship between the amount of surgery-induced gastric volume reduction and long-term weight loss and glucose tolerance. Background Data: Vertical sleeve gastrectomy (VSG) has recently surpassed gastric bypass to become the most popular surgical intervention to induce sustained weight loss. Besides inducing significant weight loss, VSG also improves glucose tolerance. Although no clear correlation has been observed between the size of the residual stomach and sustained weight loss, this begs the question whether less aggressive gastric volume reduction may provide sufficient efficacy when weight loss is not the major goal of the surgical intervention. Methods: A series of strategies to reduce gastric volume were developed and tested in Long Evans male rats, namely: VSG, Fundal (F)-Resection, Gastric Sleeve Plication (GSP), Fundal-Plication, and Fundal-Constrained. Results: All surgical interventions resulted in a reduction of gastric volume relative to sham, but none of the interventions were as effective as the VSG. Gastric volume was linearly correlated to increased gastric emptying rate as well as increased GLP-1 response. Overall, cumulative food intake was the strongest correlate to weight loss and was logarithmically related to gastric volume. Regression modeling revealed a nonlinear inverse relation between body weight reduction and gastric volume, confirming that VSG is the only effective long-term weight loss strategy among the experimental operations tested. Conclusions: The data suggest a minimum threshold volume of the residual stomach that is necessary to induce sustained weight loss. Although all gastric volume interventions increased the GLP-1 response, none of the interventions, except VSG, significantly improved glucose tolerance. In conclusion, if weight loss is the primary goal of surgical intervention, significant volume reduction is required, and this most likely requires excising gastric tissue.

In recent years, the role of the kidney in glucose homeostasis has gained global interest. The kidneys are innervated by renal nerves, and renal denervation studies to control hypertension have shown improved glucose regulation. We hypothesized that kilohertz frequency stimulation, which can block propagation of action potentials, applied to renal nerves would reduce blood glucose concentration levels by increasing urinary glucose excretion. We performed experiments on 8 anesthetized, streptozotocin-induced diabetic rats. The renal nerves were encircled by a cuff electrode. Blood glucose concentrations were obtained from tail blood samples. Urine glucose concentrations were obtained from bilateral cannulation of the ureters. Electrical stimulation (50 kHz, 15 V) was applied for 60 minutes. The average blood glucose concentration rate was lower during stimulation (-0.78 +/- 1.20 mg/dL/min), compared to before stimulation (+1.14 +/- 1.83 mg/dL/min; p < 0.05) and after stimulation (+0.63 +/- 1.32 mg/dL/min). The average area under the curve for urine glucose concentration was higher during stimulation (7687.4 +/- 4006.1 mg/dL) compared to before (6466.9 +/- 2772.8 mg/dL) and after stimulation (5277.2 +/- 3381.5 mg/dL). Overall, our results show that kilohertz frequency stimulation of renal nerves is a possible approach for the modulation of blood glucose concentration and may introduce an alternative treatment modality for glycemic control in patients with diabetes.

The role of the kidney in glucose homeostasis has gained global interest. Kidneys are innervated by renal nerves, and renal denervation animal models have shown improved glucose regulation. We hypothesized that stimulation of renal nerves at kilohertz frequencies, which can block propagation of action potentials, would increase urine glucose excretion. Conversely, we hypothesized that low frequency stimulation, which has been shown to increase renal nerve activity, would decrease urine glucose excretion.

Bariatric surgeries, including vertical sleeve gastrectomy (VSG), resolve diabetes in 40–50% of patients. Studies examining the molecular mechanisms underlying this effect have centered on the role of the insulinotropic glucagon-like peptide 1 (GLP-1), in great part because of the ∼10-fold rise in its circulating levels after surgery. However, there is currently debate over the role of direct β-cell signaling by GLP-1 to mediate improved glucose tolerance following surgery. In order to assess the importance of β-cell GLP-1 receptor (GLP-1R) for improving glucose control after VSG, a mouse model of this procedure was developed and combined with a genetically modified mouse line allowing an inducible, β-cell–specific Glp1r knockdown (Glp1rβ-cell-ko). Mice with VSG lost ∼20% of body weight over 30 days compared with sham-operated controls and had a ∼60% improvement in glucose tolerance. Isolated islets from VSG mice had significantly greater insulin responses to glucose than controls. Glp1r knockdown in β-cells caused glucose intolerance in diet-induced obese mice compared with obese controls, but VSG improved glycemic profiles to similar levels during oral and intraperitoneal glucose challenges in Glp1rβ-cell-ko and Glp1rWT mice. Therefore, even though the β-cell GLP-1R seems to be important for maintaining glucose tolerance in obese mice, in these experiments it is dispensable for the improvement in glucose tolerance after VSG. Moreover, the metabolic physiology activated by VSG can overcome the deficits in glucose regulation caused by lack of β-cell GLP-1 signaling in obesity.

Background: Although vertical sleeve gastrectomy (VSG) is fashioned in humans by applying multiple staple loads, rodent VSG is generally created through a single -staple load application. Objectives: To investigate the impact of a 2-staple load VSG rat model more closely resembling the multistaple load operation done in humans on weight, metabolic outcomes, and the microbiome and how these compare with those obtained with the standard one -staple load model. Setting: University research facility, United States. Methods: High-fat diet induced obese male rats were randomized to single -staple load VSG (VSG1), 2-staple load VSG (VSG2), or sham operation (Sham). Outcomes included weight and composition, food intake, glucose metabolism, lipids, bile acids, and intestinal microbiome. Statistical comparisons were performed using analysis of variance. Results: Both procedures resulted in substantial weight and body fat loss compared with Sham treated animals. Weight loss was modestly greater for VSG2 compared with VSG1. Food intake was reduced in both procedures and accounted for the observed weight reduction. Glucose tolerance and plasma and hepatic lipid profiles were improved comparably in VSG1 and VSG2 relative to Sham. Bile acids were higher for VSG2 compared with Sham but not significantly different between VSG1 and VSG2. Neither procedure impacted intestinal microbiome richness and diversity compared with Sham across multiple intestinal sections. Colonic Actinobacteria was more abundant in VSG2 than in Sham. Relative abundances of bacterial phyla did not differ among VSG1, VSG2, and Sham across the remaining intestinal sections. Conclusions: Although VSG1 or VSG2 offer effective and overall comparable platforms for the study of obesity, VSG2 resulted in superior weight loss. (C) 2018 American Society for Bariatric Surgery. Published by Elsevier Inc. All rights reserved.

Provision of liquid enteral nutrition (LEN) during the perioperative period is standard practice for rodents undergoing bariatric surgery, yet these diets are associated with several challenges, including coagulation of the liquid diet within the delivery system and decreased postoperative consumption. We investigated the use of a commercially available high-calorie dietary gel supplement (DG) as an alternative food source for mice during the perioperative period. C57BL/6J male mice were fed high-fat diet for 8 to 10 wk prior to surgery. The study groups were: vertical sleeve gastrectomy (VSG) +DG, VSG+LEN, sham surgery+DG, and sham+LEN. Food and water intakes, body weight, and body fat composition was monitored throughout the study. Mice that received DG lost significantly more weight preoperatively than those fed LEN. However, during the postoperative period, body weight, body fat composition, and water and caloric intake were similar among all experimental diet groups. Three mice in the VSG+LEN group were euthanized due to clinical illness during the course of the study. In summary, feeding a high-calorie DG to mice undergoing VSG surgery is a viable alternative to LEN, given that DG does not significantly affect the surgical model of weight loss or result in adverse clinical outcomes. We recommend additional metabolic characterization of DG supplementation to ensure that this novel diet does not confound specific research goals in the murine VSG model.

Background: Obesity negatively impacts basal-like breast cancer (BLBC) prognosis, but the reversibility of these pro-cancer effects via weight loss remains unclear. However, there is consistent evidence suggesting that weight loss via bariatric surgery reduces breast cancer risk. This may be related to the reductions in metabolic perturbations and inflammation that follow bariatric surgery, effects that could be mediated by epigenetic reprogramming and/or changes in the gut microbiome. Purpose: We previously demonstrated that mammary tumor growth and inflammation remain elevated in formerly obese mice, in concordance with aberrant methylation of inflammation-related genes. Here we aim to determine the differential effects of surgical versus non-surgical weight loss on inflammation, DNA methylation, the gut microbiome, and tumor burden in a mouse model of BLBC. Methods: Mice were fed a low fat control (n=25) or high fat diet-induced obesity (DIO, n=75) regimen for 15 weeks to model chronic obesity. DIO mice were then randomized to remain on DIO (Obese) or receive either a surgical (sleeve gastrectomy) or dietary (low fat control diet) weight loss intervention, resulting in formerly obese (FOb)-Surg or FOb-Diet mice, respectively. The Control mice were maintained on the low fat diet throughout study. Four weeks after weight stabilization in the FOb mice, all mice were orthotopically injected with E0771 mammary tumor cells, which model BLBC. Stool samples were collected at baseline and prior to tumor cell injection. Results: The average weight and percent body fat of the FOb-Surg and FOb-Diet mice were equivalent to Control and significantly lower than Obese mice at study endpoint. Average tumor weight in FOb-Surg mice was statistically equivalent to Control mice, but tumor weight in FOb-Diet mice was significantly greater than Control mice and statistically equivalent to Obese mice. Furthermore, FOb-Surg mice had significantly lower serum tumor necrosis factor alpha, mammary gland interleukin-6 expression, and tumor-infiltrating adipocyte area in comparison to FOb-Diet. To further define the effects of surgical versus non-surgical weight loss, characterization of the gut microbiota as well as global mammary tissue gene expression and DNA methylation via paired RNA sequencing and reduced representation bisulfide sequencing is in progress. Conclusions: Our results suggest that the strong anti-cancer benefits seen with bariatric surgery may be related to a significant reduction in systemic and local inflammation, which did not occur with non-surgical weight loss. Identification of the mechanisms mediating the protective effects of bariatric surgery against breast cancer could help identify new targets and strategies for breaking the obesity-cancer link. Citation Format: Laura W. Bowers, Emily L. Rossi, Subreen A. Khatib, Steven Doerstling, Alfor Lewis, Randy J. Seeley, Stephen D. Hursting. The protumorigenic, proinflammatory effects of obesity are reversed by weight loss via bariatric surgery, but not a low-fat diet [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2689. doi:10.1158/1538-7445.AM2017-2689

To investigate the impact of a two-staple line vertical sleeve gastrectomy (VSG), a model more closely resembling the multi-staple line done in humans, on weight, metabolic outcomes, and the microbiome in rats.

Background: One-anastomosis gastric bypass (OAGB) and single-anastomosis duodenal switch (SADS) have become increasingly popular weight loss strategies. However, data directly comparing the effectiveness of these procedures with Roux-en-Y gastric bypass (RYGB) and vertical sleeve gastrectomy (SG) are limited. Objectives: To examine the metabolic outcomes of OAGB, SADS, RYGB, and SG in a controlled rodent model. Setting: Academic research laboratory, United States. Methods: Surgeries were performed in diet-induced obese Long-Evans rats, and metabolic outcomes were monitored before and for 15 weeks after surgery. Results: All bariatric procedures induced weight loss compared with sham that lasted throughout the course of the study. The highest percent fat loss occurred after OAGB and RYGB. All bariatric procedures had improved glucose dynamics associated with an increase in insulin (notably OAGB and SADS) and/or glucagon-like protein-1 secretion. Circulating cholesterol was reduced in OAGB, SG, and RYGB. OAGB and SG additionally decreased circulating triglycerides. Liver triglycerides were most profoundly reduced after OAGB and RYGB. Circulating iron levels were decreased in all surgical groups, associated with a decreased hematocrit value and increased reticulocyte count. The fecal microbiome communities of OAGB, SADS, and RYGB were significantly altered; however, SG exhibited no change in microbiome diversity or composition. Conclusions: These data support the use of the rat for modeling bariatric surgical procedures and highlight the ability of the OAGB to meet or exceed the metabolic improvements of RYGB. These data point to the likelihood that each surgery accomplishes metabolic improvements through both overlapping and distinct mechanisms and warrants further research. (C) 2018 American Society for Bariatric Surgery. Published by Elsevier Inc.