Gastric mucosal devitalization improves blood pressure, renin and cardiovascular lipid deposition in a rat model of obesity.

Background and study aims In lieu of the drawbacks of metabolic surgery, a method of mimicking resection of the gastric mucosa could be of value to those with obesity-related cardiovascular disease (CVD). Our study aims to investigate the effect of gastric mucosal devitalization (GMD) on blood pressure (BP) and cardiovascular lipid deposition in a rat model of obesity. Methods GMD of 70 % of the stomach was achieved by argon plasma coagulation. GMD was compared to sleeve gastrectomy (SG) and sham (SH) in a high-fat-diet-induced rat model of obesity (48 rats). At 8 weeks, we measured noninvasive BP, renin, vessel relaxation and ghrelin receptor regulation in the aorta. In addition, we quantified cardiac lipid deposition and lipid droplet deposition in cardiac muscle and aorta. Results GMD and SG were observed to have similar reductions in body weight, visceral adiposity, and serum lipid profile compared to SH rats. GMD resulted in a significant reduction in arterial BP compared to SH. Furthermore, there were significant reductions in plasma renin activity and percentage of phenylnephrine constriction to acetylcholine at the aortic ring in GMD rats compared to SH, providing insights into the mechanisms behind the reduced BP. Interestingly, the reduced BP occurred despite a reduction in endothelial ghrelin recteptor activation. Cardiac lipid content was significantly reduced in GMD rats. Lipid deposition, as illustrated by Nile Red stain, was reduced in cardiac muscle and the aorta. Conclusion GMD resulted in a significant improvement in BP, renin and cardiovascular lipid deposition. GMD deserves further attention as a method of treating obesity-related CVD.

Gastric mucosal devitalization is safe and effective in reducing body weight and visceral adiposity in a porcine model.

BACKGROUND AND AIMS: The early improvement in metabolic profile after sleeve gastrectomy (SG) indicates that the significant benefits of metabolic surgery are gastric in origin. We have previously demonstrated that devitalization of the gastric mucosa (without a reduction in gastric volume) in metabolically disturbed obese rats results in an improvement of obesity and its associated comorbidities. The aims of this study were to assess the technical feasibility, efficacy, and safety of gastric mucosal devitalization (GMD) in a large animal (porcine) model. METHODS: A 3-arm (GMD versus SG versus sham [SH]) prospective randomized controlled trial with an 8-week follow-up period was performed. The primary endpoint was relative weight loss. Secondary endpoints were absolute body weight, abdominal visceral adiposity, abdominal subcutaneous adiposity, organ lipid content, and serum ghrelin level. RESULTS: GMD resulted in a significant relative weight loss of 36% over SH at 8 weeks (P < .05). There was no significant difference in relative weight loss between GMD and SG at 4 weeks; however, SG resulted in a 29% superior relative weight loss at 8 weeks (P < .05). With regard to visceral adiposity, there was a significant benefit of GMD over SH at 8 weeks. Despite differences in relative weight loss, there was no significant difference in visceral adiposity between SG and GMD at 8 weeks. Significant improvements in GMD over SH were noted with regard to skeletal and heart muscle lipid content. GMD pigs at 8 weeks demonstrated regeneration of the gastric mucosa without ulceration or significant scarring. Despite mucosal regeneration, the abundance of serum ghrelin was significantly lower in the GMD cohort compared with the SG and SH cohorts. CONCLUSIONS: GMD was technically feasible and resulted in relative weight loss and an improvement in visceral adiposity. The benefits noted were out of proportion to what would be expected with weight loss alone.

Gastric mucosal devitalization reduces adiposity and improves lipid and glucose metabolism in obese rats.

BACKGROUND AND AIMS: The gastric mucosa is an endocrine organ that regulates satiation pathways by expression of orexigenic and anorexigenic hormones. Vertical sleeve gastrectomy (VSG) excludes gastric mucosa and reduces gastric volume. Our study aimed to investigate the independent effects of altering gastric mucosa on obesity and its related comorbidities. METHODS: Gastric mucosa devitalization (GMD) of 70% of the stomach was achieved by argon plasma coagulation in a high-fat diet rat model and was compared with VSG and sham surgery. In an 8-week follow-up study, we quantified body weight, visceral adiposity, insulin resistance index, cholesterol profiles, and free fatty acid profiles by enzyme-linked immunosorbent assay (ELISA). Following a 2-hour oral glucose tolerance test, the kinetics of ghrelin, glucagon-like peptide-1, peptide YY, and serum and liver bile acid levels were measured. Liver lipid content was quantified by ELISA. RESULTS: GMD resulted in significant reductions in body weight, visceral and subcutaneous adipose tissue, and hepatic steatosis as well as an improvement in lipid metabolism. GMD resulted in significant reductions in food intake and intestinal malabsorption of free fatty acids, both contributing to improved body composition and metabolic profile. Mechanistically, GMD resulted in a significant reduction in serum palmitate levels as well as an increase in serum and liver bile acid levels, known to alter glucose and lipid metabolism. Similar changes were noted when VSG rats were compared with sham surgery rats. CONCLUSIONS: Devitalization of gastric mucosa, independent of altering gastric volume, was able to reduce obesity-related comorbidities. The gastric mucosa may be a potential target for treating obesity and its associated comorbidities.

Establishing a reliable multiple reaction monitoring-based method for the quantification of obesity-associated comorbidities in serum and adipose tissue requires intensive clinical validation.

Multiple reaction monitoring (MRM)-based mass spectrometric quantification of peptides and their corresponding proteins has been successfully applied for biomarker validation in serum. The option of multiplexing offers the chance to analyze various proteins in parallel, which is especially important in obesity research. Here, biomarkers that reflect multiple comorbidities and allow monitoring of therapy outcomes are required. Besides the suitability of established MRM assays for serum protein quantification, it is also feasible for analysis of tissues secreting the markers of interest. Surprisingly, studies comparing MRM data sets with established methods are rare, and therefore the biological and clinical value of most analytes remains questionable. A MRM method using nano-UPLC-MS/MS for the quantification of obesity related surrogate markers for several comorbidities in serum, plasma, visceral and subcutaneous adipose tissue was established. Proteotypic peptides for complement C3, adiponectin, angiotensinogen, and plasma retinol binding protein (RBP4) were quantified using isotopic dilution analysis and compared to the standard ELISA method. MRM method variabilities were mainly below 10%. The comparison with other MS-based approaches showed a good correlation. However, large differences in absolute quantification for complement C3 and adiponectin were obtained compared to ELISA, while less marked differences were observed for angiotensinogen and RBP4. The verification of MRM in obesity was performed to discriminate first lean and obese phenotype and second to monitor excessive weight loss after gastric bypass surgery in a seven-month follow-up. The presented MRM assay was able to discriminate obese phenotype from lean and monitor weight loss related changes of surrogate markers. However, inclusion of additional biomarkers was necessary to interpret the MRM data on obesity phenotype properly. In summary, the development of disease-related MRMs should include a step of matching the MRM data with clinically approved standard methods and defining reference values in well-sized representative age, gender, and disease-matched cohorts.

Free fatty acid palmitate impairs the vitality and function of cultured human bladder smooth muscle cells.

BACKGROUND: Incidence of urinary tract infections is elevated in patients with diabetes mellitus. Those patients show increased levels of the saturated free fatty acid palmitate. As recently shown metabolic alterations induced by palmitate include production and secretion of the pro-inflammatory cytokine interleukine-6 (IL-6) in cultured human bladder smooth muscle cells (hBSMC). Here we studied the influence of palmitate on vital cell properties, for example, regulation of cell proliferation, mitochondrial enzyme activity and antioxidant capacity in hBSMC, and analyzed the involvement of major cytokine signaling pathways. METHODOLOGY/PRINCIPAL FINDINGS: HBSMC cultures were set up from bladder tissue of patients undergoing cystectomy and stimulated with palmitate. We analyzed cell proliferation, mitochondrial enzyme activity, and antioxidant capacity by ELISA and confocal immunofluorescence. In signal transduction inhibition experiments we evaluated the involvement of NF-κB, JAK/STAT, MEK1, PI3K, and JNK in major cytokine signaling pathway regulation. We found: (i) palmitate decreased cell proliferation, increased mitochondrial enzyme activity and antioxidant capacity; (ii) direct inhibition of cytokine receptor by AG490 even more strongly suppressed cell proliferation in palmitate-stimulated cells, while counteracting palmitate-induced increase of antioxidant capacity; (iii) in contrast knockdown of the STAT3 inhibitor SOCS3 increased cell proliferation and antioxidant capacity; (iv) further downstream JAK/STAT3 signaling cascade the inhibition of PI3K or JNK enhanced palmitate induced suppression of cell proliferation; (v) increase of mitochondrial enzyme activity by palmitate was enhanced by inhibition of PI3K but counteracted by inhibition of MEK1. CONCLUSIONS/SIGNIFICANCE: Saturated free fatty acids (e.g., palmitate) cause massive alterations in vital cell functions of cultured hBSMC involving distinct major cytokine signaling pathways. Thereby, certain cytokines might counteract the palmitate-induced downregulation of cell proliferation and vitality. This could be an important link to clinical findings of increased risk of metabolic related bladder diseases such as overactive bladder (OAB) and bladder pain syndrome/interstitial cystitis (BPS/IC).

Cytokine effects on gap junction communication and connexin expression in human bladder smooth muscle cells and suburothelial myofibroblasts.

BACKGROUND: The last decade identified cytokines as one group of major local cell signaling molecules related to bladder dysfunction like interstitial cystitis (IC) and overactive bladder syndrome (OAB). Gap junctional intercellular communication (GJIC) is essential for the coordination of normal bladder function and has been found to be altered in bladder dysfunction. Connexin (Cx) 43 and Cx45 are the most important gap junction proteins in bladder smooth muscle cells (hBSMC) and suburothelial myofibroblasts (hsMF). Modulation of connexin expression by cytokines has been demonstrated in various tissues. Therefore, we investigate the effect of interleukin (IL) 4, IL6, IL10, tumor necrosis factor-alpha (TNFα) and transforming growth factor-beta1 (TGFß1) on GJIC, and Cx43 and Cx45 expression in cultured human bladder smooth muscle cells (hBSMC) and human suburothelial myofibroblasts (hsMF). METHODOLOGY/PRINCIPAL FINDINGS: HBSMC and hsMF cultures were set up from bladder tissue of patients undergoing cystectomy. In cytokine stimulated cultured hBSMC and hsMF GJIC was analyzed via Fluorescence Recovery after Photo-bleaching (FRAP). Cx43 and Cx45 expression was assessed by quantitative PCR and confocal immunofluorescence. Membrane protein fraction of Cx43 and Cx45 was quantified by Dot Blot. Upregulation of cell-cell-communication was found after IL6 stimulation in both cell types. In hBSMC IL4 and TGFß1 decreased both, GJIC and Cx43 protein expression, while TNFα did not alter communication in FRAP-experiments but increased Cx43 expression. GJ plaques size correlated with coupling efficacy measured, while Cx45 expression did not correlate with modulation of GJIC. CONCLUSIONS/SIGNIFICANCE: Our finding of specific cytokine effects on GJIC support the notion that cytokines play a pivotal role for pathophysiology of OAB and IC. Interestingly, the effects were independent from the classical definition of pro- and antiinflammatory cytokines. We conclude, that connexin regulation involves genomic and/or post-translational events, and that GJIC in hBSMC and hsMF depend of Cx43 rather than on Cx45.

TGF-beta1 inhibits Cx43 expression and formation of functional syncytia in cultured smooth muscle cells from human detrusor.

BACKGROUND: Human detrusor smooth muscle cells (hBSMCs) are coupled by connexin 43 (Cx43)-positive gap junctions to form functional syncytia. Gap junctional communication likely is necessary for synchronised detrusor contractions and is supposed to be altered in voiding disturbances. Other authors have shown that the pleiotropic cytokine TGF-beta1 upregulates Cx43 expression in human aortic smooth muscle cells. OBJECTIVE: In this study, we examined the TGF-beta1 effects on Cx43 expression in cultured hBSMCs. DESIGN, SETTING, AND PARTICIPANTS: hBSMC cultures, established from patients undergoing cystectomy, were treated with recombinant human TGF-beta1. MEASUREMENTS: Cx43 expression was then examined by Western blotting, real-time PCR, and immunocytochemistry. Dye-injection experiments were used to study the size of functional syncytia. RESULTS AND LIMITATIONS: Dye-coupling experiments revealed stable formation of functional syncytia in passaged cell cultures (P1-P4). Stimulation with TGF-beta1 led to significant reduction of Cx43 immunoreactivity and coupling. Cx43 protein expression was significantly downregulated and Cx43 mRNA was only 30% of the control level. Interestingly, low phosphorylation species of Cx43 were particularly affected. CONCLUSIONS: Our experiments demonstrated a significant down regulation of connexin 43 by TGF-beta1 in cultured hBSMCs. These findings support the view that TGF-beta1 is involved in the pathophysiology of urinary bladder dysfunction.