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Aim: In the present study, we investigated the involvement of NLRP3 inflammasome in the intestinal epithelial barrier (IEB) changes associated with obesity, and its role in the interplay between enteric glia and intestinal epithelial cells (IECs)., Methods: Wild-type C57BL/6J and NLRP3-KO ( -/- ) mice were fed with high-fat diet (HFD) or standard diet for 8 weeks. Colonic IEB integrity and inflammasome activation were assessed. Immunolocalization of colonic mucosal GFAP- and NLRP3-positive cells along with in vitro coculture experiments with enteric glial cells (EGCs) and IECs allowed to investigate the potential link between altered IEB, enteric gliosis, and NLRP3 activation., Results: HFD mice showed increased body weight, altered IEB integrity, increased GFAP-positive glial cells, and NLRP3 inflammasome hyperactivation. HFD-NLRP3 -/- mice showed a lower increase in body weight, an improvement in IEB integrity and an absence of enteric gliosis. Coculture experiments showed that palmitate and lipopolysaccharide contribute to IEB damage and promote enteric gliosis with consequent hyperactivation of enteric glial NLRP3/caspase-1/IL-1β signaling. Enteric glial-derived IL-1β release exacerbates the IEB alterations. Such an effect was abrogated upon incubation with anakinra (IL-1β receptor antagonist) and with conditioned medium derived from silenced-NLRP3 glial cells., Conclusion: HFD intake elicits mucosal enteric gliotic processes characterized by a hyperactivation of NLRP3/caspase-1/IL-1β signaling pathway, that contributes to further exacerbate the disruption of intestinal mucosal barrier integrity. However, we cannot rule out the contribution of NLRP3 inflammasome activation from other cells, such as immune cells, in IEB alterations associated with obesity. Overall, our results suggest that enteric glial NLRP3 inflammasome might represent an interesting molecular target for the development of novel pharmacological approaches aimed at managing the enteric inflammation and intestinal mucosal dysfunctions associated with obesity., (© 2024 The Author(s). Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Intestinal barrier alterations represent a primum movens in obesity and related intestinal dysfunctions. However, whether gut barrier remodeling represents prodromal events in obesity before weight gain, metabolic alterations, and systemic inflammation remains unclear. Herein, we examined morphologic changes in the gut barrier in a mouse model of high-fat diet (HFD) since the earliest phases of diet assumption. C57BL/6J mice were fed with standard diet (SD) or HFD for 1, 2, 4, or 8 weeks. Remodeling of intestinal epithelial barrier, inflammatory infiltrate, and collagen deposition in the colonic wall was assessed by histochemistry and immunofluorescence analysis. Obese mice displayed increased body and epididymal fat weight along with increased plasma resistin, IL-1β, and IL-6 levels after 8 weeks of HFD. Starting from 1 week of HFD, mice displayed (1) a decreased claudin-1 expression in lining epithelial cells, (2) an altered mucus in goblet cells, (3) an increase in proliferating epithelial cells in colonic crypts, (4) eosinophil infiltration along with an increase in vascular P-selectin, and (5) deposition of collagen fibers. HFD intake is associated with morphologic changes in the large bowel at mucosal and submucosal levels. In particular, the main changes include alterations in the mucous layer and intestinal epithelial barrier integrity and activation of mucosal defense-enhanced fibrotic deposition. These changes represent early events occurring before the development of obesity condition that could contribute to compromising the intestinal mucosal barrier and functions, opening the way for systemic dissemination., (Copyright © 2023 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Experimental evidence suggests a key role of SIRT1 (silent information regulator 1) in age- and metabolic-related vascular dysfunction. Whether these effects hold true in the human microvasculature is unknown. We aimed to investigate the SIRT1 role in very early stages of age- and obesity-related microvascular dysfunction in humans., Methods: Ninety-five subjects undergoing elective laparoscopic surgery were recruited and stratified based on their body mass index status (above or below 30 kg/m 2 ) and age (above or below 40 years) in 4 groups: Young Nonobese, Young Obese, Old Nonobese, and Old Obese. We measured small resistance arteries' endothelial function by pressurized micromyography before and after incubation with a SIRT1 agonist (SRT1720) and a mitochondria reactive oxygen species (mtROS) scavenger (MitoTEMPO). We assessed vascular levels of mtROS and nitric oxide availability by confocal microscopy and vascular gene expression of SIRT1 and mitochondrial proteins by qPCR. Chromatin immunoprecipitation assay was employed to investigate SIRT1-dependent epigenetic regulation of mitochondrial proteins., Results: Compared with Young Nonobese, obese and older patients showed lower vascular expression of SIRT1 and antioxidant proteins (FOXO3 [forkhead box protein O3] and SOD2) and higher expression of pro-oxidant and aging mitochondria proteins p66 Shc and Arginase II. Old Obese, Young Obese and Old Nonobese groups endothelial dysfunction was rescued by SRT1720. The restoration was comparable to the one obtained with mitoTEMPO. These effects were explained by SIRT1-dependent chromatin changes leading to reduced p66 Shc expression and upregulation of proteins involved in mitochondria respiratory chain., Conclusions: SIRT1 is a novel central modulator of the earliest microvascular damage induced by age and obesity. Through a complex epigenetic control mainly involving p66 Shc and Arginase II, it influences mtROS levels, NO availability, and the expression of proteins of the mitochondria respiratory chain. Therapeutic modulation of SIRT1 restores obesity- and age-related endothelial dysfunction. Early targeting of SIRT1 might represent a crucial strategy to prevent age- and obesity-related microvascular dysfunction.

Background and Purpose: Enteric neurogenic/inflammation contributes to bowel dysmotility in obesity. We examined the role of NLRP3 in colonic neuromuscular dysfunctions in mice with high-fat diet (HFD)-induced obesity., Experimental Approach: Wild-type C57BL/6J and NLRP3-KO (Nlrp3 -/- ) mice were fed with HFD or standard diet for 8 weeks. The activation of inflammasome pathways in colonic tissues from obese mice was assessed. The role of NLRP3 in in vivo colonic transit and in vitro tachykininergic contractions and substance P distribution was evaluated. The effect of substance P on NLRP3 signalling was tested in cultured cells., Key Results: HFD mice displayed increased body and epididymal fat weight, cholesterol levels, plasma resistin levels and plasma and colonic IL-1β levels, colonic inflammasome adaptor protein apoptosis-associated speck-like protein containing caspase-recruitment domain (ASC) and caspase-1 mRNA expression and ASC immunopositivity in macrophages. Colonic tachykininergic contractions were enhanced in HFD mice. HFD NLRP3 -/- mice developed lower increase in body and epididymal fat weight, cholesterol levels, systemic and bowel inflammation. In HFD Nlrp3 -/- mice, the functional alterations of tachykinergic pathways and faecal output were normalized. In THP-1 cells, substance P promoted IL-1β release. This effect was inhibited upon incubation with caspase-1 inhibitor or NK 1 antagonist and not observed in ASC -/- cells., Conclusion and Implications: In obesity, NLRP3 regulates an interplay between the shaping of enteric immune/inflammatory responses and the activation of substance P/NK 1 pathways underlying the onset of colonic dysmotility. Identifying NLRP3 as a therapeutic target for the treatment of bowel symptoms related to obesity., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background: The murine model of high fat diet (HFD)-induced obesity is characterized by an increment of intestinal permeability, secondary to an impairment of mucosal epithelial barrier and enteric inflammation, followed by morphofunctional rearrangement of the enteric nervous system. The present study investigated the involvement of abdominal macrophages in the mechanisms underlying the development of enteric dysmotility associated with obesity., Methods: Wild type C57BL/6J mice were fed with HFD (60% kcal from fat) or normocaloric diet (NCD, 18% kcal from fat) for 8 weeks. Groups of mice fed with NCD or HFD were treated with clodronate encapsulated into liposomes to deplete abdominal macrophages. Tachykininergic contractions, elicited by electrical stimulation or exogenous substance P (SP), were recorded in vitro from longitudinal muscle colonic preparations. Substance P distribution was examined by confocal immunohistochemistry. The density of macrophages in the colonic wall was examined by immunohistochemical analysis. Malondialdehyde (MDA, colorimetric assay) and IL-1β (ELISA assay) levels were also evaluated., Results: MDA and IL-1β levels were increased in colonic tissues from HFD-treated animals. In colonic preparations, electrically evoked tachykininergic contractions were enhanced in HFD mice. Immunohistochemistry displayed an increase in substance P immunoreactivity in myenteric ganglia, as well as in the muscular layers of colonic cryosections from obese mice. Macrophage depletion in HFD mice was associated with a significant reduction of colonic inflammation. In addition, the decrease in macrophage density attenuated the morphofunctional alterations of tachykininergic pathways observed in obese mice., Conclusion: Obesity elicited by HFD determines a condition of colonic inflammation, followed by a marked rearrangement of motor excitatory tachykininergic enteric nerves. Macrophage depletion counteracted the morphofunctional changes of colonic neuromuscular compartment, suggesting a critical role for these immune cells in the onset of enteric dysmotility associated with obesity.

Objective- Arginase can reduce NO availability. In this study, we explored arginase as a determinant of endothelial dysfunction in small arteries from obese patients and its relationship with aging and microvascular remodeling. Approach and Results- Small arteries were dissected after subcutaneous fat biopsies and evaluated on a pressurized micromyograph. Endothelium-dependent vasodilation was assessed by acetylcholine, repeated under L-NAME ( N  G -nitro-L-arginine-methyl ester), N(ω)-hydroxy-nor-l-arginine (arginase inhibitor) and gp91ds-tat (NADPH [nicotinamide adenine dinucleotide phosphate oxidase] oxidase inhibitor) in vessels from young (age <30 years) control and obese and old (>30 years) control and obese subjects. Media-lumen ratio and amount of vascular wall fibrosis were used as markers of vascular remodeling. Amount of vascular superoxide anions and NO production were determined with immunofluorescence, whereas arginase expression was quantified using Western blot and quantitative polymerase chain reaction. Obese and older age groups had lower vascular NO, as well as higher media-lumen ratio, wall fibrosis, intravascular superoxide, and blunted inhibitory effect of L-NAME on acetylcholine versus controls and younger age groups. N(ω)-hydroxy-nor-l-arginine restored the acetylcholine-induced vasodilation in young and, to a lesser extent, in old obese subjects. This effect was abolished by addition of L-NAME. Gp91ds-tat increased the vasodilatory response to N(ω)-hydroxy-nor-l-arginine in old obese. Superoxide anions and arginase I/II levels were higher in the vascular wall of obese versus controls. Conclusions- Arginase contributes to microvascular endothelial dysfunction in obesity. Its impact is reduced by aging because of higher levels of vascular oxidative stress. Obesity is accompanied by accelerated microvascular remodeling, the extent of which is related to the amount of arginase in the vascular wall.

Background and Purpose: Apigenin can exert beneficial actions in the prevention of obesity. However, its putative action on obesity-associated bowel motor dysfunctions is unknown. This study examined the effects of apigenin on colonic inflammatory and motor abnormalities in a mouse model of diet-induced obesity., Experimental Approach: Male C57BL/6J mice were fed with standard diet (SD) or high-fat diet (HFD). SD or HFD mice were treated with apigenin (10 mg/Kg/day). After 8 weeks, body and epididymal fat weight, as well as cholesterol, triglycerides and glucose levels were evaluated. Malondialdehyde (MDA), IL-1β and IL-6 levels, and let-7f expression were also examined. Colonic infiltration by eosinophils, as well as substance P (SP) and inducible nitric oxide synthase (iNOS) expressions were evaluated. Motor responses elicited under blockade of NOS and tachykininergic contractions were recorded in vitro from colonic longitudinal muscle preparations., Key Results: When compared to SD mice, HFD animals displayed increased body weight, epididymal fat weight and metabolic indexes. HFD mice showed increments in colonic MDA, IL-1β and IL-6 levels, as well as a decrease in let-7f expression in both colonic and epididymal tissues. HFD mice displayed an increase in colonic eosinophil infiltration. Immunohistochemistry revealed an increase in SP and iNOS expression in myenteric ganglia of HFD mice. In preparations from HFD mice, electrically evoked contractions upon NOS blockade or mediated by tachykininergic stimulation were enhanced. In HFD mice, Apigenin counteracted the increase in body and epididymal fat weight, as well as the alterations of metabolic indexes. Apigenin reduced also MDA, IL-1β and IL-6 colonic levels as well as eosinophil infiltration, SP and iNOS expression, along with a normalization of electrically evoked tachykininergic and nitrergic contractions. In addition, apigenin normalized let-7f expression in epididymal fat tissues, but not in colonic specimens., Conclusions and Implications: Apigenin prevents systemic metabolic alterations, counteracts enteric inflammation and normalizes colonic dysmotility associated with obesity.

Adenosine A 2B receptors (A 2B R) regulate several enteric functions. However, their implication in the pathophysiology of intestinal dysmotility associated with high-fat diet (HFD)-induced obesity has not been elucidated. We investigated the expression of A 2B R in mouse colon and their role in the mechanisms underlying the development of enteric dysmotility associated with obesity. Wild-type C57BL/6J mice were fed with HFD (60% kcal from fat) or normocaloric diet (NCD; 18% kcal from fat) for 8 weeks. Colonic A 2B R localization was examined by immunofluorescence. The role of A 2B R in the control of colonic motility was examined in functional experiments on longitudinal muscle preparations (LMPs). In NCD mice, A 2B R were predominantly located in myenteric neurons; in HFD animals, their expression increased throughout the neuromuscular layer. Functionally, the A 2B R antagonist MRS1754 enhanced electrically induced NK 1 -mediated tachykininergic contractions in LMPs from HFD mice, while it was less effective in tissues from NCD mice. The A 2B receptor agonist BAY 60-6583 decreased colonic tachykininergic contractions in LMPs, with higher efficacy in preparations from obese mice. Both A 2B R ligands did not affect contractions elicited by exogenous substance P. Obesity is related with a condition of colonic inflammation, leading to an increase of A 2B R expression. A 2B R, modulating the activity of excitatory tachykininergic nerves, participate to the enteric dysmotility associated with obesity.

Adenosine A receptors (AR) regulate several enteric functions. However, their implication in the pathophysiology of intestinal dysmotility associated with high-fat diet (HFD)-induced obesity has not been elucidated. We investigated the expression of AR in mouse colon and their role in the mechanisms underlying the development of enteric dysmotility associated with obesity. Wild-type C57BL/6J mice were fed with HFD (60% kcal from fat) or normocaloric diet (NCD; 18% kcal from fat) for 8 weeks. Colonic AR localization was examined by immunofluorescence. The role of AR in the control of colonic motility was examined in functional experiments on longitudinal muscle preparations (LMPs). In NCD mice, AR were predominantly located in myenteric neurons; in HFD animals, their expression increased throughout the neuromuscular layer. Functionally, the AR antagonist MRS1754 enhanced electrically induced NK-mediated tachykininergic contractions in LMPs from HFD mice, while it was less effective in tissues from NCD mice. The A receptor agonist BAY 60-6583 decreased colonic tachykininergic contractions in LMPs, with higher efficacy in preparations from obese mice. Both AR ligands did not affect contractions elicited by exogenous substance P. Obesity is related with a condition of colonic inflammation, leading to an increase of AR expression. AR, modulating the activity of excitatory tachykininergic nerves, participate to the enteric dysmotility associated with obesity. [ABSTRACT FROM AUTHOR]

The article discusses a study conducted to examine mucosal permeability, immune/inflammatory responses and colonic motility in mice with high-fat diet (HFD)-induced obesity. In the setting of obesity, the activation of NLRP3 inflammasome in tissue-resident macrophages contributes to colonic dysmotility, through the modulation of tachykininergic neurogenic responses, thus identifying NLRP3 as a therapeutic target for the treatment of bowel symptoms related to obesity.

Background: Nucleotide-binding oligomerization domain leucine rich repeat and pyrin domain containing protein 3 (NLRP3) inflammasome activation in immune/inflammatory cells contributes to enteric inflammation and bowel dysmotility in obesity (1). However, the involvement of NLRP3 inflammasome in enteric glial activation and gut barrier alterations associated with obesity has not yet been investigated. Aim: This study examined the interplay among enteric glia NLRP3 inflammasome activation and intestinal barrier impairments in high-fat diet (HFD)-induced obesity Methods: Wild-type C57BL/6J mice were fed with HFD or standard diet for 8 weeks. The remodelling of intestinal epithelial barrier in the colonic wall were assessed by standard histological staining, immunohistochemistry and immunofluorescence and western blot. Double-immunofluorescence of ASC and GFAP-positive glial cells was performed to characterize inflammasome activation in enteric glial cells. The molecular mechanisms underlying the interplay between altered intestinal barrier, enteric gliosis and NLRP3 activation in the setting of obesity were investigated with in vitro co-culture experiments enteric glial cells (EGCs) and intestinal epithelial cells (IECs). Results: After 8 weeks of HFD, animals displayed a remodeling of mucus layer, decrease in expression of tight junction proteins along with an increase in proliferating epithelial cells in colonic crypts. HFD animals were also characterized by increased ASC immunopositivity in GFAP-positive glial cells. In co-culture experiments, exposure to palmitate and lipopolysaccharide, besides to induce intestinal epithelial barrier alterations, promotes enteric gliosis with consequent hyperactivation of enteric glial NLRP3/caspase-1/IL-1β signaling pathway. The glial-derived IL-1β release contributes to exacerbate the disruption of IEB. Such an effect was abrogated upon incubation with IL-1β receptor antagonist, anakinra. Conclusions: Glial NLRP3 inflammasome acts as a regulatory hub linking EGCs and IECs, representing a promising therapeutic target for the treatment of gut barrier alterations related to obesity. [ABSTRACT FROM AUTHOR]

Background and Aim. Obesity is a metabolic disorder with an increasing incidence in Western countries and childhood. It is characterized by low-grade systemic inflammation and several comorbidities, including alterations of gastrointestinal (GI) functions, which impact negatively on patients' quality of life. There is currently limited information on the morpho-functional features of the GI tract in obese subjects. Of note, the intestinal barrier function has been found to be altered in obese subjects, even before the occurrence of body weight increase [1]. In this light, the present study was carried out to assess, in a mouse model of diet-induced obesity, whether high fat diet (HFD) is associated with morphological alterations of the colonic mucosal barrier. Methods. C57BL/6 mice (n=5/group) were fed with standard diet (SD, 18% calories from fat) or HFD (60% calories from fat). After 8 weeks, body weight, and levels of blood cholesterol, triglycerides and glucose were evaluated. Malondialdehyde (MDA, colorimetric assay), IL-1β and IL-6 levels (ELISA assays) were examined in colonic tissues. Morphological features of colonic mucosal structures (lining epithelial cells, goblet cells, inflammatory infiltrates and enteric glia) were examined by histochemistry and immunohistochemistry. Results. HFD mice displayed significant differences at both molecular and histomorphological level, as compared with SD animals: increased body weight and blood metabolic indexes; increased MDA, IL-1β and IL-6 levels in colonic tissues; altered pattern of claudin-1 expression along with upregulation of transmembrane 16A protein and induced nitric oxide synthase in the enteric epithelium facing the lumen; increased proliferation rate of crypts; altered composition of goblet cell mucous; mucosal gliosis and infiltrates with mixed inflammatory cells. Conclusions. After 8 weeks, HFD intake led to significant alterations of systemic metabolic indexes, colonic tissue inflammation, and colonic mucosal barrier in obese mice, as compared with controls. Morphological studies can be useful to allow the characterization of histopathological patterns of colonic wall remodelling and inflammation underlying bowel motor dysfunctions associated with obesity. [ABSTRACT FROM AUTHOR]