The effects of obesity superimposed with aging in female mouse model

BACKGROUND AND AIM: People worldwide are living longer and the prevalence of overweight and obesity is growing at an alarming rate. Moreover, obesity has proved to be typically more prevalent among women, who usually live longer than men. Based on these evidences and considering that obesity leads to body health consequences in a way resembling aging, the aim of this study is to evaluate whether obesity superimposed with aging worsens the agedependent changes at peripheral, systemic and central level in female mice. METHODS: The 4-week-old C57BL/6J female mice were fed with standard diet (SD, 10% of energy from fat) or high fat diet (HFD, 60% of energy from fat) for 8, 20, or 36 weeks. After the exposure to the diet, animals were weighted and fasting metabolic parameters (glucose, triglycerides, cholesterol, insulin, leptin) were measured in blood. The gastrointestinal transit was analyzed by the intestinal distribution of high molecular weight fluorescein isothiocyanate dextran (FITC-dextran 70 kDa). The number of fecal pellets was evaluated during 1-hour collection period, and then the fecal water content was calculated. The integrity of intestinal barrier was assessed functionally by plasma level measurement of low molecular weight FITC-dextran 4 kDa after oral gavage and by evaluation of tight junctions occludin (western blot) and zonulin-1 (ELISA) expression level. To investigate the systemic inflammation, the following serum parameters were measured by ELISA: IL-1β, IL-6, IL-23, IL-10. Levels of Aβ1-42 amyloid (ELISA), p-Tau, SIRT1, occludin and zonulin-1 (western blot) were evaluated in hippocampus. RESULTS: In female mice, long-term HFD consumption resulted in an obese phenotype and accelerated age-dependent changes in cholesterol, glucose, insulin and leptin serum levels. Obese aged mice showed delayed intestinal transit, decreased gastric emptying, constipation, reduction in fecal water and increased intestinal permeability earlier and in an enhanced extent compared to SD aged mice. Moreover, obesity caused a further release of systemic inflammatory cytokines, previously observed during aging. Finally, HFD exposition had detrimental effects on brain barrier integrity, increased levels of Aβ1-42 amyloid and decreased SIRT1 expression in hippocampus. CONCLUSION: Our results demonstrated that chronic HFD exposure worsened metabolic alterations, gastrointestinal dysfunctions and systemic inflammation observed in aged SD animals. Moreover, HFD intake caused alterations of brain barrier integrity at early time when compared to old SD mice, possibly accelerating comorbidities at central nervous system. In conclusion, obesity superimposed with aging would accelerate or aggravate the process of aging itsel

Retinal defects in mice lacking the autism-associated gene Engrailed-2

Defective cortical processing of visual stimuli and altered retinal function have been described in autism spectrum disorder (ASD)patients. In keeping with these findings, anatomical and functional defects have been found in the visual cortex and retina of mice bearing mutations for ASD-associated genes. Here we sought to investigate the anatomy and function of the adult retina of Engrailed 2 knockout (En2 −/− )mice, a model for ASD. Our results showed that En2 is expressed in all three nuclear layers of the adult retina. When compared to age-matched En2 +/+ controls, En2 −/− adult retinas showed a significant decrease in the number of calbindin + horizontal cells, and a significant increase in calbindin + amacrine/ganglion cells. The total number of ganglion cells was not altered in the adult En2 −/− retina, as shown by Brn3a + cell counts. In addition, En2 −/− adult mice showed a significant reduction of photoreceptor (rhodopsin)and bipolar cell (Pcp2, PKCα)markers. Functional defects were also present in the retina of En2 mutants, as indicated by electroretinogram recordings showing a significant reduction in both a-wave and b-wave amplitude in En2 −/− mice as compared to controls. These data show for the first time that anatomical and functional defects are present in the retina of the En2 ASD mouse model

TGFβ upregulates PAR-1 expression and signalling responses in A549 lung adenocarcinoma cells

The major high-affinity thrombin receptor, proteinase activated receptor-1 (PAR-1) is expressed at low levels by the normal epithelium but is upregulated in many types of cancer, including lung cancer. The thrombin-PAR-1 signalling axis contributes to the activation of latent TGFβ in response to tissue injury via an avβ6 integrin-mediated mechanism. TGFβ is a pleiotropic cytokine that acts as a tumour suppressor in normal and dysplastic cells but switches into a tumour promoter in advanced tumours. In this study we demonstrate that TGFβ is a positive regulator of PAR-1 expression in A549 lung adenocarcinoma cells, which in turn increases the sensitivity of these cells to thrombin signalling. We further demonstrate that this effect is Smad3-, ERK1/2-and Sp1-dependent. We also show that TGFβ-mediated PAR-1 upregulation is accompanied by increased expression of integrin av and β6 subunits. Finally, TGFβ pre-stimulation promotes increased migratory potential of A549 to thrombin. These data have important implications for our understanding of the interplay between coagulation and TGFβ signalling responses in lung cancer

Different patterns of H2S/NO activity and cross-talk in the control of the coronary vascular bed under normotensive or hypertensive conditions

Hydrogen sulfide (H2S) and nitric oxide (NO) play pivotal roles in the cardiovascular system. Conflicting results have been reported about their cross-talk. This study investigated their interplays in coronary bed of normotensive (NTRs) and spontaneously hypertensive rats (SHRs). The effects of H2S- (NaHS) and NO-donors (sodium nitroprusside, SNP) on coronary flow (CF) were measured in Langendorff-perfused hearts of NTRs and SHRs, in the absence or in the presence of propargylglycine (PAG, inhibitor of H2S biosynthesis), L-NAME (inhibitor of NO biosynthesis), ODQ (inhibitor of guanylate cyclase), L-Cysteine (substrate for H2S biosynthesis) or L-Arginine (substrate for NO biosynthesis). In NTRs, NaHS and SNP increased CF; their effects were particularly evident in Angiotensin II (AngII)-contracted coronary arteries. The dilatory effects of NaHS were abolished by L-NAME and ODQ; conversely, PAG abolished the effects of SNP. In SHRs, high levels of myocardial ROS production were observed. NaHS and SNP did not reduce the oxidative stress, but produced clear increases of the basal CF. In contrast, in AngII-contracted coronary arteries of SHRs, significant hyporeactivity to NaHS and SNP was observed. In SHRs, the vasodilatory effects of NaHS were only modestly affected by L-NAME and ODQ; PAG poorly influenced the effects of SNP. Then, in NTRs, the vascular actions of H2S required NO and vice versa. By contrast, in SHRs, the H2S-induced actions scarcely depend on NO release; as well, the NO effects are largely H2S-independent. These results represent the first step for understanding pathophysiological mechanisms of NO/H2S interplays under both normotensive and hypertensive conditions

Colonic dysmotility and inflammation associated with high fat diet-induced obesity: role of the enteric glia

AbstractIntroductionEnteric glial cells (EGCs) contribute to the regulation of bowel motility, and have been implicated in the onset and development of several digestive disorders. However, the involvement of EGCs in obesity-related intestinal dysmotility is unknown. Accordingly, this study examined the role of EGCs in colonic neuromuscular dysfunctions in a mouse model of diet-induced obesity.Materials and MethodsC57BL/6 male mice (n = 6 per group) were fed with standard diet (SD) or high fat diet (HFD) for 8 weeks. Body and epididymal fat weight, and blood fasting glucose levels were evaluated the day before sacrifice. Colonic longitudinal muscle strips were set up in organ baths with Krebs solution and connected to isometric transducers. The effects of fluorocitrate (FC, gliotoxin) were tested on contractile responses mediated by NK1 tachykininergic receptors upon application of electrical stimuli (0.5 ms, 28 V, 10 Hz) [incubation with atropine, guanethidine, L-NAME, GR159897 and SB218795 (NK2 and NK3 antagonists, respectively)] or exogenous substance P (SP). Colonic levels of interleukin (IL)-1β, IL-6, malondialdehyde (MDA) and occludin (a tight junction protein involved the maintenance of mucosal barrier) were measured. Cultured rat EGCs were exposed to palmitate and lipopolysaccharide (LPS), either alone or in combination, to mimic the exposure to HFD. IL-1β and SP levels were then assessed in cell supernatants, while toll-like receptor 4 (TLR4) expression was evaluated in cell lysates.ResultsHFD-mice displayed increments of body weight, epididymal fat weight and blood glucose levels. In in vitro experiments, electrically induced colonic tachykininergic contractions were enhanced in HFD mice, as compared with SD animals. No differences were observed when comparing contractions to exogenous SP. The increase in electrically evoked tachykininergic contractions was blunted upon incubation with the gliotoxin FC. Exogenous SP-induced contractions were not affected by FC. HFD mice displayed an increase in colonic IL-1β, IL-6 and MDA levels and a reduced occludin expression, as compared with SD mice. Exposure of EGCs to palmitate, alone or in combination with LPS, resulted in a significant increase in TLR4 expression, while LPS alone was without effects. The combination of palmitate and LPS increased significantly IL-1β and SP levels in cell supernatants, while single treatments were without effects.DiscussionHFD is characterized by colonic dysmotility along with bowel inflammation, oxidative stress, and an impairment of mucosal barrier integrity. In this setting, the hyperactivation of EGCs, likely via TLR4, appears to contribute to inflammation and colonic tachykininergic motor dysfunctions

A Comparative Study on the Efficacy of NLRP3 Inflammasome Signaling Inhibitors in a Pre-clinical Model of Bowel Inflammation

Nucleotide-binding oligomerization domain leucine rich repeat and pyrin domain-containing protein 3 (NLRP3) inflammasome is pivotal in maintaining intestinal homeostasis and sustaining enteric immune responses in the setting of inflammatory bowel diseases. Drugs acting as NLRP3 blockers could represent innovative strategies for treatment of bowel inflammation. This study was performed in rats with dinitrobenzenesulfonic acid (DNBS)-induced colitis, to investigate how the direct blockade of NLRP3 inflammasome with an irreversible inhibitor (INF39) compares with Ac-YVAD-cmk (YVAD, caspase-1 inhibitor) and anakinra (IL-1β receptor antagonist), acting downstream on NLRP3 signaling. Animals with DNBS-colitis received YVAD (3 mg/kg) or anakinra (100 mg/Kg) intraperitoneally, and INF39 (25 mg/kg) or dexamethasone (DEX, 1 mg/kg) orally for 6 days, starting on the same day of colitis induction. Under colitis, there was a body weight decrease, which was attenuated by YVAD, anakinra or INF39, but not DEX. All test drugs counteracted the increase in spleen weight. The colonic shortening and morphological colonic alterations associated with colitis were counteracted by INF39, anakinra and DEX, while YVAD was without effects. Tissue increments of myeloperoxidase, tumor necrosis factor and interleukin-1β were more effectively counteracted by INF39 and DEX, than YVAD and anakinra. These findings indicate that: (1) direct inhibition of NLRP3 inflammasome with INF39 is more effective than caspase-1 inhibition or IL-1β receptor blockade in reducing systemic and bowel inflammatory alterations; (2) direct NLRP3 inhibition can be a suitable strategy for treatment of bowel inflammation

Enteric α-synuclein impairs intestinal epithelial barrier through caspase-1-inflammasome signaling in Parkinson's disease before brain pathology

Bowel inflammation, impaired intestinal epithelial barrier (IEB), and gut dysbiosis could represent early events in Parkinson’s disease (PD). This study examined, in a descriptive manner, the correlation among enteric α-synuclein, bowel inflammation, impairments of IEB and alterations of enteric bacteria in a transgenic (Tg) model of PD before brain pathology. Human A53T α-synuclein Tg mice were sacrificed at 3, 6, and 9 months of age to evaluate concomitance of enteric inflammation, IEB impairments, and enteric bacterial metabolite alterations during the early phases of α-synucleinopathy. The molecular mechanisms underlying the interplay between α-synuclein, activation of immune/inflammatory responses and IEB alterations were investigated with in vitro experiments in cell cultures. Tg mice displayed an increase in colonic levels of IL-1β, TNF, caspase-1 activity and enteric glia activation since 3 months of age. Colonic TLR-2 and zonulin-1 expression were altered in Tg mice as compared with controls. Lipopolysaccharide levels were increased in Tg animals at 3 months, while fecal butyrate and propionate levels were decreased. Co-treatment with lipopolysaccharide and α-synuclein promoted IL-1β release in the supernatant of THP-1 cells. When applied to Caco-2 cells, the THP- 1-derived supernatant decreased zonulin-1 and occludin expression. Such an effect was abrogated when THP-1 cells were incubated with YVAD (caspase-1 inhibitor) or when Caco-2 were incubated with anakinra, while butyrate incubation did not prevent such decrease. Taken together, early enteric α-synuclein accumulation contributes to compromise IEB through the direct activation of canonical caspase-1-dependent inflammasome signaling. These changes could contribute both to bowel symptoms as well as central pathology.Bowel inflammation, impaired intestinal epithelial barrier (IEB), and gut dysbiosis could represent early events in Parkinson’s disease (PD). This study examined, in a descriptive manner, the correlation among enteric α-synuclein, bowel inflammation, impairments of IEB and alterations of enteric bacteria in a transgenic (Tg) model of PD before brain pathology. Human A53T α-synuclein Tg mice were sacrificed at 3, 6, and 9 months of age to evaluate concomitance of enteric inflammation, IEB impairments, and enteric bacterial metabolite alterations during the early phases of α-synucleinopathy. The molecular mechanisms underlying the interplay between α-synuclein, activation of immune/inflammatory responses and IEB alterations were investigated with in vitro experiments in cell cultures. Tg mice displayed an increase in colonic levels of IL-1β, TNF, caspase-1 activity and enteric glia activation since 3 months of age. Colonic TLR-2 and zonulin-1 expression were altered in Tg mice as compared with controls. Lipopolysaccharide levels were increased in Tg animals at 3 months, while fecal butyrate and propionate levels were decreased. Co-treatment with lipopolysaccharide and α-synuclein promoted IL-1β release in the supernatant of THP-1 cells. When applied to Caco-2 cells, the THP-1-derived supernatant decreased zonulin-1 and occludin expression. Such an effect was abrogated when THP-1 cells were incubated with YVAD (caspase-1 inhibitor) or when Caco-2 were incubated with anakinra, while butyrate incubation did not prevent such decrease. Taken together, early enteric α-synuclein accumulation contributes to compromise IEB through the direct activation of canonical caspase-1-dependent inflammasome signaling. These changes could contribute both to bowel symptoms as well as central pathology

Studio funzionale sugli effetti vasomotori di H2S sui distretti mesenterico e coronarico e sul cross-talk H2S-NO.

E’ ormai dimostrato che l’H2S, considerato a lungo unicamente come gas tossico, è un importante modulatore endogeno che mostra i tipici benefici dell’NO, senza portare alla produzione di specie reattive dell’ossigeno (ROS). Al contrario, risulta essere uno “scavenger” dei ROS. L’H2S nei tessuti dei mammiferi viene sintetizzato, partendo dall’amminoacido L-Cisteina, ad opera di due enzimi: la cistationina β sintetasi (CBS) e la cistationina γ liasi (CSE). L’H2S espleta la sua azione in diversi distretti, e dalle conoscenze scientifiche odierne sembra che agisca tramite attivazione di canali ionici, come i canali del potassio ATP-dipendenti (KATP). A livello cardio-vascolare, l’H2S evoca una risposta vasorilassante in modo concentrazione-dipendente. Sebbene la vasodilatazione indotta dall’H2S possa essere ottenuta tramite effetti diretti endotelio-indipendenti sulle cellule di muscolatura liscia, è stato riportato che l’utilizzo di inibitori dell’NO-sintetasi o la rimozione dell’endotelio possano attenuare il rilasciamento indotto dall’H2S. Pertanto è stato ipotizzato che possa esistere un cross-talk tra la via L-arginina/NO e L-cisteina/ H2S. Visto che le conoscenze scientifiche circa il ruolo fisiologico e patologico dell’H2S sono ancora molto frammentarie, questa tesi si è posta l’obiettivo di approfondire le conoscenze farmacodinamiche sul ruolo di H2S a livello di arterie di resistenza, ed in particolare: • valutare l’effetto dell’NaHS (fonte esogena di H2S) a livello dei distretti coronarico e mesenterico; • verificare il cross-talk tra la via L-arginina/NO e L-cisteina/H2S in questi distretti; MATERIALI E METODI: Il cuore è stato allestito tramite metodo alla Langendorf e il flusso coronarico è stato misurato volumetricamente. Le arterie mesenteriche sono state allestite utilizzando un micro miografo a pressione e l’effetto vasomotore delle arterie mesenteriche è stato valutato tramite misurazione della variazione del lume. RISULTATI: L’H2S è dotato di effetti vasodilatatori sia a livello mesenterico sia a livello coronarico che vanno a contromodulare gli effetti vasospastici prodotti da importanti mediatori endogeni come Noradrenalina e Angiotensina II. Gli effetti vasodilatatori di NaHS sono in qualche modo interconnessi con l’NO attraverso vie di cross-talk complesse e probabilmente eterogenee che saranno oggetto di indagini sperimentali specifiche

Pathological Remodeling of the Gut Barrier as a Prodromal Event of High-Fat Diet-Induced Obesity

Intestinal barrier alterations represent a primum movens in obesity and related intestinal dys-functions. 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-1b, 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 asso-ciated 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.& COPY; 2023 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved

Enteric Alpha-Synuclein Inclusions, Colonic Inflammation, Increased Mucosal Permeability and Alterations of Bowel Neuromuscular Functions Precede Central Neurodegeneration in a Transgenic Mouse Model of Parkinson's Disease

No abstract availabl