Intracellular molecular effects of insulin resistance in patients with metabolic syndrome

<p>Abstract</p> <p>Aim of the study</p> <p>Patients with metabolic syndrome (MetS) have an increased risk of cardiovascular disease. Data obtained from muscle biopsies have demonstrated altered insulin signaling (IS) in patients with MetS. The IS regulates critical cell functions including molecular-regulated cellular metabolite fluxes, protein and energetic metabolism, cell proliferation and apoptosis with consequent regulation of cell life including endothelial homeostasis and blood coagulation. However, little is known about blood cell IS in MetS patients. The aim of this study was to develop a method to evaluate IS in peripheral lymphocytes to identify altered intracellular molecules in patients with MetS to use as risk biomarkers of vascular thrombosis.</p> <p>Patients and Methods</p> <p>We investigated 40 patients with MetS and 20 controls. MetS was defined according to guidelines from the US National Cholesterol Education Program Adult Treatment Panel III. Blood samples were taken from all participants. Total mononuclear cells were isolated from peripheral blood using density gradient centrifugation. IS molecules were evaluated using Western blot analysis followed by computer-assisted densitometer evaluation.</p> <p>Results</p> <p>Lymphocytes of MetS patients showed a reduced mTOR expression (the mammalian target of rapamycin) which is a fundamental molecule of IS. Major impairment of IS was confirmed by reduced upstream and downstream mTOR molecules which regulate fundamental cells metabolic functions.</p> <p>Conclusions</p> <p>In patients with MetS, we found a reduction of mTOR and other mTOR-related molecules involved in insulin resistance, cell repair, coagulation and vasculogenesis. A reduced expression of mTOR may reflect an increased risk of vascular thrombosis.</p

DDS-induced colorectal fibrosis in mice: anti-fibrotic effects of GED 0507-34 levo, a novel PPARγ ligand

Intestinal fibrosis is a progressive process characterized by de novo synthesis and uncontrolled deposition of extracellular matrix components (ECM) following a tissue chronic inflammation mainly regulated by Transforming Growth Factor (TGF)β/ Smads pathway. Frequently associated to Inflammatory Bowel Disease (IBD), intestinal fibrosis may lead to stenosis and obstructions that require surgery up to 75% of patients as drugs currently used in IBD are unable to improve fibrostenosis lesions (1). Peroxisome proliferator-activated receptor (PPAR)-γ is able to antagonize (TGF) β/Smads and could be an crucial target to develop novel antifibrotic therapeutic strategies (2). Aim of this study is to evaluate the antifibrotic action of a novel PPARγ agonist, GED 0507-34 levo, in colonic fibrosis in mice. Immunohistochemistry and immunoblotting evaluations, TGFβ1, CTGF, Collagen types I-III, Smad3, α-SMA, were performed in in three groups of C57BL/6 mice: Dextran Sulphate Sodium (DSS) colitis group, DSS+GED group and controls. Evident macroscopic and microscopic lesions in the most of colons of DSS treated mice were observed compared to DSS+GED mice and controls. The tissue levels of the main markers of fibrosis resulted significantly increased in DSS mice and restored by administration of GED. GED seems to prevents ECM colonic deposition and to improve the intestinal fibrotic lesions by its ability in controlling TGFβ/Smads pathway signalling activation

Anti-fibrotic effect of a novel PPAR-γ ligand, GED-0507-34 LEVO, in DSS induced colonic fibrosis in mice

Intestinal fibrosis is a common complication of inflammatory bowel disease (IBD) in which chronic inflammation leads to abnormal deposition of extracellular matrix components (ECM) causing obstruction and loss of function of the intestinal tract involved (1). Fibrogenesis is mainly regulates by trasforming growth factor (TGF) β/Smad pathway and a key antagonist of this signaling is represented by peroxisome proliferator-activated receptor (PPAR)-γ. As the anti-inflammatory drugs currently used in IBD are unable to improve intestinal fibrosis the exploration of new therapeutical approaches has now became crucial (2). Aim of this study is to evaluate the antifibrotic action of a novel PPARγ agonist, GED-0507-34-LEVO (GED), in colon fibrosis in mice. Chronic colitis and fibrosis were induced in C57BL/6 mice by administration of 2,5% (w/v) dextrane sulphate sodium (DSS) in drinking water for 5 days followed by 7 days of water for 3 cycles. Mice were divided into 3 groups: DSS, DSS+GED and control. 30mg/Kg/mice of GED was daily administrated by oral gavage starting from the second DSS cycle. Samples from colon were excised and processed to assess macroscopic lesions, histological and morphometrical aspects and immunohistochemical and immunoblotting analysis for TGFβ1, CTGF, collagen types I-III, Smad3,α-SMA. Evident shortening and dilation in the most of colons of DSS treated mice were observed. Macroscopic and microscopic findings were significantly improved in DSS mice+GED compared with control mice. The tissue levels of collagen and α-SMA, specific markers of fibrosis, resulted significantly increased in mice receving DSS compared to control mice, as well as the expression of TGFβ1, CTGF, Smad3. DSS+GED group showed reduced expression of all markers involved. GED significantly improves the intestinal fibrotic lesions in DSS chronic colitis murine model and controls the pivotal molecular events leading to fibrosis

Interaction between sphingosine kinase/sphingosine 1 phosphate and transforming growth factor-β/Smads pathways in experimental intestinal fibrosis. An in vivo immunohistochemical study

A concomitant action of multiple profibrotic mediators appears crucial in the development and progression of fibrosis. Sphingosine kinase/sphingosine 1 phosphate and transforming growth factor-β/Smads pathways are both involved in pathogenesis of fibrosis in several organs by controlling differentiation of fibroblasts to myofibroblasts and the epithelial to-mesenchymal transition. However, their direct involvement in chronic colitis-associated fibrosis it is not yet known. In this study we evaluated the immunohistochemical expression of some proteins implicated in sphingosine kinase/sphingosine 1 phosphate and transforming growth factor-β/Smads pathways in Dextrane Sodium Sulphate (DSS)-induced colorectal fibrosis in mice. Compared to control mice, DSS-induced chronic colitis mice developed a marked intestinal fibrosis associated with a concomitant overexpression of TGF-β, p-Smad3, α-SMA, collagen I-III, SPHK1, RhoA, PI3K, Akt, p-Akt, p-mTOR. This study highlights the relationship between the two pathways and the possible role of SPHK1 in the intestinal fibrosis.  These results, if confirmed by in vitro studies, may have important clinical implications in the development of new therapeutical approaches in inflammatory bowel disease

Soluble markers of B cell activation suggest a role of B cells in the pathogenesis of systemic sclerosis-associated pulmonary arterial hypertension

IntroductionSoluble markers of B cell activation are interesting diagnostic and prognostic tools in autoimmune diseases. Data in systemic sclerosis (SSc) are scarce and few studies focused on their association with disease characteristics.Methods1. Serum levels of 14 B cell biomarkers (β2-microglobulin, rheumatoid factor (RF), immunoglobulins (Ig) G, IgA, IgM, BAFF, APRIL, soluble (s)TACI, sBCMA sCD21, sCD23, sCD25, sCD27, CXCL13) were measured in SSc patients and healthy controls (HC). 2. Associations between these biomarkers and SSc characteristics were assessed. 3. The pathophysiological relevance of identified associations was explored by studying protein production in B cell culture supernatant.ResultsIn a discovery panel of 80 SSc patients encompassing the broad spectrum of disease manifestations, we observed a higher frequency of RF positivity, and increased levels of β2-microglobulin, IgG and CXCL13 compared with HC. We found significant associations between several biomarkers and SSc characteristics related to disease phenotype, activity and severity. Especially, serum IgG levels were associated with pulmonary hypertension (PH); β2-microglobulin with Nt-pro-BNP and DLCO; and BAFF with peak tricuspid regurgitation velocity (TRV). In a validation cohort of limited cutaneous SSc patients without extensive ILD, we observed lower serum IgG levels, and higher β2-microglobulin, sBCMA, sCD23 and sCD27 levels in patients with pulmonary arterial hypertension (PAH). BAFF levels strongly correlated with Nt-pro-BNP levels, FVC/DLCO ratio and peak TRV in SSc-PAH patients. Cultured SSc B cells showed increased production of various angiogenic factors (angiogenin, angiopoietin-1, VEGFR-1, PDGF-AA, MMP-8, TIMP-1, L-selectin) and decreased production of angiopoietin-2 compared to HC.ConclusionSoluble markers of B cell activation could be relevant tools to assess organ involvements, activity and severity in SSc. Their associations with PAH could plead for a role of B cell activation in the pathogenesis of pulmonary microangiopathy. B cells may contribute to SSc vasculopathy through production of angiogenic mediators

The Expression of the Short Isoform of Thymic Stromal Lymphopoietin in the Colon Is Regulated by the Nuclear Receptor Peroxisome Proliferator Activated Receptor-Gamma and Is Impaired during Ulcerative Colitis

The etiology of inflammatory bowel diseases remains largely unknown. We previously demonstrated that the expression of the peroxisome proliferator activated receptor-gamma (PPARγ) is downregulated in colonic epithelial cells of patients with ulcerative colitis (UC). PPARγ is a nuclear receptor that modulates inflammation. We hypothesized that its deficiency may play a role in the loss of intestinal homeostasis through the control of immunomodulatory factors. We found that thymic stromal lymphopoietin (TSLP), an epithelial cytokine with pleiotropic functions, is regulated by PPARγ. While this cytokine possesses two isoforms, only the short form (sfTSLP) was regulated by PPARγ. sfTSLP mRNA expression was decreased both in PPARγ knock-down Caco2 cells and cells treated with PPARγ antagonist, whereas PPARγ agonists induced the expression of sfTSLP in Caco2 and T-84 cells. The response element activated by PPARγ was identified in the promoter of the sfTSLP gene by chromatin immunoprecipitation and gene reporter assays. The expression of sfTSLP was significantly decreased in the colonic mucosa of UC patients compared to controls and was correlated with PPARγ expression. Our results identified sfTSLP as a new PPARγ-target gene and support the hypothesis that, in UC, PPARγ deficiency in colonic mucosa could play a role in the loss of intestinal tolerance through an impaired sfTSLP expression

The Expression of the Short Isoform of Thymic Stromal Lymphopoietin in the Colon Is Regulated by the Nuclear Receptor Peroxisome Proliferator Activated Receptor-Gamma and Is Impaired during Ulcerative Colitis

International audienceThe etiology of inflammatory bowel diseases remains largely unknown. We previously demonstrated that the expression of the peroxisome proliferator activated receptor-gamma (PPARγ) is downregulated in colonic epithelial cells of patients with ulcerative colitis (UC). PPARγ is a nuclear receptor that modulates inflammation. We hypothesized that its deficiency may play a role in the loss of intestinal homeostasis through the control of immunomodulatory factors. We found that thymic stromal lymphopoietin (TSLP), an epithelial cytokine with pleiotropic functions, is regulated by PPARγ. While this cytokine possesses two isoforms, only the short form (sfTSLP) was regulated by PPARγ. sfTSLP mRNA expression was decreased both in PPARγ knock-down Caco2 cells and cells treated with PPARγ antagonist, whereas PPARγ agonists induced the expression of sfTSLP in Caco2 and T-84 cells. The response element activated by PPARγ was identified in the promoter of the sfTSLP gene by chromatin immunoprecipitation and gene reporter assays. The expression of sfTSLP was significantly decreased in the colonic mucosa of UC patients compared to controls and was correlated with PPARγ expression. Our results identified sfTSLP as a new PPARγ-target gene and support the hypothesis that, in UC, PPARγ deficiency in colonic mucosa could play a role in the loss of intestinal tolerance through an impaired sfTSLP expression

Essential amino acids improve insulin activation of Akt/mTOR signaling in soleus muscle of aged rats

Essential amino acids (EAA) improve basal muscle protein synthesis in the elderly. Nevertheless, in settings of prolonged supplementation, putative signal pathways of EAA are currently unknown. The purpose of this study was to test the effects of prolonged supplementation of EAA enriched mixture (12-L-Amin) on Insulin/Insulin-like Growth Factor-1 (IGF1) pathway by measuring total and phosphorylated Akt (Ser473) and its upstream (IRS1 at Ser636) and downstream (mTOR at Ser2448, p70S6K at Thr389) targets in basal conditions and following acute insulin (0.1 U/L) incubation in vitro. To this aim, soleus muscles were dissected from male Wistar rats divided in three groups of 7 each: adults (AD, 10 mo of age), elderly (EL, 22 mo of age) and elderly supplemented (EL-AA, 12-L-Amin 1.5gr/Kg die in drinking water for 3 mo). EL showed reduced basal and post-insulin mTOR and p70S6K activation and reduced post-insulin IRS1 degradation relative to AD. EL-AA showed an increase of post-insulin Akt activation, no change in basal and post-insulin phospho-mTOR, lower reduction of phospho-p70S6K and increased post-insulin IRS1 degradation relative to AD. These results demonstrate that chronic 12-LAmin administration exerts anti-ageing effects on the activation/inactivation of the Insulin/IGF1/mTOR pathway which is identified as putative target of EAA in the elderly

Adherent invasive escherichia coli (aiec) strain lf82, but not candida albicans, plays a profibrogenic role in the intestine

International audienceBACKGROUND: Intestinal fibrosis is a frequent complication of Crohn's disease. However, the factors that cause chronicity and promote fibrogenesis are not yet understood.OBJECTIVE: In the present study, we evaluated the profibrotic effects of adherent-invasive Escherichia coli (AIEC) LF82 strain and Candida albicans in the gut.METHODS: Colonic fibrosis was induced in C57BL/6 mice by administration of three cycles of 2.5% (w/v) dextran sulfate sodium (DSS) for 5 weeks. LF82 and C. albicans were administered orally once at the start of each week or each cycle, respectively. Expression of markers of myofibroblast activation was determined in TGF-β1-stimulated human intestinal epithelial cells (IECs).RESULTS: LF82 administration exacerbated fibrosis in DSS-treated mice, revealed by increased colonic collagen deposition and expression of the profibrotic genes Col1a1, Col3a1, Fn1 and Vim. This was accompanied by enhanced gene expression of proinflammatory cytokines and chemokines, as well as more recruited inflammatory cells into the intestine. LF82 also potentiated TGF-β1-stimulated epithelial-mesenchymal transition and myofibroblast activation in IECs, by further inducing gene expression of the main mesenchymal cell markers FN1 and VIM and downregulating the IEC marker OCLN. Proinflammatory cytokines were overexpressed with LF82 in TGF-β1-stimulated IECs. Conversely, C. albicans did not affect intestinal fibrosis progression in DSS-treated mice or myofibroblast activation in TGF-β1-stimulated IECs.CONCLUSIONS: These results demonstrate that AIEC strain LF82, but not C. albicans, may play a major profibrogenic role in the gut