[Show abstract][Hide abstract]ABSTRACT: Maternal obesity increases the risk of obesity and/or obesity-related diseases in the offspring of animal models. The aim of this study was to identify metabolic dysfunctions that could represent an enhanced risk for human obesity or obesity-related diseases in newborn or in adult life, similar to what occurs in animal models. To this aim, we studied the proteome of 12 obese (Ob-) and 6 non-obese (Co-) human amniotic mesenchymal stem cells (hA-MSCs) obtained from women at delivery by cesarean section (pre-pregnancy body mass index [mean ± SD]: 42.7 ± 7.7 and 21.3 ± 3.3 kg/m2, respectively). The proteome, investigated by two-dimensional fluorescence difference gel electrophoresis/mass spectrometry, revealed 62 differently expressed proteins in Ob- vs Co-hA-MSCs (P < 0.05), nine of which were confirmed by western blotting. Bioinformatics analysis showed that these 62 proteins are involved in several statistically significant pathways (P < 0.05), including the stress response, cytoskeleton and metabolic pathways. Oxidative stress was shown to be an early triggering factor of tissue fat accumulation and obesity-related disorders in the offspring of obese animal models. Our finding of a reduced stress response in Ob-hA-MSCs suggests that a similar mechanism could occur also in humans. Long-term follow-up studies of newborns of obese mothers are required to verify this hypothesis.
[Show abstract][Hide abstract]ABSTRACT: Mitochondrial DNA (mtDNA) haplogroups have been associated with the expression of mitochondrial-related diseases and with metabolic alterations, but their role has not yet been investigated in morbid obese Caucasian subjects. Therefore, we investigated the association between mitochondrial haplogroups and morbid obesity in patients from southern Italy. The mtDNA D-loop of morbid obese patients (n = 500; BMI > 40 kg/m(2)) and controls (n = 216; BMI < 25 kg/m(2)) was sequenced to determine the mtDNA haplogroups. The T and J haplogroup frequencies were higher and lower, respectively, in obese subjects than in controls. Women bearing haplogroup T or J had twice or half the risk of obesity. Binomial logistic regression analysis showed that haplogroup T and systolic blood pressure are risk factors for a high degree of morbid obesity, namely, BMI > 45 kg/m(2) and in fact together account for 8% of the BMI. In conclusion, our finding that haplogroup T increases the risk of obesity by about two-fold, suggests that, besides nuclear genome variations and environmental factors, the T haplogroup plays a role in morbid obesity in our study population from southern Italy.
[Show abstract][Hide abstract]ABSTRACT: Maternal obesity and nutrient excess in utero increase the risk of future metabolic diseases. The mechanisms underlying this process are poorly understood, but probably include genetic, epigenetic alterations and changes in fetal nutrient supply. We have studied the microRNA (miRNA) expression profile in amnion from obese and control women at delivery to investigate if a specific miRNA-signature is associated with obesity. The expression profile of 365 human miRNAs was evaluated with the TaqMan Array in amnion from 10 obese and 5 control (pre-pregnancy BMI>30 and <25 kg/m(2), respectively) women at delivery. Target genes and miRNA-regulated pathways were predicted by bioinformatics. Anthropometric and biochemical parameters were also measured in mothers and newborns. Seven miRNAs were expressed only in obese women (miR-422b, miR-219, miR-575, miR-523, miR-579, miR-618, miR-659), while 13 miRNAs were expressed at a higher level and 12 at a lower level in obese women than in controls. MicroRNAs significantly downregulated the neurotrophin, cancer/ErbB, mTOR, insulin, adipocytokine, actin cytoskeleton and MAPK signaling pathways. In conclusion, we show that the miRNA profile is altered in amnion during obesity and hypothesize that this could affect pathways important for placental growth and function thereby contributing to increase the newborn's risk of future metabolic diseases.International Journal of Obesity accepted article preview online, 2 July 2013; doi:10.1038/ijo.2013.121.
Article · Jul 2013 · International journal of obesity (2005)
[Show abstract][Hide abstract]ABSTRACT: Maternal obesity is associated to increased fetal risk of obesity and other metabolic diseases. Human amniotic mesenchymal stem cells (hA-MSC) have not been characterized in obese women. The aim of this study was to isolate and compare hA-MSC immunophenotypes from obese (Ob-) and normal weight control (Co-) women to identify alterations possibly predisposing the fetus to obesity. We enrolled 16 Ob- and 7 Co-women at delivery (mean/SEM pre-pregnancy BMI: 40.3/1.8 kg/m<sup>2</sup> and 22.4/1.0 kg/m<sup>2</sup>, respectively) and 32 not pregnant women. hA-MSCs were phenotyped by flow cytometry; several maternal and newborn clinical and biochemical parameters were also measured. The expression of membrane antigen CD13 was higher on Ob-hA-MSCs than on Co-hA-MSCs (P=0.0043). Also serum levels of CD13 at delivery were higher in Ob- versus Co-pregnant women and correlated with CD13 antigen expression on Ob-hA-MSCs (r<sup>2</sup>=0.84, P<0.0001). Adipogenesis induction experiments revealed that Ob-hA-MSCs had a higher adipogenic potential than Co-hA-MSCs as witnessed by higher PPARγ and aP2 mRNA levels (P=0.02 and P=0.03, respectively) at post-induction day 14 associated with increased CD13 mRNA levels from baseline to day 4 post-induction (P<0.05). Adipogenesis was similar in the two sets of hA-MSCs after CD13 silencing, whereas it was increased in Co-hA-MSCs after CD13 overexpression. CD13 expression was high also in Ob-h-MSCs from umbilical cords or visceral adipose tissue of not pregnant women. In conclusion, antigen CD13, by influencing the adipogenic potential of hA-MSCs could be an in-utero risk factor for obesity. Our data strengthen the hypothesis that high levels of serum and MSC CD13 are obesity markers.
[Show abstract][Hide abstract]ABSTRACT: Type 2 Maturity Onset Diabetes of the Young (MODY2) is a monogenic autosomal disease characterized by a primary defect in insulin secretion and hyperglycemia. It results from GCK gene mutations that impair enzyme activity. Between 2006 and 2010, we investigated GCK mutations in 66 diabetic children from southern Italy with suspected MODY2. Denaturing High Performance Liquid Chromatography (DHPLC) and sequence analysis revealed 19 GCK mutations in 28 children, six of which were novel: p.Glu40Asp, p.Val154Leu, p.Arg447Glyfs, p.Lys458_Cys461del, p.Glu395_Arg397del and c.580-2A>T. We evaluated the effect of these 19 mutations using bioinformatic tools such as Polymorphism Phenotyping (Polyphen), Sorting Intolerant From Tolerant (SIFT) and in silico modelling. We also conducted a functional study to evaluate the pathogenic significance of seven mutations that are among the most severe mutations found in our population, and have never been characterized: p.Glu70Asp, p.His137Asp, p.Phe150Tyr, p.Val154Leu, p.Gly162Asp, p.Arg303Trp and p.Arg392Ser. These seven mutations, by altering one or more kinetic parameters, reduced enzyme catalytic activity by >40%. All mutations except p.Glu70Asp displayed thermal-instability, indeed >50% of enzyme activity was lost at 50°C/30 min. Thus, these seven mutations play a pathogenic role in MODY2 insurgence. In conclusion, this report revealed six novel GCK mutations and sheds some light on the structure-function relationship of human GCK mutations and MODY2.
[Show abstract][Hide abstract]ABSTRACT: Close-up view of the p.Lys420Glu mutation at the inter-domain interface. The small and large domains are drawn in cyan and red, respectively. Helix 13 is shown in orange. Lys420 (red stick) forms a salt-bridge with Glu440 (yellow stick) which is located in a loop connecting the two domains.
[Show abstract][Hide abstract]ABSTRACT: Adipose tissues show selective gene expression patterns, to whom microRNAs (miRNAs) may contribute. We evaluated in visceral adipose tissue (VAT) from obese and nonobese females, both miRNA and protein expression profiles, to identify miRNA/protein target pairs associated with obesity (metabolic pathways miRNA-deregulated during obesity). Obese and nonobese females [BMI 42.2 ± 1.6 and 23.7 ± 1.2 kg/m(2) (mean ± SEM), respectively] were enrolled in this study. Notably, most miRNAs were down-expressed in obese tissues, whereas most of the proteins from the investigated spots were up-expressed. Bioinformatics integration of miRNA expression and proteomic data highlighted two potential miRNA/protein target pairs: miR-141/YWHAG (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, gamma polypeptide) and miR-520e/RAB11A (Ras-related protein RAB-11A); the functional interaction between these miRNAs and their target sequences on the corresponding mRNAs was confirmed by luciferase assays. Both RAB11A and YWHAG proteins are involved in glucose homeostasis; YWHAG is also involved in lipid metabolism. Hence, the identified miRNA/protein target pairs are potential players in the obese phenotype.
[Show abstract][Hide abstract]ABSTRACT: Decreased KLF4 and increased Ki67 expression in small intestine from CD patients compared with controls. A. KLF4 staining of small intestinal villi in GFD patients and Controls (Original magnification 20×). A statistically significant reduced KLF4-positive cells/villi were counted in GFD patients than in controls, respectively 29.0±5.0 vs 79.0±3.0 (mean±SEM) (p<0.0001). B. Increased Ki67 signal is present in small intestinal crypts of active CD, GFD patients than in controls (Original magnification 20×). (CTRL: controls; GFD: gluten free diet; CD: celiac disease).
[Show abstract][Hide abstract]ABSTRACT: Automated Counts of NOTCH1 and HES1 stained/unstained cells. A. Automated counts of NOTCH1 stained/unstained cells (reported in Figure 2) in small intestine from CD patients (6 active CD and 6 GFD patients) and from controls (n = 4). Data are expressed as mean percent of intensely stained, low-moderately stained and unstained cells of the total intraepithelial cells (IECs) counted in ten crypts. The numbers of intensely stained and unstained cells were significantly (p = 0.02) higher and lower, respectively, in CTRL than in active CD and in GFD patients. B. Automated counts of HES1 stained/unstained cells (reported in Figure 3) in small intestine from CD patients (6 active CD and 6 GFD patients) and from controls (n = 4). Data are expressed as mean percent of intensely stained, low-moderately stained and unstained cells of the total intraepithelial cells (IECs) counted in ten crypts. The number of intensely stained cells was significantly higher in controls versus CD and GFD patients (p = 0.02) and the number of unstained cells was significantly lower in CTRL than in active CD patients (p = 0.03). (CTRL: controls; GFD: gluten free diet; CD: celiac disease).
[Show abstract][Hide abstract]ABSTRACT: The luciferase assay confirms that miR-449a inhibits the expression of NOTCH1 and KLF4. In HEK293 cells co-transfected or with pRL-NOTCH1 vector (panel A) or with pRL-KLF4 vector (panel B), a pre-miR-449a concentration of 100 nmol/L was sufficient to significantly reduce (respectively, p = 0.001 and p = 0.002) Renilla luciferase activity versus control values. No inhibition of the Renilla luciferase expression was observed in mutant 3′UTR of KLF4-mRNA with miR-449a, so confirming the miR-449a/3′UTR KLF4-mRNA direct interaction (panel B). We didn't verify the interaction miR-449a/3′UTR NOTCH1 being this latter recently validated by Marcet B et al .
[Show abstract][Hide abstract]ABSTRACT: Other examples of NOTCH1 immunohistochemistry in CD patients. Examples of NOTCH1 immunohistochemistry in 4 CD patients (2 active CD: TIII Marsh stage and 2 GFD: TI and T0 Marsh stage) and 2 controls (T0 Marsh stage). The images show that the low expression levels of NOTCH1 in intestinal mucosa from CD patients were always present from TIII to T0 Marsh stage. (CTRL: controls; GFD: gluten free diet; CD: celiac disease).
[Show abstract][Hide abstract]ABSTRACT: Specificity of NOTCH1 and HES1 signals by immunohistochemistry. Specificity controls of NOTCH1 and HES1 antibodies. Positive NOTCH1 (A) and HES1 (B) immunostaining signals obtained in human colon cancer and negative NOTCH1 (C) and HES1 (D) immunostaining signals obtained in human endothelial wall.
[Show abstract][Hide abstract]ABSTRACT: MiRNAs play a relevant role in regulating gene expression in a variety of physiological and pathological conditions including autoimmune disorders. MiRNAs are also important in the differentiation and function of the mouse intestinal epithelium. Our study was aimed to look for miRNA-based modulation of gene expression in celiac small intestine, and particularly for genes involved in cell intestinal differentiation/proliferation mechanisms. A cohort of 40 children (20 with active CD, 9 on a gluten-free diet (GFD), and 11 controls), were recruited at the Paediatrics Department (University of Naples Federico II). The expression of 365 human miRNAs was quantified by TaqMan low-density arrays. We used bioinformatics to predict putative target genes of miRNAs and to select biological pathways. The presence of NOTCH1, HES1, KLF4, MUC-2, Ki67 and beta-catenin proteins in the small intestine of CD and control children was tested by immunohistochemistry. The expression of about 20% of the miRNAs tested differed between CD and control children. We found that high miR-449a levels targeted and reduced both NOTCH1 and KLF4 in HEK-293 cells. NOTCH1, KLF4 signals and the number of goblet cells were lower in small intestine of children with active CD and in those on a GFD than in controls, whereas more nuclear beta-catenin staining, as a sign of the WNT pathway activation, and more Ki67 staining, as sign of proliferation, were present in crypts from CD patients than in controls.
In conclusion we first demonstrate a miRNA mediated gene regulation in small intestine of CD patients. We also highlighted a reduced NOTCH1 pathway in our patients, irrespective of whether the disease was active or not. We suggest that NOTCH pathway could be constitutively altered in the celiac small intestine and could drive the increased proliferation and the decreased differentiation of intestinal cells towards the secretory goblet cell lineage.
[Show abstract][Hide abstract]ABSTRACT: Other examples of HES1 immunohistochemistry in CD patients. Examples of HES1 immunohistochemistry in 4 CD patients (2 active CD: TIII Marsh stage and 2 GFD: TI and T0 Marsh stage) and 2 controls (T0 Marsh stage). The images show that the low expression levels of HES1 in intestinal mucosa from CD patients were always present from TIII to T0 Marsh stage. (CTRL: controls; GFD: gluten free diet; CD: celiac disease).
[Show abstract][Hide abstract]ABSTRACT: Bioinformatics analysis of miR-449a putative target genes. miR-449a putative target genes with most favorable context score, selected by bioinformatics, were sorted into pathways using GOTM (http://bioinfo.vanderbilt.edu/webgestalt/) and then combined into functional groups. (http://mirecords.biolead.org/interactions.php?species=Homosapiens&mirna_acc=hsa-miR-449a&targetgene_type=refseq_acc&targetgene_info=&v=yes&search_int=Search) (http://www.targetscan.org/cgibin/targetscan/vert_50/targetscan.cgi?species=Human&gid=&mir_sc=&mir_c=&mir_nc=&mirg=hsa-miR-449a). In each functional group are reported the genes belonging to NOTCH pathway/total gene number.
[Show abstract][Hide abstract]ABSTRACT: Increased expression of beta-catenin in small intestine from CD patients compared with controls. Immunostaining with beta-catenin in small intestinal crypts from active CD, GFD and controls. We counted the beta-catenin labeled nuclei. Similar counts of beta-catenin labelled nuclei were detected in the crypts of the small intestine in all groups. However, higher even if not statistical significant mean percentage counts (beta-catenin positive nuclei/crypt) were obtained in active CD and GFD than in controls, respectively 57.0±11.5 and 37.0±4.6 vs 27.0±4.6 (Original magnification 63×). (CTRL: controls; GFD: gluten free diet; CD: celiac disease).