ABSTRACT
With the continuous rise in the worldwide prevalence of obesity and type 2 diabetes, developing therapies regulating body weight and glycemia has become a matter of great concern. Among the current treatments, evidence now shows that the use of intestinal hormone analogs (e.g., GLP1 analogs and others) helps to control glycemia and reduces body weight. Indeed, intestinal endocrine cells produce a large variety of hormones regulating metabolism, including appetite, digestion, and glucose homeostasis. Herein, we discuss how the enteroendocrine system is affected by local environmental and metabolic signals. These signals include those arising from unbalanced diet, gut microbiota, and the host metabolic organs and their complex cross-talk with the intestinal barrier integrity.
Subject(s)
Diabetes Mellitus, Type 2 , Gastrointestinal Hormones , Gastrointestinal Microbiome , Metabolic Diseases , Blood Glucose , Diabetes Mellitus, Type 2/metabolism , Gastrointestinal Microbiome/physiology , Humans , Obesity/metabolismABSTRACT
Carbohydrates and sweeteners are detected by the sweet taste receptor in enteroendocrine cells (EECs). This receptor is coupled to the gustducin G-protein, which α-subunit is encoded by GNAT3 gene. In intestine, the activation of sweet taste receptor triggers a signaling pathway leading to GLP-1 secretion, an incretin hormone. In metabolic diseases, GLP-1 concentration and incretin effect are reduced while partly restored after Roux-en-Y gastric bypass (RYGB). We wondered if the decreased GLP-1 secretion in metabolic diseases is caused by an intestinal defect in sweet taste transduction pathway. In our RNA-sequencing of EECs, GNAT3 expression is decreased in patients with obesity and type 2 diabetes compared with normoglycemic obese patients. This prompted us to explore sweet taste signaling pathway in mice with metabolic deteriorations. During obesity onset in mice, Gnat3 expression was downregulated in EECs. After metabolic improvement with enterogastro anastomosis surgery in mice (a surrogate of the RYGB in humans), the expression of Gnat3 increased in the new alimentary tract and glucose-induced GLP-1 secretion was improved. To evaluate if high-fat diet-induced dysbiotic intestinal microbiota could explain the changes in the expression of sweet taste α-subunit G-protein, we performed a fecal microbiota transfer in mice. However, we could not conclude if dysbiotic microbiota impacted or not intestinal Gnat3 expression. Our data highlight that metabolic disorders were associated with altered gene expression of sweet taste signaling in intestine. This could contribute to impaired GLP-1 secretion that is partly rescued after metabolic improvement.NEW & NOTEWORTHY Our data highlighted 1) the sweet taste transduction pathway in EECs plays pivotal role for glucose homeostasis at least at gene expression level; 2) metabolic disorders lead to altered gene expression of sweet taste signaling pathway in intestine contributing to impaired GLP-1 secretion; and 3) after surgical intestinal modifications, increased expression of GNAT3, encoding α-gustducin contributed to metabolic improvement.
Subject(s)
Diabetes Mellitus, Type 2/metabolism , Obesity/metabolism , Signal Transduction , Taste , Transducin/metabolism , Animals , Dysbiosis/metabolism , Enteroendocrine Cells/metabolism , Gastrointestinal Microbiome , Humans , Male , Mice, Inbred C57BLABSTRACT
OBJECTIVES: Altered enteroendocrine cell (EEC) function in obesity and type 2 diabetes is not fully understood. Understanding the transcriptional program that controls EEC differentiation is important because some EEC types harbor significant therapeutic potential for type 2 diabetes. METHODS: EEC isolation from jejunum of obese individuals with (ObD) or without (Ob) type 2 diabetes was obtained with a new method of cell sorting. EEC transcriptional profiles were established by RNA-sequencing in a first group of 14 Ob and 13 ObD individuals. EEC lineage and densities were studied in the jejunum of a second independent group of 37 Ob, 21 ObD and 22 non obese (NOb) individuals. RESULTS: The RNA seq analysis revealed a distinctive transcriptomic signature and a decreased differentiation program in isolated EEC from ObD compared to Ob individuals. In the second independent group of ObD, Ob and NOb individuals a decreased GLP-1 cell lineage and GLP-1 maturation from proglucagon, were observed in ObD compared to Ob individuals. Furthermore, jejunal density of GLP-1-positive cells was significantly reduced in ObD compared to Ob individuals. CONCLUSIONS: These results highlight that the transcriptomic signature of EEC discriminate obese subjects according to their diabetic status. Furthermore, type 2 diabetes is associated with reduced GLP-1 cell differentiation and proglucagon maturation leading to low GLP-1-cell density in human obesity. These mechanisms could account for the decrease plasma GLP-1 observed in metabolic diseases.
Subject(s)
Diabetes Mellitus, Type 2 , Enteroendocrine Cells/metabolism , Jejunum/cytology , Obesity , Adult , Cells, Cultured , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/epidemiology , Diabetes Mellitus, Type 2/metabolism , Enteroendocrine Cells/cytology , Female , Humans , Male , Middle Aged , Obesity/complications , Obesity/epidemiology , Obesity/metabolismABSTRACT
Intestinal organoids accurately recapitulate epithelial homeostasis in vivo, thereby representing a powerful in vitro system to investigate lineage specification and cellular differentiation. Here, we applied a multi-omics framework on stem cell-enriched and stem cell-depleted mouse intestinal organoids to obtain a holistic view of the molecular mechanisms that drive differential gene expression during adult intestinal stem cell differentiation. Our data revealed a global rewiring of the transcriptome and proteome between intestinal stem cells and enterocytes, with the majority of dynamic protein expression being transcription-driven. Integrating absolute mRNA and protein copy numbers revealed post-transcriptional regulation of gene expression. Probing the epigenetic landscape identified a large number of cell-type-specific regulatory elements, which revealed Hnf4g as a major driver of enterocyte differentiation. In summary, by applying an integrative systems biology approach, we uncovered multiple layers of gene expression regulation, which contribute to lineage specification and plasticity of the mouse small intestinal epithelium.
Subject(s)
Computational Biology , Intestines/cytology , Organogenesis , Organoids/cytology , Animals , Gene Expression Regulation , Mice , Organogenesis/genetics , Stem CellsABSTRACT
The diagnosis of a uterine smooth muscle lesion is, in the majority of cases, straightforward. However, in a small number of cases, the morphological criteria used in such lesions cannot differentiate with certainty a benign from a malignant lesion and a diagnosis of smooth muscle tumor with uncertain malignant potential (STUMP) is made. Uterine leiomyosarcomas are often easy to diagnose but it is difficult or even impossible to identify a prognostic factor at the moment of the diagnosis with the exception of the stage. We hypothesize, for uterine smooth muscle lesions, that there is a gradient of genomic complexity that correlates to outcome. We first tested this hypothesis on STUMP lesions in a previous study and demonstrated that this 'gray category' could be split according to genomic index into two groups. A benign group, with a low to moderate alteration rate without recurrence and a malignant group, with a highly rearranged profile akin to uterine leiomyosarcomas. Here, we analyzed a large series of 77 uterine smooth muscle lesions (from 76 patients) morphologically classified as 19 leiomyomas, 14 STUMP and 44 leiomyosarcomas with clinicopathological and genomic correlations. We confirmed that genomic index with a cut-off=10 is a predictor of recurrence (P<0.0001) and with a cut-off=35 is a marker for poor overall survival (P=0.035). For the tumors confined to the uterus, stage as a prognostic factor was not useful in survival prediction. At stage I, among the tumors reclassified as molecular leiomyosarcomas (ie, genomic index ≥10), the poor prognostic markers were: 5p gain (overall survival P=0.0008), genomic index at cut-off=35 (overall survival P=0.0193), 13p loss including RB1 (overall survival P=0.0096) and 17p gain including MYOCD gain (overall survival P=0.0425). Based on these findings (and the feasibility of genomic profiling by array-comparative genomic hybridization), genomic index, 5p and 17p gains prognostic value could be evaluated in future prospective chemotherapy trials.
Subject(s)
Gene Expression Profiling , Oligonucleotide Array Sequence Analysis , Smooth Muscle Tumor/diagnosis , Smooth Muscle Tumor/genetics , Uterine Neoplasms/diagnosis , Uterine Neoplasms/genetics , Adult , Aged , Aged, 80 and over , Chromosomes, Human, Pair 13/genetics , Chromosomes, Human, Pair 17/genetics , Chromosomes, Human, Pair 5/genetics , Female , Humans , Leiomyoma/diagnosis , Leiomyoma/genetics , Leiomyoma/pathology , Leiomyosarcoma/diagnosis , Leiomyosarcoma/genetics , Leiomyosarcoma/pathology , Middle Aged , Neoplasm Recurrence, Local , Prognosis , Smooth Muscle Tumor/pathology , Survival Analysis , Uterine Neoplasms/pathologyABSTRACT
The worldwide prevalence of metabolic diseases is increasing, and there are global recommendations to limit consumption of certain nutrients, especially saturated lipids. Insulin resistance, a common trait occurring in obesity and type 2 diabetes, is associated with intestinal lipoprotein overproduction. However, the mechanisms by which the intestine develops insulin resistance in response to lipid overload remain unknown. Here, we show that insulin inhibits triglyceride secretion and intestinal microsomal triglyceride transfer protein expression in vivo in healthy mice force-fed monounsaturated fatty acid-rich olive oil but not in mice force-fed saturated fatty acid-rich palm oil. Moreover, when mouse intestine and human Caco-2/TC7 enterocytes were treated with the saturated fatty acid, palmitic acid, the insulin-signaling pathway was impaired. We show that palmitic acid or palm oil increases ceramide production in intestinal cells and that treatment with a ceramide analogue partially reproduces the effects of palmitic acid on insulin signaling. In Caco-2/TC7 enterocytes, ceramide effects on insulin-dependent AKT phosphorylation are mediated by protein kinase C but not by protein phosphatase 2A. Finally, inhibiting de novo ceramide synthesis improves the response of palmitic acid-treated Caco-2/TC7 enterocytes to insulin. These results demonstrate that a palmitic acid-ceramide pathway accounts for impaired intestinal insulin sensitivity, which occurs within several hours following initial lipid exposure.
Subject(s)
Ceramides/biosynthesis , Enterocytes/metabolism , Insulin/metabolism , Intestinal Mucosa/metabolism , Palmitic Acid/pharmacology , Signal Transduction , Animals , Caco-2 Cells , Humans , Mice , Palm Oil , Palmitic Acid/metabolism , Phosphorylation/drug effects , Plant Oils/pharmacology , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
The diagnosis and management of uterine smooth muscle tumors with uncertain malignant potential (STUMP) is often challenging, and genomic data on these lesions as well as on uterine smooth muscle lesions are limited. We tested the hypothesis that genomic profile determination by array-CGH could split STUMP into a benign group with scarce chromosomal alterations akin to leiomyoma and a malignant group with high chromosomal instability akin to leiomyosarcoma. Array-CGH genomic profile analysis was conducted for a series of 29 cases of uterine STUMP. A group of ten uterine leiomyomas and ten uterine leiomyosarcomas served as controls. The mean age was 50 years (range, 24-85) and the follow-up ranged from 12 to 156 months (average 70 months). Since STUMP is a heterogenous group of tumors with genomic profiles that can harbor few to many chromosomal alterations, we compared genomic indices in leiomyomas and leiomyosarcomas and set a genomic index=10 threshold. Tumors with a genomic index <10 were classified as nonrecurring STUMPs and those with a genomic index >10 represented STUMPs with recurrences and unfavorable outcomes. Hence, the genomic index threshold splits the STUMP category into two groups of tumors with different outcomes: a group comparable to leiomyomas and another similar to leiomyosarcomas, but more indolent. In our STUMP series, genomic analysis by array-CGH is an innovative diagnostic tool for problematic smooth muscle uterine lesions, complementary to the morphological evaluation approach. We provide an improved classification method for distinguishing truly malignant tumors from benign lesions within the category of STUMP, especially those with equivocal morphological features.
Subject(s)
Leiomyoma/diagnosis , Smooth Muscle Tumor/diagnosis , Uterine Neoplasms/diagnosis , Adult , Aged , Aged, 80 and over , Comparative Genomic Hybridization , Female , Humans , Leiomyoma/genetics , Leiomyoma/pathology , Middle Aged , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/pathology , Smooth Muscle Tumor/genetics , Smooth Muscle Tumor/pathology , Uterine Neoplasms/genetics , Uterine Neoplasms/pathology , Young AdultABSTRACT
Epithelioid sarcomas (ES) are mesenchymal neoplasms subclassified into distal and proximal subtypes based on their distinct clinical presentations and histologic features. Consistent loss of SMARCB1 nuclear expression has been considered as the hallmark abnormality for both subtypes, a feature shared with atypical teratoid/rhabdoid tumor of infancy (ATRT). While virtually all ATRTs harbor underlying SMARCB1 somatic or germline alterations, mechanisms of SMARCB1 inactivation in ES are less well defined. To further define mechanisms of SMARCB1 inactivation a detailed molecular analysis was performed on 40 ES (25 proximal and 15 distal ES, with classic morphology and negative SMARCB1 expression) for their genomic status of SMARCB1 and related genes encoding the SWI/SNF subunits (PBRM1, BRG1, BRM, SMARCC1/2 and ARID1A) by FISH using custom BAC probes. An additional control group was included spanning a variety of 41 soft tissue neoplasms with either rhabdoid/epithelioid features or selected histotypes previously shown to lack SMARCB1 by IHC. Furthermore, 12 ES were studied by array CGH (aCGH) and an independent TMA containing 50 additional ES cases was screened for Aurora Kinase A (AURKA) and cyclin D1 immunoexpression. Homozygous SMARCB1 deletions were found by FISH in 36/40 ES (21/25 proximal-type). One of the distal-type ES displayed homozygous SMARCB1 deletion in the tumor cells, along with a heterozygous deletion within normal tissue, finding confirmed by array CGH. None of the proximal ES lacking homozygous SMARCB1 deletions displayed alterations in other SWI/SNF subunits gene members. Among controls, only the SMARCB1-immunonegative myoepithelial carcinomas displayed SMARCB1 homozygous deletions in 3/5 cases, while no gene specific abnormalities were seen among all other histologic subtypes of sarcomas tested regardless of the SMARCB1 protein status. There was no consistent pattern of AURKA and Cyclin D1 expression. The array CGH was successful in 9/12 ES, confirming the SMARCB1 and other SWI/SNF genes copy numbers detected by FISH. Our study confirms the shared pathogenesis of proximal and distal ES, showing consistent SMARCB1 homozygous deletions. Additionally we report the first ES case associated with a SMARCB1 constitutional deletion, establishing a previously undocumented link with ATRT. Alternative mechanisms of SMARCB1 inactivation in SMARCB1-disomic ES remain to be identified, but appear unrelated to large genomic abnormalities in other SWI/SNF subunits.
Subject(s)
Carcinoma/genetics , Chromosomal Proteins, Non-Histone/genetics , DNA-Binding Proteins/genetics , Gene Deletion , Homozygote , Sarcoma/genetics , Tissue Banks , Transcription Factors/genetics , Adolescent , Adult , Aged , Carcinoma/pathology , Case-Control Studies , Child , Female , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Male , Middle Aged , Retrospective Studies , SMARCB1 Protein , Sarcoma/pathology , Young AdultABSTRACT
Neuroblastoma, a tumour derived from the peripheral sympathetic nervous system, is one of the most frequent solid tumours in childhood. It usually occurs sporadically but familial cases are observed, with a subset of cases occurring in association with congenital malformations of the neural crest being linked to germline mutations of the PHOX2B gene. Here we conducted genome-wide comparative genomic hybridization analysis on a large series of neuroblastomas. Copy number increase at the locus encoding the anaplastic lymphoma kinase (ALK) tyrosine kinase receptor was observed recurrently. One particularly informative case presented a high-level gene amplification that was strictly limited to ALK, indicating that this gene may contribute on its own to neuroblastoma development. Through subsequent direct sequencing of cell lines and primary tumour DNAs we identified somatic mutations of the ALK kinase domain that mainly clustered in two hotspots. Germline mutations were observed in two neuroblastoma families, indicating that ALK is a neuroblastoma predisposition gene. Mutated ALK proteins were overexpressed, hyperphosphorylated and showed constitutive kinase activity. The knockdown of ALK expression in ALK-mutated cells, but also in cell lines overexpressing a wild-type ALK, led to a marked decrease of cell proliferation. Altogether, these data identify ALK as a critical player in neuroblastoma development that may hence represent a very attractive therapeutic target in this disease that is still frequently fatal with current treatments.
Subject(s)
Germ-Line Mutation/genetics , Neuroblastoma/genetics , Point Mutation/genetics , Protein-Tyrosine Kinases/genetics , Anaplastic Lymphoma Kinase , Cell Division , Cell Line , Cell Line, Tumor , Child , Gene Dosage , Genome, Human/genetics , Humans , Neuroblastoma/enzymology , Nucleic Acid Hybridization , Phosphorylation , Polymorphism, Single Nucleotide/genetics , Protein-Tyrosine Kinases/chemistry , Protein-Tyrosine Kinases/deficiency , Protein-Tyrosine Kinases/metabolism , Receptor Protein-Tyrosine KinasesABSTRACT
The study of the gut microbiome holds great promise for understanding and treating metabolic diseases, as its functions and derived metabolites can influence the metabolic status of the host. While research on the fecal microbiome has provided valuable insights, it tells us only part of the story. This limitation arises from the substantial variations in microorganism distribution throughout the gastrointestinal tract due to changes in physicochemical conditions. Thus, relying solely on the fecal microbiome may not be sufficient to draw comprehensive conclusions about metabolic diseases. The proximal part of the small intestine, particularly the jejunum, indeed, serves as the crucial site for digestion and absorption of nutrients, suggesting a potential role of its microbiome in metabolic regulation. Unfortunately, it remains relatively underexplored due to limited accessibility. This review presents current evidence regarding the relationships between the microbiome in the upper small intestine and various phenotypes, focusing on obesity and type 2 diabetes, in both humans and rodents. Research on humans is still limited with variability in the population and methods used. Accordingly, to better understand the role of the whole gut microbiome in metabolic diseases, studies exploring the human microbiome in different niches are needed.
Subject(s)
Diabetes Mellitus, Type 2 , Metabolic Diseases , Microbiota , Humans , Metabolic Diseases/metabolism , Obesity/therapy , Intestine, Small/metabolismABSTRACT
Endometrial stromal sarcomas represent the second most common mesenchymal uterine tumor. The 2003 WHO classification distinguishes low-grade and undifferentiated endometrial stromal sarcomas with different prognoses. Endometrial stromal sarcomas are a genetically heterogeneous group of sarcomas harboring different cytogenetic anomalies. Recently, a fusion between the YWHAE and FAM22A/B genes subsequent to a t(10;17) (q22;p13) has been described in endometrial sarcomas with high-grade histology. We examined YWHAE rearrangements by FISH break-apart and RT-PCR in a series of 27 undifferentiated uterine stromal sarcoma without JAZF1 rearrangements. Immunohistochemistry (IHC) was carried out with a panel of antibodies (estrogen (ER) and progesterone (PR) receptors, CD10, Cyclin D1, ß-catenin, p53, and Ki-67). We identified a subgroup of endometrial sarcomas with high-grade histology and uniform morphology harboring YWHAE rearrangements. FISH break-apart was interpretable in 20 cases (74%). Twelve cases (60%) showed <10% of tumor cells with a YWHAE rearrangement, 4 cases (20%) showed between 10 and ≤20%, and 4 (20%) >20%. RT-PCR was tested on 24/27 cases (88%) and 19 cases were interpretable (79%). Five cases (26%) showed a specific fusion transcript YWHAE-FAM22A/B sequence. The best concordance rate between FISH and RT-PCR (94%) was obtained with the threshold of 20% of cells with a YWHAE rearrangement. The YWHAE-rearranged cases showed high-grade morphology with uniform appearance, spindle or round epithelioid cells, low ER and PR, CD10 expression, and a high and diffuse positivity for Cyclin D1, p53, and nuclear ß-catenin negativity. Cyclin D1 was the most sensitive marker for high-grade endometrial sarcomas with YWHAE rearrangement. All undifferentiated uterine sarcomas with pleomorphic appearances did not harbor any YWHAE rearrangements, except for one case. Overall, for endometrial sarcoma cases with high-grade morphology we recommend to test for YWHAE rearrangements by FISH break-apart, a cost- and time-efficient method, and to complete the investigation by RT-PCR in borderline cases.
Subject(s)
14-3-3 Proteins/genetics , Endometrial Neoplasms/genetics , Gene Rearrangement , Sarcoma, Endometrial Stromal/genetics , 14-3-3 Proteins/metabolism , Adult , Aged , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Endometrial Neoplasms/metabolism , Endometrial Neoplasms/pathology , Female , Humans , In Situ Hybridization, Fluorescence , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sarcoma, Endometrial Stromal/metabolism , Sarcoma, Endometrial Stromal/pathologyABSTRACT
BACKGROUND & AIMS: The four and a half LIM-only protein 2 (FHL2) is upregulated in diverse pathological conditions. Here, we analyzed the effects of FHL2 overexpression in the liver of FHL2 transgenic mice (Apo-FHL2). METHODS: We first examined cell proliferation and apoptosis in Apo-FHL2 livers and performed partial hepatectomy to investigate high FHL2 expression in liver regeneration. Expression of FHL2 was then analyzed by real time PCR in human hepatocellular carcinoma and adjacent non-tumorous livers. Finally, the role of FHL2 in hepatocarcinogenesis was assessed using Apo-FHL2;Apc(lox/lox) mice. RESULTS: Six-fold increase in cell proliferation in transgenic livers was associated with concomitant apoptosis, resulting in normal liver mass. In Apo-FHL2 livers, both cyclin D1 and p53 were markedly increased. Evidence supporting a p53-dependent cell death mechanism was provided by the findings that FHL2 bound to and activated the p53 promoter, and that a dominant negative p53 mutant compromised FHL2-induced apoptosis in hepatic cells. Following partial hepatectomy in Apo-FHL2 mice, hepatocytes displayed advanced G1 phase entry and DNA synthesis leading to accelerated liver weight restoration. Interestingly, FHL2 upregulation in human liver specimens showed significant association with increasing inflammation score and cirrhosis. Finally, while Apo-FHL2 mice developed no tumors, the FHL2 transgene enhanced hepatocarcinogenesis induced by liver-specific deletion of the adenomatous polyposis coli gene and aberrant Wnt/ß-catenin signaling in Apc(lox/lox) animals. CONCLUSIONS: Our results implicate FHL2 in the regulation of signaling pathways that couple proliferation and cell death machineries, and underscore the important role of FHL2 in liver homeostasis and carcinogenesis.
Subject(s)
Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Homeostasis/physiology , LIM-Homeodomain Proteins/metabolism , Liver/metabolism , Liver/pathology , Muscle Proteins/metabolism , Transcription Factors/metabolism , Animals , Apoptosis/physiology , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/surgery , Cell Proliferation , Cyclin D1/metabolism , Disease Models, Animal , Female , Hepatectomy , Humans , LIM-Homeodomain Proteins/genetics , Liver/surgery , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Liver Neoplasms/surgery , Liver Regeneration/physiology , Male , Mice , Mice, Transgenic , Muscle Proteins/genetics , Transcription Factors/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
With an excessive postprandial accumulation of intestine-derived, triglyceride-rich lipoproteins being a risk factor of cardiovascular diseases, it is essential to characterize the mechanisms controlling the intestinal absorption of dietary lipids. Our aim was to investigate the role of the transcription factor hepatocyte nuclear factor (HNF)-4α in this process. We used transgenic mice with a specific and inducible intestinal knockout of Hnf-4α gene. One hour after a lipid bolus, in the presence of the lipase inhibitor tyloxapol, lower amounts of triglycerides were found in both plasma and intestinal epithelium of the intestine-specific Hnf-4α knockout (Hnf-4α(intΔ)) mice compared with the Hnf-4α(loxP/loxP) control mice. These discrepancies were due to a net decrease of the intestinal uptake of fatty acid in Hnf-4α(intΔ) mice compared with Hnf-4α(loxP/loxP) mice, as assessed by the amount of radioactivity that was recovered in intestine and plasma after gavage with labeled triolein or oleic acid, or in intestinal epithelial cells isolated from jejunum after a supply of labeled oleic acid-containing micelles. This decreased fatty acid uptake was associated with significant lower levels of the fatty acid transport protein-4 mRNA and protein along the intestinal tract and with a lower acyl-CoA synthetase activity in Hnf-4α(intΔ) mice compared with the control mice. We conclude that the transcription factor HNF-4α is a key factor of the intestinal absorption of dietary lipids, which controls this process as early as in the initial step of fatty acid uptake by enterocytes.
Subject(s)
Dietary Fats/metabolism , Fatty Acids/metabolism , Hepatocyte Nuclear Factor 4/metabolism , Intestinal Absorption/genetics , Intestinal Mucosa/metabolism , Animals , Coenzyme A Ligases/genetics , Coenzyme A Ligases/metabolism , Enterocytes/drug effects , Enterocytes/metabolism , Fatty Acid Transport Proteins/genetics , Fatty Acid Transport Proteins/metabolism , Hepatocyte Nuclear Factor 4/genetics , Intestinal Absorption/drug effects , Intestinal Mucosa/drug effects , Intestines/drug effects , Mice , Mice, Knockout , Polyethylene Glycols/pharmacology , Postprandial Period/physiologyABSTRACT
BACKGROUND: Retrospective studies have demonstrated the prognostic impact of genomic profiles in neuroblastoma (NB). Segmental chromosome alterations have been found useful for identifying tumors with a high risk of relapse. As the gain of chromosome arm 17q is the most frequent chromosome alteration reported in NB primary tumors, we evaluated the presence of this 17q gain in the peripheral blood of patients with NB. PROCEDURE: Using duplex quantitative real-time PCR, we quantified simultaneously MPO (17q.23.1) and a reference gene, p53, and Survivin (17q25) and p53. MPO and Survivin copy numbers were evaluated as MPO/p53 and Survivin/p53 ratios in 142 serum or plasma samples in which 17q status had been determined by array-based comparative genomic hybridization (aCGH) or multiplex ligation-dependent probe amplification (MLPA). RESULTS: In patients <18 months of age, serum-based determination of 17q gain in DNA sequences had good specificity (94.4%) and 58.8% sensitivity (P < 0.001). In contrast, for patients over 18 months of age, the approach exhibited moderate specificity (71.4%) and 51.2% sensitivity (P = ns). Similar results were observed in patients with tumors without MYCN amplification. CONCLUSION: Our results show that 17q gain determination in circulating DNA is possible and suggest that this non-invasive test could be useful for very young children when no reliable information on genomic alterations is obtained by aCGH or MPLA analysis of tumor samples This test is complementary to previously developed techniques for detecting circulating MYCN DNA sequences.
Subject(s)
Biomarkers, Tumor/blood , Chromosomes, Human, Pair 17/genetics , DNA, Neoplasm/blood , Neuroblastoma/genetics , Biomarkers, Tumor/genetics , Case-Control Studies , Chromosome Aberrations , Comparative Genomic Hybridization , Female , Gene Amplification , Granulocyte Colony-Stimulating Factor/blood , Granulocyte Colony-Stimulating Factor/genetics , Humans , In Situ Hybridization, Fluorescence , Infant , Inhibitor of Apoptosis Proteins/blood , Inhibitor of Apoptosis Proteins/genetics , Interleukin-3/blood , Interleukin-3/genetics , Male , Neuroblastoma/blood , Neuroblastoma/pathology , Polymerase Chain Reaction , Prognosis , Recombinant Fusion Proteins/blood , Recombinant Fusion Proteins/genetics , Recombinant Proteins , Retrospective Studies , Sensitivity and Specificity , Survival Rate , Survivin , Tumor Suppressor Protein p53/blood , Tumor Suppressor Protein p53/geneticsABSTRACT
Changes in dietary habits have occurred concomitantly with a rise of type 2 diabetes (T2D) and obesity. Intestine is the first organ facing nutrient ingestion and has to adapt its metabolism with these dietary changes. HNF-4γ, a transcription factor member of the nuclear receptor superfamily and mainly expressed in intestine, has been suggested to be involved in susceptibility to T2D. Our aim was to investigate the role of HNF-4γ in metabolic disorders and related mechanisms. Hnf4g-/- mice were fed high-fat/high-fructose (HF-HF) diet for 6 weeks to induce obesity and T2D. Glucose homeostasis, energy homeostasis in metabolic cages, body composition and stool energy composition, as well as gene expression analysis in the jejunum were analyzed. Despite an absence of decrease in calorie intake, of increase in locomotor activity or energy expenditure, Hnf4g-/- mice fed with HF-HF are protected against weight gain after 6 weeks of HF-HF diet. We showed that Hnf4g-/- mice fed HF-HF display an increase in fecal calorie loss, mainly due to intestinal lipid malabsorption. Gene expression of lipid transporters, Fatp4 and Scarb1 and of triglyceride-rich lipoprotein secretion proteins, Mttp and ApoB are decreased in gut epithelium of Hnf4g-/- mice fed HF-HF, showing the HNF-4γ role in intestine lipid absorption. Furthermore, plasma GLP-1 and jejunal GLP-1 content are increased in Hnf4g-/- mice fed HF-HF, which could contribute to the glucose intolerance protection. The loss of HNF-4γ leads to a protection against a diet-induced weight gain and to a deregulated glucose homeostasis, associated with lipid malabsorption.
Subject(s)
Hepatocyte Nuclear Factor 4/genetics , Intestinal Absorption/genetics , Lipid Metabolism/genetics , Obesity/genetics , Animals , Cells, Cultured , Diet, High-Fat/adverse effects , Female , Fructose/adverse effects , Gene Deletion , Glucose Intolerance/etiology , Glucose Intolerance/genetics , Glucose Intolerance/metabolism , Intestines/metabolism , Malabsorption Syndromes/genetics , Malabsorption Syndromes/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Obesity/etiology , Obesity/metabolism , Triglycerides/metabolism , Weight Gain/geneticsABSTRACT
The morphology of malignant cells distinguishes between undifferentiated, poorly differentiated and differentiating neuroblastomas and constitutes a strong prognostic factor. Spontaneous or treatment-induced maturation characterizes a subset of neuroblastomas. It constitutes the basis of retinoic acid treatment to improve survival in aggressive neuroblastomas. However, the molecular events that drive differentiation are poorly understood. In the present study we have investigated the relationships between gene expression profiles and differentiation criteria in stroma-poor neuroblastomas. This study included three undifferentiated (UN), 20 poorly differentiated (PDN) and 11 differentiating (DN) neuroblastomas. These groups could be clearly separated using unsupervised clustering methods, which further enabled a major classification impact of genes involved in neural development, differentiation and function to be identified. UNs are characterized by high ASCL1, high PHOX2B, low GATA2, low TH and low DBH expressions. Most PDNs harbour a clear adrenergic phenotype, even in the presence of missense PHOX2B mutations. Finally, all DN tumours demonstrate cholinergic features. Depending upon their association with adrenergic characteristics, this enables dual 'cholinergic/adrenergic' and 'fully cholinergic' neuroblastomas to be defined. This suggests that the cholinergic switch, a final specification process that occurs physiologically in a minority of sympathetic neurons, is a critical step of differentiation in some neuroblastic tumours. This switch is associated with a down regulation of DBH that is apparently not strictly dependent upon PHOX2B. Conversely, GATA2 and TFAP2B may play critical roles in maintaining adrenergic features in poorly differentiated tumours.
Subject(s)
Cholinergic Fibers/pathology , Neuroblastoma/pathology , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Differentiation/genetics , Dopamine beta-Hydroxylase/metabolism , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , Homeodomain Proteins/metabolism , Humans , Neoplasm Proteins/metabolism , Neoplasm Staging , Neuroblastoma/metabolism , Phenotype , Transcription Factors/metabolismABSTRACT
BACKGROUND & AIMS: Recent evidence has suggested that the intact intestinal epithelial barrier protects our body from a range of immune-mediated diseases. The epithelial layer has an impressive ability to reconstitute and repair upon damage and this process of repair increasingly is seen as a therapeutic target. In vitro models to study this process in primary intestinal cells are lacking. METHODS: We established and characterized an in vitro model of intestinal damage and repair by applying γ-radiation on small-intestinal organoids. We then used this model to identify novel regulators of intestinal regeneration. RESULTS: We identified hepatocyte nuclear factor 4α (HNF4α) as a pivotal upstream regulator of the intestinal regenerative response. Organoids lacking Hnf4a were not able to propagate in vitro. Importantly, intestinal Hnf4a knock-out mice showed impaired regeneration after whole-body irradiation, confirming intestinal organoids as a valuable alternative to in vivo studies. CONCLUSIONS: In conclusion, we established and validated an in vitro damage-repair model and identified HNF4α as a crucial regulator of intestinal regeneration. Transcript profiling: GSE141515 and GSE141518.
Subject(s)
Hepatocyte Nuclear Factor 4/metabolism , Intestinal Mucosa/pathology , Intestine, Small/pathology , Regeneration , Animals , Cells, Cultured , Hepatocyte Nuclear Factor 4/genetics , Intestinal Mucosa/radiation effects , Intestine, Small/radiation effects , Male , Mice , Mice, Knockout , Organoids , Primary Cell Culture , Radiation Injuries, ExperimentalABSTRACT
Somatically acquired chromosomal imbalances are a key feature of neuroblastoma, a heterogeneous pediatric solid tumor. Among these alterations, genomic amplification targeting the MYCN oncogene and observed in about 25-30% of the cases, strongly correlates with advanced stage and poor outcome. In this work, we have used BAC and SNP arrays as well as gene expression arrays to characterize amplifications in neuroblastoma. Eighty-eight distinct BACs defining high-level amplification events were identified in 65 samples, including 43 tumors and 22 cell lines. Although the highest recurrence was observed on chromosome 2, clones on chromosomes 8, 12, 16, and 17 also revealed genomic amplification in several samples. A detailed analysis of the 2p22-2p25 MYCN containing region indicated highly complex patterns in a number of cases. Coamplifications involving MYCN and other regions were explored by FISH in three cell lines. High-resolution arrays then allowed us to further refine the mapping of 25 amplicons in 19 samples, either reducing the size of a single continuous amplicon or increasing the complexity by highlighting multiple noncontiguous regions of amplification. Combined analysis of gene expression profiling and array-CGH data indicated that 12 to 25% of the genes that are targeted by genomic amplification are actually over-expressed in tumor cells, several of them having already been implicated in cancer. Finally, our results suggest that the presence of amplicons localized outside of chromosome 2, in addition to MYCN amplification, may be linked to a particularly severe outcome in neuroblastoma patients.
Subject(s)
Chromosome Aberrations , DNA, Neoplasm/genetics , Gene Amplification , Neuroblastoma/genetics , Nuclear Proteins/genetics , Child , Chromosome Mapping , Chromosomes, Artificial, Bacterial , Chromosomes, Human/genetics , Gene Expression Profiling , Humans , In Situ Hybridization, Fluorescence , N-Myc Proto-Oncogene Protein , Oligonucleotide Array Sequence Analysis , Oncogene Proteins/genetics , Polymorphism, Single Nucleotide/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Survival Rate , Tumor Cells, CulturedABSTRACT
In neuroblastoma, the most frequent genetic alterations are unbalanced translocations involving chromosome 17. To gain insights into these rearrangements, we have characterized a previously identified der(1)t(1;17) of the CLB-Bar cell line. The 17q breakpoint was mapped by FISH. Subsequently, a rearranged fragment was identified by Southern analysis, cloned in a lambda vector and sequenced. The chromosome rearrangement is more complex than expected due to the presence of an interstitial 4p telomeric sequence between chromosome 1p and 17q. Three different genes, which may play a role in neuroblastoma development, are disrupted by the translocation breakpoints. Indeed, the 3'UTR of the PIP5K2B gene on chromosome 17q is directly fused to the (TTAGGG)n repeat of the chromosome 4p telomere, and the (1;4) fusion disrupts the MACF1 (microtubule-actin crosslinking factor 1) and POLN genes, respectively. Interestingly, the (1;4) fusion was present at diagnosis and at relapse, whereas the (4;17) fusion was detected at relapse only, leading to a secondary 17q gain confirmed by array CGH therefore indicating that 17q gain may not be a primary event in neuroblastoma. Finally, screening of a panel of neuroblastoma cell lines identified interstitial telomeric sequences in three other cases, suggesting that this may be a recurrent mechanism leading to unbalanced translocations in neuroblastoma.
Subject(s)
Chromosomes, Human, Pair 17 , Neuroblastoma/genetics , Telomere/ultrastructure , Translocation, Genetic , Base Sequence , Blotting, Southern , Chromosome Aberrations , Chromosome Mapping , Chromosomes, Human, Pair 1 , Chromosomes, Human, Pair 4 , Cloning, Molecular , Humans , In Situ Hybridization, Fluorescence , Models, Genetic , Molecular Sequence Data , Neuroblastoma/metabolism , Polymerase Chain ReactionABSTRACT
In the small intestine, the expression of the apolipoprotein (apo) C-III and A-IV genes is restricted to the enterocytes of the villi. We have previously shown that, in transgenic mice, specific expression of the human apo C-III requires a hormone-responsive element (HRE) located in the distal region of the human apoA-IV promoter. This HRE binds the hepatic nuclear factors (HNF)-4alpha and gamma. Here, intraduodenal injections in mice and infections of human enterocytic Caco-2/TC7 cells with an adenovirus expressing a dominant-negative form of HNF-4alpha repress the expression of the apoA-IV gene, demonstrating that HNF-4 controls the apoA-IV gene expression in enterocytes. We show that HNF-4alpha and gamma functionally interact with a second HRE present in the proximal region of the human apoA-IV promoter. New sets of transgenic mice expressing mutated forms of the promoter, combined with the human apo C-III enhancer, demonstrate that, whereas a single HRE is sufficient to reproduce the physiological cephalo-caudal gradient of apoA-IV gene expression, both HREs are required for expression that is restricted to villi. The combination of multiple HREs may specifically recruit regulatory complexes associating HNF-4 and either coactivators in villi or corepressors in crypts.