ABSTRACT
BACKGROUND & AIMS: In one-third of hepatocellular carcinomas (HCCs), cancer cells have mutations that activate ß-catenin pathway. These cells have alterations in glutamine, bile, and lipid metabolism. We investigated whether positron emission tomography (PET) imaging allows identification of altered metabolic pathways that might be targeted therapeutically. METHODS: We studied mice with activation of ß-catenin in liver (Apcko-liv mice) and male C57Bl/6 mice given injections of diethylnitrosamine, which each develop HCCs. Mice were fed a conventional or a methionine- and choline-deficient diet or a choline-deficient (CD) diet. Choline uptake and metabolism in HCCs were analyzed by micro-PET imaging of mice; livers were collected and analyzed by histologic, metabolomic, messenger RNA quantification, and RNA-sequencing analyses. Fifty-two patients with HCC underwent PET imaging with 18F-fluorodeoxyglucose, followed by 18F-fluorocholine tracer metabolites. Human HCC specimens were analyzed by immunohistochemistry, quantitative polymerase chain reaction, and DNA sequencing. We used hepatocytes and mouse tumor explants for studies of incorporation of radiolabeled choline into phospholipids and its contribution to DNA methylation. We analyzed HCC progression in mice fed a CD diet. RESULTS: Livers and tumors from Apcko-liv mice had increased uptake of dietary choline, which contributes to phospholipid formation and DNA methylation in hepatocytes. In patients and in mice, HCCs with activated ß-catenin were positive in 18F-fluorocholine PET, but not 18F-fluorodeoxyglucose PET, and they overexpressed the choline transporter organic cation transporter 3. The HCC cells from Apcko-liv mice incorporated radiolabeled methyl groups of choline into phospholipids and DNA. In Apcko-liv mice, the methionine- and choline-deficient diet reduced proliferation and DNA hypermethylation of hepatocytes and HCC cells, and the CD diet reduced long-term progression of tumors. CONCLUSIONS: In mice and humans, HCCs with mutations that activate ß-catenin are characterized by increased uptake of a fluorocholine tracer, but not 18F-fluorodeoxyglucose, revealed by PET. The increased uptake of choline by HCCs promotes phospholipid formation, DNA hypermethylation, and hepatocyte proliferation. In mice, the CD diet reverses these effects and promotes regression of HCCs that overexpress ß-catenin.
Subject(s)
Carcinoma, Hepatocellular/diagnostic imaging , Carcinoma, Hepatocellular/genetics , Liver Neoplasms/diagnostic imaging , Liver Neoplasms/genetics , Mutation , Positron-Emission Tomography , beta Catenin/genetics , Animals , Carcinoma, Hepatocellular/chemically induced , Carcinoma, Hepatocellular/pathology , Cell Proliferation , Choline/administration & dosage , Choline/analogs & derivatives , Choline Deficiency/complications , DNA Methylation , Diethylnitrosamine , Disease Models, Animal , Genes, APC , Genetic Predisposition to Disease , Humans , Liver Neoplasms/chemically induced , Liver Neoplasms/pathology , Male , Methionine/deficiency , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Phospholipids/metabolism , Predictive Value of Tests , Radiopharmaceuticals/administration & dosage , beta Catenin/metabolismABSTRACT
OBJECTIVES: CTNNB1-mutated hepatocellular carcinomas (HCCs) constitute a major part of human HCC and are largely inaccessible to target therapy. Yet, little is known about the metabolic reprogramming induced by ß-catenin oncogenic activation in the liver. We aimed to decipher such reprogramming and assess whether it may represent a new avenue for targeted therapy of CTNNB1-mutated HCC. DESIGN: We used mice with hepatocyte-specific oncogenic activation of ß-catenin to evaluate metabolic reprogramming using metabolic fluxes on tumourous explants and primary hepatocytes. We assess the role of Pparα in knock-out mice and analysed the consequences of fatty acid oxidation (FAO) using etomoxir. We explored the expression of the FAO pathway in an annotated human HCC dataset. RESULTS: ß-catenin-activated HCC were not glycolytic but intensively oxidised fatty acids. We found that Pparα is a ß-catenin target involved in FAO metabolic reprograming. Deletion of Pparα was sufficient to block the initiation and progression of ß-catenin-dependent HCC development. FAO was also enriched in human CTNNB1-mutated HCC, under the control of the transcription factor PPARα. CONCLUSIONS: FAO induced by ß-catenin oncogenic activation in the liver is the driving force of the ß-catenin-induced HCC. Inhibiting FAO by genetic and pharmacological approaches blocks HCC development, showing that inhibition of FAO is a suitable therapeutic approach for CTNNB1-mutated HCC.
Subject(s)
Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Fatty Acids/metabolism , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , beta Catenin/metabolism , Animals , Epoxy Compounds/pharmacology , Gene Expression Regulation, Neoplastic , Humans , Mice, Knockout , Mutation , Oxidation-Reduction , PPAR alpha/physiology , beta Catenin/geneticsABSTRACT
BACKGROUND & AIMS: The Wnt/ß-catenin pathway is the most frequently deregulated pathway in hepatocellular carcinoma (HCC). Inactivating mutations of the gene encoding AXIN1, a known negative regulator of the Wnt/ß-catenin signaling pathway, are observed in about 10% of HCCs. Whole-genome studies usually place HCC with AXIN1 mutations and CTNNB1 mutations in the group of tumors with Wnt/ß-catenin activated program. However, it has been shown that HCCs with activating CTNNB1 mutations form a group of HCCs, with a different histology, prognosis and genomic signature to those with inactivating biallelic AXIN1 mutations. We aimed to elucidate the relationship between CTNNB1 mutations, AXIN1 mutations and the activation level of the Wnt/ß-catenin program. METHODS: We evaluated two independent human HCC datasets for the expression of a 23-ß-catenin target genes program. We modeled Axin1 loss of function tumorigenesis in two engineered mouse models and performed gene expression profiling. RESULTS: Based on gene expression, we defined three levels of ß-catenin program activation: strong, weak or no activation. While more than 80% CTNNB1-mutated tumors were found in the strong or in the weak activation program, most of the AXIN1-mutated tumors (>70%) were found in the subgroup with no activation. We validated this result by demonstrating that mice with a hepatocyte specific AXIN1 deletion developed HCC in the absence of ß-catenin induction. We defined a 329-gene signature common in human and mouse AXIN1 mutated HCC that is highly enriched in Notch and YAP oncogenic signatures. CONCLUSIONS: AXIN1-mutated HCCs occur independently of the Wnt/ß-catenin pathway and involve Notch and YAP pathways. These pathways constitute potentially interesting targets for the treatment of HCC caused by AXIN1 mutations. LAY SUMMARY: Liver cancer has a poor prognosis. Defining the molecular pathways involved is important for developing new therapeutic approaches. The Wnt/ß-catenin pathway is the most frequently deregulated pathway in hepatocellular carcinoma (HCC). Mutations of AXIN1, a member of this pathway, represent about 10% of HCC mutations. Using both human HCC collections and engineered mouse models of liver cancers with AXIN1 mutation or deletion, we defined a common signature of liver tumors mutated for AXIN1 and demonstrate that these tumors occur independently of the activation of the Wnt/ß-catenin pathway.
Subject(s)
Axin Protein/deficiency , Carcinoma, Hepatocellular/etiology , Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/etiology , Liver Neoplasms/metabolism , Animals , Axin Protein/genetics , Carcinogenesis/genetics , Carcinogenesis/metabolism , Carcinoma, Hepatocellular/genetics , Hepatocytes/metabolism , Humans , Liver Neoplasms/genetics , Liver Neoplasms, Experimental/etiology , Liver Neoplasms, Experimental/genetics , Liver Neoplasms, Experimental/metabolism , Male , Mice , Mice, Knockout , Mutation , Prognosis , Receptors, Notch/genetics , Receptors, Notch/metabolism , Wnt Signaling Pathway/genetics , beta Catenin/metabolismABSTRACT
The nuclear receptor Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII) is an important coordinator of glucose homeostasis through its function in different organs such as the endocrine pancreas, adipose tissue, skeletal muscle, and liver. Recently we have demonstrated that COUP-TFII expression in the hypothalamus is restricted to a subpopulation of neurons expressing the steroidogenic factor 1 transcription factor, known to play a crucial role in glucose homeostasis. To understand the functional significance of COUP-TFII expression in the steroidogenic factor 1 neurons, we generated hypothalamic ventromedial nucleus-specific COUP-TFII KO mice using the cyclization recombination/locus of X-overP1 technology. The heterozygous mutant mice display insulin hypersensitivity and a leaner phenotype associated with increased energy expenditure and similar food intake. These mutant mice also present a defective counterregulation to hypoglycemia with altered glucagon secretion. Moreover, the mutant mice are more likely to develop hypoglycemia-associated autonomic failure in response to recurrent hypoglycemic or glucopenic events. Therefore, COUP-TFII expression levels in the ventromedial nucleus are keys in the ability to resist the onset of hypoglycemia-associated autonomic failure.
Subject(s)
COUP Transcription Factor II/biosynthesis , Glucose/metabolism , Hypoglycemia/metabolism , Nerve Tissue Proteins/biosynthesis , Neurons/metabolism , Ventromedial Hypothalamic Nucleus/metabolism , Animals , Autonomic Nervous System Diseases/etiology , Autonomic Nervous System Diseases/genetics , Autonomic Nervous System Diseases/metabolism , Autonomic Nervous System Diseases/pathology , COUP Transcription Factor II/genetics , Chickens , Glucose/genetics , Heterozygote , Hypoglycemia/complications , Hypoglycemia/genetics , Hypoglycemia/pathology , Mice , Mice, Transgenic , Nerve Tissue Proteins/genetics , Neurons/pathology , Organ Specificity/genetics , Steroidogenic Factor 1/genetics , Steroidogenic Factor 1/metabolism , Ventromedial Hypothalamic Nucleus/pathologyABSTRACT
Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is an orphan nuclear receptor involved in the control of numerous functions in various organs (organogenesis, differentiation, metabolic homeostasis, etc.). The aim of the present work was to characterize the regulation and contribution of COUP-TFII in the control of hepatic fatty acid and glucose metabolisms in newborn mice. Our data show that postnatal increase in COUP-TFII mRNA levels is enhanced by glucagon (via cAMP) and PPARα. To characterize COUP-TFII function in the liver of suckling mice, we used a functional (dominant negative form; COUP-TFII-DN) and a genetic (shRNA) approach. Adenoviral COUP-TFII-DN injection induces a profound hypoglycemia due to the inhibition of gluconeogenesis and fatty acid oxidation secondarily to reduced PEPCK, Gl-6-Pase, CPT I, and mHMG-CoA synthase gene expression. Using the crossover plot technique, we show that gluconeogenesis is inhibited at two different levels: 1) pyruvate carboxylation and 2) trioses phosphate synthesis. This could result from a decreased availability in fatty acid oxidation arising cofactors such as acetyl-CoA and reduced equivalents. Similar results are observed using the shRNA approach. Indeed, when fatty acid oxidation is rescued in response to Wy-14643-induced PPARα target genes (CPT I and mHMG-CoA synthase), blood glucose is normalized in COUP-TFII-DN mice. In conclusion, this work demonstrates that postnatal increase in hepatic COUP-TFII gene expression is involved in the regulation of liver fatty acid oxidation, which in turn sustains an active hepatic gluconeogenesis that is essential to maintain an appropriate blood glucose level required for newborn mice survival.
Subject(s)
COUP Transcription Factor II/physiology , Fatty Acids/metabolism , Gluconeogenesis/genetics , Lipid Metabolism/genetics , Liver/metabolism , Animals , Animals, Newborn , Cells, Cultured , Female , Fetus/metabolism , Hepatocytes/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Oxidation-Reduction , PPAR alpha/genetics , PregnancyABSTRACT
BACKGROUND & AIMS: Axin1 is a negative regulator of wingless-type MMTV integration site family, member 1 (Wnt)/ß-catenin signaling with tumor-suppressor function. The Wnt pathway has a critical role in the intestine, both during homeostasis and cancer, but the role of Axin1 remains elusive. METHODS: We assessed the role of Axin1 in normal intestinal homeostasis, with control, epithelial-specific, Axin1-knockout mice (Axin1ΔIEC) and Axin2-knockout mice. We evaluated the tumor-suppressor function of Axin1 during chemically induced colorectal tumorigenesis and dextran sulfate sodium-induced colitis, and performed comparative gene expression profiling by whole-genome RNA sequencing. The clinical relevance of the Axin1-dependent gene expression signature then was tested in a database of 2239 clinical colorectal cancer (CRC) samples. RESULTS: We found that Axin1 was dispensable for normal intestinal homeostasis and redundant with Axin2 for Wnt pathway down-regulation. Axin1 deficiency in intestinal epithelial cells rendered mice more susceptible to chemically induced colon carcinogenesis, but reduced dextran sulfate sodium-induced colitis by attenuating the induction of a proinflammatory program. RNA-seq analyses identified an interferon γ/T-helper1 immune program controlled by Axin1 that enhances the inflammatory response and protects against CRC. The Axin1-dependent gene expression signature was applied to human CRC samples and identified a group of patients with potential vulnerability to immune checkpoint blockade therapies. CONCLUSIONS: Our study establishes, in vivo, that Axin1 has redundant function with Axin2 for Wnt down-regulation and infers a new role for Axin1. Physiologically, Axin1 stimulates gut inflammation via an interferon γ/Th1 program that prevents tumor growth. Linked to its T-cell-mediated effect, the colonic Axin1 signature offers therapeutic perspectives for CRC.
Subject(s)
Colitis , Interferon-gamma , Mice , Animals , Humans , Dextran Sulfate/toxicity , Carcinogenesis/genetics , Colitis/chemically induced , Wnt Signaling Pathway/genetics , Mice, Knockout , Axin Protein/genetics , Axin Protein/metabolismABSTRACT
The transcription factor carbohydrate-responsive element-binding protein (ChREBP) has emerged as a central regulator of lipid synthesis in liver because it is required for glucose-induced expression of the glycolytic enzyme liver-pyruvate kinase (L-PK) and acts in synergy with SREBP to induce lipogenic genes such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). Liver X receptors (LXRs) are also important regulators of the lipogenic pathway, and the recent finding that ChREBP is a direct target of LXRs and that glucose itself can bind and activate LXRs prompted us to study the role of LXRs in the induction of glucose-regulated genes in liver. Using an LXR agonist in wild-type mice, we found that LXR stimulation did not promote ChREBP phosphorylation or nuclear localization in the absence of an increased intrahepatic glucose flux. Furthermore, the induction of ChREBP, L-PK, and ACC by glucose or high-carbohydrate diet was similar in LXRalpha/beta knockout compared with wild-type mice, suggesting that the activation of these genes by glucose occurs by an LXR-independent mechanism. We used fluorescence resonance energy transfer analysis to demonstrate that glucose failed to promote the interaction of LXRalpha/beta with specific cofactors. Finally, siRNA silencing of ChREBP in LXRalpha/beta knockout hepatocytes abrogated glucose-induced expression of L-PK and ACC, further demonstrating the central role of ChREBP in glucose signaling. Taken together, our results demonstrate that glucose is required for ChREBP functional activity and that LXRs are not necessary for the induction of glucose-regulated genes in liver.
Subject(s)
DNA-Binding Proteins/physiology , Gene Expression Regulation , Glucose/metabolism , Liver/metabolism , Nuclear Proteins/physiology , Receptors, Cytoplasmic and Nuclear/physiology , Transcription Factors/physiology , Acetyl-CoA Carboxylase/genetics , Active Transport, Cell Nucleus , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Liver X Receptors , Male , Mice , Mice, Inbred C57BL , Orphan Nuclear Receptors , Phosphorylation , Pyruvate Kinase/geneticsABSTRACT
CTNNB1 (catenin beta 1)-mutated hepatocellular carcinomas (HCCs) account for a large proportion of human HCCs. They display high levels of respiratory chain activity. As metabolism and redox balance are closely linked, tumor cells must maintain their redox status during these metabolic alterations. We investigated the redox balance of these HCCs and the feasibility of targeting this balance as an avenue for targeted therapy. We assessed the expression of the nuclear erythroid 2 p45-related factor 2 (NRF2) detoxification pathway in an annotated human HCC data set and reported an enrichment of the NRF2 program in human HCCs with CTNNB1 mutations, largely independent of NFE2L2 (nuclear factor, erythroid 2 like 2) or KEAP1 (Kelch-like ECH-associated protein 1) mutations. We then used mice with hepatocyte-specific oncogenic ß-catenin activation to evaluate the redox status associated with ß-catenin activation in preneoplastic livers and tumors. We challenged them with various oxidative stressors and observed that the ß-catenin pathway activation increased transcription of Nfe2l2, which protects ß-catenin-activated hepatocytes from oxidative damage and supports tumor development. Moreover, outside of its effects on reactive oxygen species scavenging, we found out that Nrf2 itself contributes to the metabolic activity of ß-catenin-activated cells. We then challenged ß-catenin activated tumors pharmacologically to create a redox imbalance and found that pharmacological inactivation of Nrf2 was sufficient to considerably decrease the progression of ß-catenin-dependent HCC development. Conclusion: These results demonstrate cooperation between oncogenic ß-catenin signaling and the NRF2 pathway in CTNNB1-mediated HCC tumorigenesis, and we provide evidence for the relevance of redox balance targeting as a therapeutic strategy in CTNNB1-mutated HCC.
ABSTRACT
Erythropoietin (EPO) is a key regulator of erythropoiesis. The embryonic liver is the main site of erythropoietin synthesis, after which the kidney takes over. The adult liver retains the ability to express EPO, and we discovered here new players of this transcription, distinct from the classical hypoxia-inducible factor pathway. In mice, genetically invalidated in hepatocytes for the chromatin remodeler Arid1a, and for Apc, the major silencer of Wnt pathway, chromatin was more accessible and histone marks turned into active ones at the Epo downstream enhancer. Activating ß-catenin signaling increased binding of Tcf4/ß-catenin complex and upregulated its enhancer function. The loss of Arid1a together with ß-catenin signaling, resulted in cell-autonomous EPO transcription in mouse and human hepatocytes. In mice with Apc-Arid1a gene invalidations in single hepatocytes, Epo de novo synthesis led to its secretion, to splenic erythropoiesis and to dramatic erythrocytosis. Thus, we identified new hepatic EPO regulation mechanism stimulating erythropoiesis.
Subject(s)
DNA-Binding Proteins/metabolism , Erythropoietin/metabolism , Hepatocytes/metabolism , Transcription Factors/metabolism , beta Catenin/metabolism , Adult , Animals , Erythropoiesis , Female , Humans , In Situ Hybridization , Male , Mice , Wnt Signaling PathwayABSTRACT
Excessive glucose production by the liver is a key factor in the hyperglycemia observed in type 2 diabetes mellitus (T2DM). Here, we highlight a novel role of liver kinase B1 (Lkb1) in this regulation. We show that mice with a hepatocyte-specific deletion of Lkb1 have higher levels of hepatic amino acid catabolism, driving gluconeogenesis. This effect is observed during both fasting and the postprandial period, identifying Lkb1 as a critical suppressor of postprandial hepatic gluconeogenesis. Hepatic Lkb1 deletion is associated with major changes in whole-body metabolism, leading to a lower lean body mass and, in the longer term, sarcopenia and cachexia, as a consequence of the diversion of amino acids to liver metabolism at the expense of muscle. Using genetic, proteomic and pharmacological approaches, we identify the aminotransferases and specifically Agxt as effectors of the suppressor function of Lkb1 in amino acid-driven gluconeogenesis.
Subject(s)
Amino Acids/metabolism , Gluconeogenesis/physiology , Liver/metabolism , Protein Serine-Threonine Kinases/metabolism , AMP-Activated Protein Kinases , Animals , Cachexia , Diabetes Mellitus, Type 2/metabolism , Fasting , Female , Glucose/metabolism , Hepatocytes/metabolism , Hyperglycemia/metabolism , Male , Mice , Mice, Knockout , Protein Serine-Threonine Kinases/genetics , Proteomics , Sarcopenia , Transaminases/metabolismABSTRACT
OBJECTIVES: This study examines the role of insulin and angiotensin II in high-density lipoprotein (HDL) metabolism by focusing on the regulation and function of scavenger receptor type-BI (SR-BI) in adipose tissue. METHODS AND RESULTS: Insulin or angiotensin II injection in wild-type mice induced a decrease in circulating HDL and it was associated with the translocation of SR-BI from intracellular sites to the plasma membrane of adipose tissue. Refeeding upregulated adipose HDL selective cholesteryl esters uptake and SR-BI proteins through transcriptional and posttranscriptional mechanisms. This occurred along with a decrease in serum HDL and an increase in adipose cholesterol content. Similar results were obtained with transgenic mice overexpressing locally angiotensinogen in adipose tissue. In adipose 3T3-L1 cell line, HDL induced lipogenesis by increasing liver X receptor binding activity. This mechanism was dependent of insulin and angiotensin II. CONCLUSIONS: Our results raise the possibility that adipose tissue SR-BI translocation might be a link between adipose tissue lipid storage and HDL clearance.
Subject(s)
Adipose Tissue/metabolism , Adiposity , Angiotensin II/metabolism , Cholesterol, HDL/metabolism , Insulin/metabolism , Lipogenesis , Scavenger Receptors, Class B/metabolism , 3T3-L1 Cells , ATP Binding Cassette Transporter 1 , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Adipocytes/drug effects , Adipocytes/metabolism , Adipose Tissue/cytology , Adipose Tissue/drug effects , Adiposity/drug effects , Adiposity/genetics , Angiotensin II/pharmacology , Angiotensinogen/genetics , Angiotensinogen/metabolism , Animals , Cell Membrane/metabolism , Cholesterol/analogs & derivatives , Cholesterol/metabolism , Cholesterol, HDL/blood , DNA-Binding Proteins/metabolism , Eating , Epididymis/metabolism , Homeostasis , Insulin/pharmacology , Lipogenesis/drug effects , Lipogenesis/genetics , Liver X Receptors , Male , Mice , Mice, Transgenic , Orphan Nuclear Receptors , Protein Transport , RNA, Messenger/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Scavenger Receptors, Class B/genetics , Sterol Regulatory Element Binding Protein 1/genetics , Sterol Regulatory Element Binding Protein 1/metabolism , Time Factors , Transcription, Genetic , Triglycerides/metabolismABSTRACT
A long-standing paradox in the pathophysiology of metabolic diseases is the selective insulin resistance of the liver. It is characterized by a blunted action of insulin to reduce glucose production, contributing to hyperglycemia, while de novo lipogenesis remains insulin sensitive, participating in turn to hepatic steatosis onset. The underlying molecular bases of this conundrum are not yet fully understood. Here, we established a model of selective insulin resistance in mice by silencing an inhibitor of insulin receptor catalytic activity, the growth factor receptor binding protein 14 (Grb14) in liver. Indeed, Grb14 knockdown enhanced hepatic insulin signaling but also dramatically inhibited de novo fatty acid synthesis. In the liver of obese and insulin-resistant mice, downregulation of Grb14 markedly decreased blood glucose and improved liver steatosis. Mechanistic analyses showed that upon Grb14 knockdown, the release of p62/sqstm1, a partner of Grb14, activated the transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2), which in turn repressed the lipogenic nuclear liver X receptor (LXR). Our study reveals that Grb14 acts as a new signaling node that regulates lipogenesis and modulates insulin sensitivity in the liver by acting at a crossroad between the insulin receptor and the p62-Nrf2-LXR signaling pathways.
Subject(s)
Insulin Resistance , Lipogenesis , Liver/metabolism , Proteins/genetics , Signal Transduction , Adaptor Proteins, Signal Transducing , Animals , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Gene Expression Regulation , Gene Knockdown Techniques , Liver/cytology , Liver X Receptors/metabolism , Mice , NF-E2-Related Factor 2/metabolism , Proteins/metabolism , Receptor, Insulin/metabolismABSTRACT
In mammals, the COUP-TF-family consisting of two structurally related proteins, COUP-TFI and COUP-TFII belongs to the orphan member of the steroid/thyroid hormone receptor superfamily. In an attempt to gain insights into the role of COUP-TFII, we examined developmental expression pattern of the mouse COUP-TFII focusing our studies on endoderm-derived tissues, pancreas and liver in particular. Independent lines of transgenic mice expressing Escherichia coli beta-galactosidase driven by the COUP-TFII promoter were generated. Embryonic expression of the beta-gal protein at day 9 of gestation was detected in the notochord, the ventral neural tube and, interestingly, in the gut endoderm, a site where COUP-TFII has not been detected previously. Between 9.5 and 11.5 dpc, beta-gal expression pattern that was established earlier persisted and sections revealed a staining of the common atrial chamber of the heart. At 15.5 dpc, beta-gal activity was found in all endoderm-derived tissues. We found that COUP-TFII mRNA and protein were present in fetal and adult hepatocytes. Finally, COUP-TFII expression was detected in pancreas, as judged by co-expression of the beta-gal in some of the glucagon and PDX1 positive-cells at 12.5 dpc and co-expression with insulin positive-cells at 15.5 dpc. In adult pancreas, COUP-TFII protein was present in the endocrine islet cells.
Subject(s)
DNA-Binding Proteins/biosynthesis , Homeodomain Proteins , Receptors, Steroid , Transcription Factors/biosynthesis , Animals , COUP Transcription Factor II , COUP Transcription Factors , Endoderm/metabolism , Escherichia coli/metabolism , Glucagon/metabolism , Heart/embryology , Hepatocytes/cytology , Hepatocytes/metabolism , Immunohistochemistry , Liver/embryology , Liver/metabolism , Mice , Mice, Transgenic , Myocardium/metabolism , Pancreas/embryology , Pancreas/metabolism , Plasmids/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Trans-Activators/metabolism , beta-Galactosidase/metabolismABSTRACT
Sterol regulatory element binding proteins (SREBPs) are a family of transcription factors that regulate lipid homeostasis by controlling the expression of a range of enzymes required for endogenous cholesterol, fatty acid (FA), triacylglycerol and phospholipid synthesis. The three SREBP isoforms, SREBP-1a, SREBP-1c and SREBP-2, have different roles in lipid synthesis. In vivo studies using transgenic and knockout mice suggest that SREBP-1c is involved in FA synthesis and insulin induced glucose metabolism (particularly in lipogenesis), whereas SREBP-2 is relatively specific to cholesterol synthesis. The SREBP-1a isoform seems to be implicated in both pathways. SREBP transcription factors are synthetized as inactive precursors bound to the endoplasmic reticulum (ER) membranes. Upon activation, the precursor undergoes a sequential two-step cleavage process to release the NH(2)-terminal active domain in the nucleus (designated nSREBPs). SREBP processing is mainly controlled by cellular sterol content. When sterol levels decrease, the precursor is cleaved to activate cholesterogenic genes and maintain cholesterol homeostasis. This sterol-sensitive process appears to be a major point of regulation for the SREBP-1a and SREBP-2 isoforms but not for SREBP-1c. Moreover, the SREBP-1c isoform seems to be mainly regulated at the transcriptional level by insulin. The unique regulation and activation properties of each SREBP isoform facilitate the co-ordinate regulation of lipid metabolism; however, further studies are needed to understand the detailed regulation pathways that specifically regulate each SREBP isoform.
Subject(s)
CCAAT-Enhancer-Binding Proteins/metabolism , DNA-Binding Proteins/metabolism , Homeostasis , Lipid Metabolism , Transcription Factors/metabolism , Animals , CCAAT-Enhancer-Binding Proteins/biosynthesis , CCAAT-Enhancer-Binding Proteins/genetics , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Gene Expression Regulation , Lipids/biosynthesis , Mice , Models, Biological , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Protein Isoforms/metabolism , Sterol Regulatory Element Binding Protein 1 , Sterol Regulatory Element Binding Protein 2 , Transcription Factors/biosynthesis , Transcription Factors/geneticsSubject(s)
Carcinoma, Hepatocellular , Cell Transformation, Neoplastic/metabolism , Glycolysis/physiology , Liver Neoplasms , beta Catenin/genetics , Animals , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Transformation, Neoplastic/genetics , Cellular Reprogramming/genetics , Energy Metabolism/physiology , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Mutation , Oxidation-ReductionABSTRACT
BACKGROUND: The control of the functional pancreatic ß-cell mass serves the key homeostatic function of releasing the right amount of insulin to keep blood sugar in the normal range. It is not fully understood though how ß-cell mass is determined. METHODOLOGY/PRINCIPAL FINDINGS: Conditional chicken ovalbumin upstream promoter transcription factor II (COUP-TFII)-deficient mice were generated and crossed with mice expressing Cre under the control of pancreatic duodenal homeobox 1 (pdx1) gene promoter. Ablation of COUP-TFII in pancreas resulted in glucose intolerance. Beta-cell number was reduced at 1 day and 3 weeks postnatal. Together with a reduced number of insulin-containing cells in the ductal epithelium and normal ß-cell proliferation and apoptosis, this suggests decreased ß-cell differentiation in the neonatal period. By testing islets isolated from these mice and cultured ß-cells with loss and gain of COUP-TFII function, we found that COUP-TFII induces the expression of the ß-catenin gene and its target genes such as cyclin D1 and axin 2. Moreover, induction of these genes by glucagon-like peptide 1 (GLP-1) via ß-catenin was impaired in absence of COUP-TFII. The expression of two other target genes of GLP-1 signaling, GLP-1R and PDX-1 was significantly lower in mutant islets compared to control islets, possibly contributing to reduced ß-cell mass. Finally, we demonstrated that COUP-TFII expression was activated by the Wnt signaling-associated transcription factor TCF7L2 (T-cell factor 7-like 2) in human islets and rat ß-cells providing a feedback loop. CONCLUSIONS/SIGNIFICANCE: Our findings show that COUP-TFII is a novel component of the GLP-1 signaling cascade that increases ß-cell number during the neonatal period. COUP-TFII is required for GLP-1 activation of the ß-catenin-dependent pathway and its expression is under the control of TCF7L2.
Subject(s)
COUP Transcription Factor II/physiology , Glucagon-Like Peptide 1/physiology , Insulin-Secreting Cells/cytology , Pancreas/growth & development , beta Catenin/physiology , Animals , Animals, Newborn , COUP Transcription Factor II/genetics , COUP Transcription Factor II/metabolism , Cell Count , Cells, Cultured , Embryo, Mammalian , Female , Glucagon-Like Peptide 1/genetics , Glucagon-Like Peptide 1/metabolism , Glucagon-Like Peptide 1/pharmacology , Humans , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Male , Mice , Mice, Transgenic , Models, Biological , Organ Size/drug effects , Organ Size/genetics , Pancreas/drug effects , Pancreas/embryology , Pancreas/metabolism , Rats , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/physiology , beta Catenin/genetics , beta Catenin/metabolismSubject(s)
Hepatocyte Nuclear Factor 4/physiology , Liver Failure/therapy , Animals , Cell Differentiation/genetics , Fibrosis/genetics , Fibrosis/metabolism , Gene Expression Profiling , Genetic Therapy/methods , Hepatocyte Nuclear Factor 4/genetics , Hepatocytes/metabolism , Hepatocytes/physiology , Humans , Liver Failure/genetics , Liver Failure/pathology , Liver Regeneration/genetics , RatsABSTRACT
BACKGROUND: The nuclear receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is an important coordinator of glucose homeostasis. We report, for the first time, a unique differential regulation of its expression by the nutritional status in the mouse hypothalamus compared to peripheral tissues. METHODOLOGY/PRINCIPAL FINDINGS: Using hyperinsulinemic-euglycemic clamps and insulinopenic mice, we show that insulin upregulates its expression in the hypothalamus. Immunofluorescence studies demonstrate that COUP-TFII gene expression is restricted to a subpopulation of ventromedial hypothalamic neurons expressing the melanocortin receptor. In GT1-7 hypothalamic cells, the MC4-R agonist MTII leads to a dose dependant increase of COUP-TFII gene expression secondarily to a local increase in cAMP concentrations. Transfection experiments, using a COUP-TFII promoter containing a functional cAMP responsive element, suggest a direct transcriptional activation by cAMP. Finally, we show that the fed state or intracerebroventricular injections of MTII in mice induce an increased hypothalamic COUP-TFII expression associated with a decreased hepatic and pancreatic COUP-TFII expression. CONCLUSIONS/SIGNIFICANCE: These observations strongly suggest that hypothalamic COUP-TFII gene expression could be a central integrator of insulin and melanocortin signaling pathway within the ventromedial hypothalamus. COUP-TFII could play a crucial role in brain integration of circulating signal of hunger and satiety involved in energy balance regulation.