Serum and Soleus Metabolomics Signature of Klf10 Knockout Mice to Identify Potential Biomarkers.

The transcription factor Krüppel-like factor 10 (Klf10), also known as Tieg1 for TGFß (Inducible Early Gene-1) is known to control numerous genes in many cell types that are involved in various key biological processes (differentiation, proliferation, apoptosis, inflammation), including cell metabolism and human disease. In skeletal muscle, particularly in the soleus, deletion of the Klf10 gene (Klf10 KO) resulted in ultrastructure fiber disorganization and mitochondrial metabolism deficiencies, characterized by muscular hypertrophy. To determine the metabolic profile related to loss of Klf10 expression, we analyzed blood and soleus tissue using UHPLC-Mass Spectrometry. Metabolomics analyses on both serum and soleus revealed profound differences between wild-type (WT) and KO animals. Klf10 deficient mice exhibited alterations in metabolites associated with energetic metabolism. Additionally, chemical classes of aromatic and amino-acid compounds were disrupted, together with Krebs cycle intermediates, lipids and phospholipids. From variable importance in projection (VIP) analyses, the Warburg effect, citric acid cycle, gluconeogenesis and transfer of acetyl groups into mitochondria appeared to be possible pathways involved in the metabolic alterations observed in Klf10 KO mice. These studies have revealed essential roles for Klf10 in regulating multiple metabolic pathways whose alterations may underlie the observed skeletal muscle defects as well as other diseases.

Analytical Methodology for a Metabolome Atlas of Goat's Plasma, Milk and Feces Using 1H-NMR and UHPLC-HRMS.

Metabolomics has been increasingly used in animal and food sciences. Animal health is one of the most important factor that can also alter animal integrity and welfare. Some studies have already investigated the link between health and metabolic profile of dairy animals. These studies in metabolomics often consider a single type of sample using a single analytical platform (nuclear magnetic resonance or mass spectrometry). Only few studies with multi-platform approaches are also used with a single or a multi type of sample, but they mainly consider dairy cows' metabolome although dairy goats present similar diseases, that it could be interesting to detect early to preserve animal health and milk production. This study aims to create a metabolic atlas of goat plasma, milk and feces, based on healthy animals. Our study describes a standard operating procedure for three goat matrices: blood plasma, milk, and feces using multiple platforms (NMR (1H), UHPLC (RP)-MS and UHPLC (HILIC)-MS) that follows a unique sample preparation procedure for each sample type to be analyzed on multi-platforms basis. Our method was evaluated for its robustness and allowed a better characterization of goat metabolic profile in healthy conditions.

Optimization of Sample Preparation for Metabolomics Exploration of Urine, Feces, Blood and Saliva in Humans Using Combined NMR and UHPLC-HRMS Platforms.

Currently, most clinical studies in metabolomics only consider a single type of sample such as urine, plasma, or feces and use a single analytical platform, either NMR or MS. Although some studies have already investigated metabolomics data from multiple fluids, the information is limited to a unique analytical platform. On the other hand, clinical studies investigating the human metabolome that combine multi-analytical platforms have focused on a single biofluid. Combining data from multiple sample types for one patient using a multimodal analytical approach (NMR and MS) should extend the metabolome coverage. Pre-analytical and analytical phases are time consuming. These steps need to be improved in order to move into clinical studies that deal with a large number of patient samples. Our study describes a standard operating procedure for biological specimens (urine, blood, saliva, and feces) using multiple platforms (1H-NMR, RP-UHPLC-MS, and HILIC-UHPLC-MS). Each sample type follows a unique sample preparation procedure for analysis on a multi-platform basis. Our method was evaluated for its robustness and was able to generate a representative metabolic map.

Panitumumab and cetuximab affect differently miRNA expression in colorectal cancer cells.

Background & aim: Resistance to anti-EGFR monoclonal antibodies in metastatic colorectal cancer (CRC) is frequent and prognostic biomarkers are lacking. MicroRNAs (miR) are good candidates in this context. We aimed to characterize cetuximab and panitumumab exposure influence on miR expression in colorectal cancer cells to identify those regulating the EGFR pathway and implicated in resistance to treatment. Finally, we aimed to identify miR expression in serum of patients with advanced CRC treated with cetuximab or panitumumab. Results: Cetuximab and panitumumab exposure induced significant expression variations of 17 miR out of a miRnome panel of 752. Six of those miR interacted with at least one downstream element of the EGFR pathway. Conclusion: After the bioinformatics two-phase process, five miR rarely described before could be potential actors of anti-EGFR monoclonal antibody resistance: miR-95-3p, miR-139-5p, miR-145-5p, miR-429 and miR-1247-5p. In vivo, we detected the expression of miR-139-5p and miR-145-5p in serum of patients with metastatic CRC.

A possible association of baseline serum IL-17A concentrations with progression-free survival of metastatic colorectal cancer patients treated with a bevacizumab-based regimen.

BACKGROUND: Colorectal cancer is a major public health issue worldwide. Interleukin-17 (IL-17) and Th17 (T-helper cell type 17)-related molecules are involved in tumor development and in resistance to bevacizumab, an anti-vascular endothelial growth factor monoclonal antibody used in association with chemotherapy in metastatic colorectal cancer. Some studies have previously shown that IL-17A and IL-17F polymorphisms, respectively rs2275913 and rs763780, are associated with gastric or colorectal cancer risk. Here we aimed at studying the influence of IL-17A-related individual factors on overall survival and progression-free survival in patients with metastatic colorectal cancer treated with a bevacizumab-based chemotherapy. METHODS: Pre-treatment serum biomarkers were retrospectively evaluated in 122 metastatic colorectal cancer patients treated by bevacizumab in combination with chemotherapy at 2-weeks intervals in a prospective cohort study (NCT00489697). The polymorphisms of IL-17A and IL-17F were analyzed by polymerase chain reaction - restriction fragment length polymorphism. Serum concentrations of Th17-related cytokines were measured by MultiPlex. The impact of individual parameters on overall survival and progression-free survival was assessed using multivariate Cox models. RESULTS: High baseline IL-17A serum concentrations were significantly associated with shorter progression-free survival [p = 0.043]. Other baseline serum Th17-related cytokines and polymorphisms of IL-17 were not associated with overall survival or progression-free survival. CONCLUSIONS: In this ancillary study, baseline serum IL-17A concentration is the only Th17/IL-17 related factor that was significantly associated with the response of patients with metastatic colorectal cancer to bevacizumab. But this main significant result is highly dependent on one case which, if left out, weakens the data. Other clinical studies are required to confirm this association. TRIAL REGISTRATION: NCT00489697 . June 20, 2007.

Downregulation of the neonatal Fc receptor expression in non-small cell lung cancer tissue is associated with a poor prognosis.

Lung cancer is the leading cause of cancer-related death worldwide. Although the recommended tumor, node and metastasis (TNM) classification and stage determination are important to select therapeutic options for patients with non-small cell lung carcinoma (NSCLC), additional molecular markers are required to indicate the prognosis, in particular within a specific stage, and help with the management of patients.Because neonatal Fc receptor (FcRn) has recently been involved in colon cancer immunosurveillance, we measured its expression in non-cancerous and NSCLC lung tissues and evaluated its prognostic value in overall survival for patient with NSCLC. FcRn expression was determined at both mRNA and protein levels on cancerous and adjacent non-cancerous tissues from 80 NSCLC patients. In NSCLC, FcRn was mainly found in resident and tumor infiltrating immune cells. The corresponding mRNA and protein were significantly less abundant in lung tumor than non-cancerous tissue. Moreover, analysis of our cohort and datasets from the public data bases show that FCGRT mRNA down-regulation is a robust and independent, unfavorable predictive factor of NSCLC patient survival. We conclude that FCGRT mRNA expression may be a useful additional marker for immunoscoring, reflecting tumor immune system, and help in the decision-making process for NSCLC patients.

IgG1 Allotypes Influence the Pharmacokinetics of Therapeutic Monoclonal Antibodies through FcRn Binding.

Because IgG1 allotypes might have different half-lives, their influence on infliximab (G1m17,1 allotype) pharmacokinetics was investigated in a group of spondyloarthritis patients. Infliximab was found to have a shorter half-life in patients homozygous for the G1m17,1 allotypes than in those carrying the G1m3 with no G1m1 (G1m3,-1) allotype. Because the neonatal FcR (FcRn) is involved in the pharmacokinetics of mAbs, the interaction of different IgG1 allotypes with FcRn was examined using cellular assays and surface plasmon resonance. G1m17,1 mAbs, such as infliximab and rituximab, were shown to bind more efficiently to FcRn and to be transcytosed better than the G1m3,-1 mAb cetuximab, which explains why infliximab is a better competitor for endogenous IgG1 in G1m3,-1 allotype-bearing patients. A set of four allotype variants of adalimumab (G1m17,1; G1m17,-1; G1m3,1; and G1m3,-1) was also tested for its binding to FcRn, revealing that the G1m3,1 variant, not present in commercial mAbs, binds more efficiently to FcRn and is transcytosed better than the other three variants, all of which are found in therapeutic mAbs.

Influence of FCGRT gene polymorphisms on pharmacokinetics of therapeutic antibodies.

The neonatal Fc receptor (FcRn) encoded by FCGRT is known to be involved in the pharmacokinetics (PK) of therapeutic monoclonal antibodies (mAbs). Variability in the expression of FCGRT gene and consequently in the FcRn protein level could explain differences in PK observed between patients treated with mAbs. We studied whether the previously described variable number tandem repeat (VNTR) or copy number variation (CNV) of FCGRT are associated with individual variations of PK parameters of cetuximab. VNTR and CNV were assessed on genomic DNA of 198 healthy individuals and of 94 patients treated with the therapeutic mAb. VNTR and CNV were analyzed by allele-specific PCR and duplex real-time PCR with Taqman (®) technology, respectively. The relationship between FCGRT polymorphisms (VNTR and CNV) and PK parameters of patients treated with cetuximab was studied. VNTR3 homozygote patients had a lower cetuximab distribution clearance than VNTR2/VNTR3 and VNTR3/VNTR4 patients (p = 0.021). We observed no affects of VNTR genotype on elimination clearance. One healthy person (0.5%) and 1 patient (1.1%) had 3 copies of FCGRT. The PK parameters of this patient did not differ from those of patients with 2 copies. The FCGRT promoter VNTR may influence mAbs' distribution in the body. CNV of FCGRT cannot be used as a relevant pharmacogenetic marker because of its low frequency.

Function of microRNA-375 and microRNA-124a in pancreas and brain.

In recent years, our understanding of how gene regulatory networks control cell physiology has improved dramatically. Studies have demonstrated that transcription is regulated not only by protein factors, but also by small RNA molecules, microRNAs (miRNAs). The first miRNA was discovered in 1993 as a result of a genetic screen for mutations in Caenorhabditis elegans. Since then, the use of sophisticated techniques and screening tools has promoted a more definitive understanding of the role of miRNAs in mammalian development and diseases. miRNAs have emerged as important regulators of genes involved in many biological processes, including development, cell proliferation and differentiation, apoptosis and metabolism. Over the last few years, the number of reviews dealing with miRNAs has increased at an impressive pace. In this review, we present general information on miRNA biology and focus more closely on comparing the expression, regulation and molecular functions of the two miRNAs, miR-375 and miR-124a. miR-375 and miR-124a share similar features; they are both specifically expressed in the pancreas and brain and directly bind a common target gene transcript encoding myotrophin, which regulates exocytosis and hormone release. Here, we summarize the available data obtained by our group and other laboratories and provide an overview of the specific molecular function of miR-375 and miR-124a in the pancreas and the brain, revealing a potential functional overlap for these two miRNAs and the emerging therapeutic potential of miRNAs in the treatment of human metabolic diseases.

miR-375 targets 3'-phosphoinositide-dependent protein kinase-1 and regulates glucose-induced biological responses in pancreatic beta-cells.

OBJECTIVE: MicroRNAs are short, noncoding RNAs that regulate gene expression. We hypothesized that the phosphatidylinositol 3-kinase (PI 3-kinase) cascade known to be important in beta-cell physiology could be regulated by microRNAs. Here, we focused on the pancreas-specific miR-375 as a potential regulator of its predicted target 3'-phosphoinositide-dependent protein kinase-1 (PDK1), and we analyzed its implication in the response of insulin-producing cells to elevation of glucose levels. RESEARCH DESIGN AND METHODS: We used insulinoma-1E cells to analyze the effects of miR-375 on PDK1 protein level and downstream signaling using Western blotting, glucose-induced insulin gene expression using quantitative RT-PCR, and DNA synthesis by measuring thymidine incorporation. Moreover, we analyzed the effect of glucose on miR-375 expression in both INS-1E cells and primary rat islets. Finally, miR-375 expression in isolated islets was analyzed in diabetic Goto-Kakizaki (GK) rats. RESULTS: We found that miR-375 directly targets PDK1 and reduces its protein level, resulting in decreased glucose-stimulatory action on insulin gene expression and DNA synthesis. Furthermore, glucose leads to a decrease in miR-375 precursor level and a concomitant increase in PDK1 protein. Importantly, regulation of miR-375 expression by glucose occurs in primary rat islets as well. Finally, miR-375 expression was found to be decreased in fed diabetic GK rat islets. CONCLUSIONS: Our findings provide evidence for a role of a pancreatic-specific microRNA, miR-375, in the regulation of PDK1, a key molecule in PI 3-kinase signaling in pancreatic beta-cells. The effects of glucose on miR-375 are compatible with the idea that miR-375 is involved in glucose regulation of insulin gene expression and beta-cell growth.

MicroRNA-124a regulates Foxa2 expression and intracellular signaling in pancreatic beta-cell lines.

MicroRNAs (miRNAs) are short non-coding RNAs that have been implicated in fine-tuning gene regulation, although the precise roles of many are still unknown. Pancreatic development is characterized by the complex sequential expression of a gamut of transcription factors. We have performed miRNA expression profiling at two key stages of mouse embryonic pancreas development, e14.5 and e18.5. miR-124a2 expression was strikingly increased at e18.5 compared with e14.5, suggesting a possible role in differentiated beta-cells. Among the potential miR-124a gene targets identified by biocomputation, Foxa2 is known to play a role in beta-cell differentiation. To evaluate the impact of miR-124a2 on gene expression, we overexpressed or down-regulated miR-124a2 in MIN6 beta-cells. As predicted, miR-124a2 regulated Foxa2 gene expression, and that of its downstream target, pancreatic duodenum homeobox-1 (Pdx-1). Foxa2 has been described as a master regulator of pancreatic development and also of genes involved in glucose metabolism and insulin secretion, including the ATP-sensitive K(+) (K(ATP)) channel subunits, Kir6.2 and Sur-1. Correspondingly, miR-124a2 overexpression decreased, and anti-miR-124a2 increased Kir6.2 and Sur-1 mRNA levels. Moreover, miR-124a2 modified basal and glucose- or KCl-stimulated intracellular free Ca(2+) concentrations in single MIN6 and INS-1 (832/13) beta-cells, without affecting the secretion of insulin or co-transfected human growth hormone, consistent with an altered sensitivity of the beta-cell exocytotic machinery to Ca(2+). In conclusion, whereas the precise role of microRNA-124a2 in pancreatic development remains to be deciphered, we identify it as a regulator of a key transcriptional protein network in beta-cells responsible for modulating intracellular signaling.

Apolipoprotein A-V deficiency results in marked hypertriglyceridemia attributable to decreased lipolysis of triglyceride-rich lipoproteins and removal of their remnants.

OBJECTIVE: ApoAV, a newly discovered apoprotein, affects plasma triglyceride level. To determine how this occurs, we studied triglyceride-rich lipoprotein (TRL) metabolism in mice deficient in apoAV. METHODS AND RESULTS: No significant difference in triglyceride production rate was found between apoa5(-/-) mice and controls. The presence or absence of apoAV affected TRL catabolism. After the injection of 14C-palmitate and 3H-cholesterol labeled chylomicrons and (125)I-labeled chylomicron remnants, the disappearance of 14C, 3H, and (125)I was significantly slower in apoa5(-/-) mice relative to controls. This was because of diminished lipolysis of TRL and the reduced rate of uptake of their remnants in apoa5(-/-) mice. Observed elevated cholesterol level was caused by increased high-density lipoprotein (HDL) cholesterol in apoa5(-/-) mice. VLDL from apoa5(-/-) mice were poor substrate for lipoprotein lipase, and did not bind to the low-density lipoprotein (LDL) receptor as well as normal very-low-density lipoprotein (VLDL). LDL receptor levels were slightly elevated in apoa5(-/-) mice consistent with lower remnant uptake rates. These alterations may be the result of the lower apoE-to-apoC ratio found in VLDL isolated from apoa5(-/-) mice. CONCLUSIONS: These results support the hypothesis that the absence of apoAV slows lipolysis of TRL and the removal of their remnants by regulating their apoproteins content after secretion.

Comparative genomic analysis reveals a distant liver enhancer upstream of the COUP-TFII gene.

COUP-TFII is a central nuclear hormone receptor that tightly regulates the expression of numerous target lipid metabolism genes in vertebrates. However, it remains unclear how COUP-TFII itself is transcriptionally controlled since studies with its promoter and upstream region fail to recapitulate the gene's liver expression. In an attempt to identify liver enhancers in the vicinity of COUP-TFII, we employed a comparative genomic approach. Initial comparisons between humans and mice of the 3470-kb gene-poor region surrounding COUP-TFII revealed 2023 conserved noncoding elements. To prioritize a subset of these elements for functional studies, we performed further genomic comparisons with the orthologous pufferfish (Fugu rubripes) locus and uncovered two anciently conserved noncoding sequences (CNS) upstream of COUP-TFII (CNS-62kb and CNS-66kb). Testing these two elements using reporter constructs in liver cells (HepG2) revealed that CNS-66kb, but not CNS-62kb, yielded robust in vitro enhancer activity. In addition, an in vivo reporter assay using naked DNA transfer with CNS-66kb linked to luciferase displayed strong reproducible liver expression in adult mice, further supporting its role as a liver enhancer. Together, these studies further support the utility of comparative genomics to uncover gene regulatory sequences based on evolutionary conservation and provide the substrates to better understand the regulation and expression of COUP-TFII.

Human apoA-I/C-III/A-IV gene cluster transgenic rabbits: effects of a high-cholesterol diet.

We have generated transgenic rabbits that express the entire human apoA-I/C-III/A-IV gene cluster. As in humans, h-apoA-I and h-apoC-III were expressed in liver and intestine, whereas h-apoA-IV mRNA was detected in intestine only. Transgenic rabbits had significantly higher plasma total cholesterol, HDL-cholesterol and total phospholipid concentrations than non-transgenic littermates. In contrast to similar transgenic mice previously generated, which have gross hypertriglyceridemia, triglyceride concentrations were only moderately raised in transgenic rabbits. Plasma and HDL from transgenic rabbits were more effective than those from controls in promoting cholesterol efflux from cultured hepatoma cells. They had lower LCAT, lower CETP and higher PLTP activities than non-transgenic littermates. Cholesterol-feeding produced major increases in plasma lipids. The qualitative response to the diet was not modified by cluster expression. Human apoA-I concentration was halved by cholesterol-feeding, whereas h-apoC-III and h-apoA-IV concentrations were not significantly altered. Cholesterol efflux from hepatoma cells to plasma and HDL was not altered by the diet. Since lipoprotein metabolism of rabbits closely resembles that of humans, human apoA-I/C-III/A-IV transgenic rabbits may provide a reliable model for studies of the transcriptional regulation of the cluster, and for evaluating the effects of different agents on the expression of the three genes.

Analysis of apolipoprotein A5, c3, and plasma triglyceride concentrations in genetically engineered mice.

OBJECTIVE: Both the apolipoprotein A5 and C3 genes have repeatedly been shown to play an important role in determining plasma triglyceride concentrations in humans and mice. In mice, transgenic and knockout experiments indicate that plasma triglyceride levels are strongly altered by changes in the expression of either of these 2 genes. In humans, common polymorphisms in both genes have also been associated with plasma triglyceride concentrations. These similar findings raised the issue of the relationship between these 2 genes and altered triglycerides. METHODS AND RESULTS: To address this issue, we generated independent lines of mice that either overexpressed ("double transgenic") or completely lacked ("double knockout") both apolipoprotein genes. We report that both "double transgenic" and "double knockout" mice display normal triglyceride concentrations compared with overexpression or deletion of either gene alone. Furthermore, we find that human ApoAV plasma protein levels in the "double transgenic" mice are approximately 500-fold lower than human ApoCIII levels, supporting ApoAV as a potent triglyceride modulator despite its low concentration. CONCLUSIONS: Together, these data support that APOA5 and APOC3 independently influence plasma triglyceride concentrations but in an opposing manner.

Expression and secretion of human apolipoprotein A-I in the heart.

Various studies have correlated apolipoprotein (apo) A-I, the major component high-density lipoprotein, with protection against development of cardiovascular disease. Although apoA-I expression has been previously detected in the liver and intestine, we have discovered that the human apoA-I gene is also expressed in the heart. Using transgenic (Tg) mice generated with the human apoA-I/C-III/A-IV gene cluster and Tg mice produced with just the 2.2 kb human apoA-I gene, we have detected significant levels of apoA-I expression in the heart. Furthermore, the detection of apoA-I expression in the hearts of human apoA-I Tg mice indicates that the minimal regulatory elements necessary for cardiac expression of the gene are located near its coding sequence. To determine if the apoA-I gene is also expressed in the human heart, similar analyses were performed, where apoA-I expression was found in both adult and fetal hearts. Furthermore in-depth investigation of the various regions of human and Tg mouse hearts revealed that the apoA-I mRNA was present in the ventricles and atria, but not in the aorta. In situ hybridization of Tg mouse hearts revealed that apoA-I expression was restricted to the cardiac myocyte cells. Finally, heart explants and cardiac primary culture experiments with Tg mice showed secretion of particles containing the human apoA-I protein, and metabolic labeling experiments have also detected a 28 kDa human apoA-I protein secreted from the heart. From these novel findings, new insights into the role and function of apoA-I can be extrapolated.

Myelination and motor coordination are increased in transferrin transgenic mice.

Myelin deficiency in the central nervous system (CNS) can cause severe disabling conditions. Most of the transgenic mice models overexpressing myelin components have limitations for investigators of myelin deficiency and myelin therapy as they severely alter CNS architecture. It has been postulated that transferrin (Tf) is involved in oligodendrocyte (OL) maturation and myelinogenesis. Because Tf is not an intrinsic myelin constituent, we decided to investigate if its overexpression could have an impact on the myelination process without affecting myelin integrity. We generated transgenic mice containing the complete human Tf gene specifically overexpressed in OLs. This overexpression leads to more than a 30% increase in myelin components, such as galactolipids, phospholipids, and proteins. Electron microscopy showed that myelin is structurally normal in terms of thickness and compaction. Behavior analysis showed that mice do not display significant modifications in their locomotion and cognitive and emotional abilities. Furthermore, in one of the genetic background, animals presented a significant increase in motor coordination. We did not find any modification in OL number during early postnatal development, suggesting that Tf does not act on OL proliferation. In addition, the levels of iron and ferritin remained unchanged in the brain of transgenic mice compared to control mice. Our findings indicate that, besides its known iron transport function, Tf is able to influence myelination process and induce behavioral improvements in mice.

Fructose intake increases hyperlipidemia and modifies apolipoprotein expression in apolipoprotein AI-CIII-AIV transgenic mice.

Fructose intake has increased steadily during the past two decades. The objective of this study was to determine the effect of fructose intake on lipid metabolism in apolipoprotein (apo) AI-CIII-AIV transgenic (Tg) mice that have severe hypertriglyceridemia and moderate hypercholesterolemia. Tg and control mice were fed for 9 mo a commercial nonpurified diet and had free access to water or 250 g/L fructose solution. In Tg mice, fructose intake increased triglycerides and cholesterol but did not induce insulin resistance. There were no differences in human hepatic apo AI and apo CIII mRNA levels in fructose-fed mice compared with untreated mice, but apo AIV mRNA was greater, indicating a differential expression of the apo AI and apo AIV genes in response to dietary perturbations. Interestingly, the plasma concentration of the three human apolipoproteins was enhanced in fructose-fed Tg mice compared with untreated Tg mice. Our data suggest that long-term fructose consumption had strong adverse effects in this hyperlipidemic mouse model.