Intestinal miRNAs regulated in response to dietary lipids.

The role of miRNAs in intestinal lipid metabolism is poorly described. The small intestine is constantly exposed to high amounts of dietary lipids, and it is under conditions of stress that the functions of miRNAs become especially pronounced. Approaches consisting in either a chronic exposure to cholesterol and triglyceride rich diets (for several days or weeks) or an acute lipid challenge were employed in the search for intestinal miRNAs with a potential role in lipid metabolism regulation. According to our results, changes in miRNA expression in response to fat ingestion are dependent on factors such as time upon exposure, gender and small intestine section. Classic and recent intestinal in vitro models (i.e. differentiated Caco-2 cells and murine organoids) partially mirror miRNA modulation in response to lipid challenges in vivo. Moreover, intestinal miRNAs might play a role in triglyceride absorption and produce changes in lipid accumulation in intestinal tissues as seen in a generated intestinal Dicer1-deletion murine model. Overall, despite some variability between the different experimental cohorts and in vitro models, results show that some miRNAs analysed here are modulated in response to dietary lipids, hence likely to participate in the regulation of lipid metabolism, and call for further research.

Intestinal Lipid Metabolism Genes Regulated by miRNAs.

MicroRNAs (miRNAs) crucial roles in translation repression and post-transcriptional adjustments contribute to regulate intestinal lipid metabolism. Even though their actions in different metabolic tissues have been elucidated, their intestinal activity is yet unclear. We aimed to investigate intestinal miRNA-regulated lipid metabolism-related genes, by creating an intestinal-specific Dicer1 knockout (Int-Dicer1 KO) mouse model, with a depletion of microRNAs in enterocytes. The levels of 83 cholesterol and lipoprotein metabolism-related genes were assessed in the intestinal mucosa of Int-Dicer1 KO and Wild Type C57BL/6 (WT) littermates mice at baseline and 2 h after an oral lipid challenge. Among the 18 genes selected for further validation, Hmgcs2, Acat1 and Olr1 were found to be strong candidates to be modulated by miRNAs in enterocytes and intestinal organoids. Moreover, we report that intestinal miRNAs contribute to the regulation of intestinal epithelial differentiation. Twenty-nine common miRNAs found in the intestines were analyzed for their potential to target any of the three candidate genes found and validated by miRNA-transfection assays in Caco-2 cells. MiR-31-5p, miR-99b-5p, miR-200a-5p, miR-200b-5p and miR-425-5p are major regulators of these lipid metabolism-related genes. Our data provide new evidence on the potential of intestinal miRNAs as therapeutic targets in lipid metabolism-associated pathologies.

Dietary supplementation with hybrid palm oil alters liver function in the common Marmoset.

Hybrid palm oil, which contains higher levels of oleic acid and lower saturated fatty acids in comparison with African palm oil, has been proposed to be somehow equivalent to extra virgin olive oil. However, the biological effects of its consumption are poorly described. Here we have explored the effects of its overconsumption on lipid metabolism in a non-human primate model, the common marmoset. Dietary supplementation of marmoset with hyperlipidic diet containing hybrid palm oil for 3 months did not modify plasma lipids levels, but increased glucose levels as compared to the supplementation with African palm oil. Liver volume was unexpectedly found to be more increased in marmosets consuming hybrid palm oil than in those consuming African palm oil. Hepatic total lipid content and circulating transaminases were dramatically increased in animals consuming hybrid palm oil, as well as an increased degree of fibrosis. Analysis of liver miRNAs showed a selective modulation of certain miRNAs by hybrid palm oil, some of which were predicted to target genes involved in cell adhesion molecules and peroxisomal pathways. Our data suggest that consumption of hybrid palm oil should be monitored carefully, as its overconsumption compared to that of African palm oil could involve important alterations to hepatic metabolism.

Fructose, but not glucose, impairs insulin signaling in the three major insulin-sensitive tissues.

Human studies support the relationship between high intake of fructose-sweetened beverages and type 2 diabetes, but there is a debate on whether this effect is fructose-specific or it is merely associated to an excessive caloric intake. Here we investigate the effects of 2 months' supplementation to female rats of equicaloric 10% w/v fructose or glucose solutions on insulin sensitivity in target tissues. Fructose supplementation caused hepatic deposition of triglycerides and changed the fatty acid profile of this fraction, with an increase in monounsaturated and a decrease in polyunsaturated species, but did not cause inflammation and oxidative stress. Fructose but not glucose-supplemented rats displayed an abnormal glucose tolerance test, and did not show increased phosphorylation of V-akt murine thymoma viral oncogene homolog-2 (Akt) in white adipose tissue and liver after insulin administration. In skeletal muscle, phosphorylation of Akt and of Akt substrate of 160 kDA (AS160) was not impaired but the expression of the glucose transporter type 4 (GLUT4) in the plasma membrane was reduced only in fructose-fed rats. In conclusion, fructose but not glucose supplementation causes fatty liver without inflammation and oxidative stress and impairs insulin signaling in the three major insulin-responsive tissues independently from the increase in energy intake.

Retinoic acid regulates Schwann cell migration via NEDD9 induction by transcriptional and post-translational mechanisms.

Schwann cell migration is essential during the regenerative response to nerve injury, however, the factors that regulate this phenomenon are not yet clear. Here we describe that retinoic acid (RA), whose production and signaling activity are greatly enhanced during nerve regeneration, increases Schwann cell migration. This is accompanied by the up-regulation of NEDD9, a member of the CAS family of scaffold proteins previously implicated in migratory and invasive behavior in gliomas, melanomas and the neural crest cells from which Schwann cells derive. This RA-induced NEDD9 accumulation is due to augmented mRNA levels, as well as an increase of NEDD9 protein stability. Although all NEDD9 phospho-isoforms present in Schwann cells are induced by the retinoid, the hormone also changes its phosphorylation status, thus altering the ratio between the different isoforms. Silencing NEDD9 in Schwann cells had no effect on basal migratory ability, but completely abrogated RA-induced enhanced migration. Collectively, our results indicate that RA could be a major regulator of Schwann cell migration after nerve injury, thus offering a new insight into peripheral nerve repair.

Regulation of retinoid receptors by retinoic acid and axonal contact in Schwann cells.

BACKGROUND: Schwann cells (SCs) are the cell type responsible for the formation of the myelin sheath in the peripheral nervous system (PNS). As retinoic acid (RA) and other retinoids have a profound effect as regulators of the myelination program, we sought to investigate how their nuclear receptors levels were regulated in this cell type. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, by using Schwann cells primary cultures from neonatal Wistar rat pups, as well as myelinating cocultures of Schwann cells with embryonic rat dorsal root ganglion sensory neurons, we have found that sustained expression of RXR-γ depends on the continuous presence of a labile activator, while axonal contact mimickers produced an increase in RXR-γ mRNA and protein levels, increment that could be prevented by RA. The upregulation by axonal contact mimickers and the transcriptional downregulation by RA were dependent on de novo protein synthesis and did not involve changes in mRNA stability. On the other hand, RAR-ß mRNA levels were only slightly modulated by axonal contact mimickers, while RA produced a strong transcriptional upregulation that was independent of de novo protein synthesis without changes in mRNA stability. CONCLUSIONS/SIGNIFICANCE: All together, our results show that retinoid receptors are regulated in a complex manner in Schwann cells, suggesting that they could have a prominent role as regulators of Schwann cell physiology.

Retinoic acid regulates myelin formation in the peripheral nervous system.

Understanding the mechanisms that control myelin formation is essential for the development of demyelinating diseases treatments. All-trans-retinoic acid (RA) plays an essential role during the development of the nervous system as a potent regulator of morphogenesis, cell growth, and differentiation. In this study, we show that RA is also a potent inhibitor of peripheral nervous system (PNS) myelination. RA acts through its binding to RA receptors (RAR) and retinoid X receptors (RXR), two members of the superfamily of nuclear receptors that act as ligand-dependent transcription factors. Schwann cells (SCs) express all retinoid receptors during the relevant stages of myelin formation. Through the activation of RXR, RA produces an upregulation of Krox20, a SC-specific regulatory transcription factor that plays a central role during myelination. Krox20 upregulation translates into Mbp and Mpz overexpression, therefore blocking myelin formation. This increase in myelin protein expression is accompanied by the induction of an adaptive ER stress response. At the same time, through a RAR-dependent mechanism, RA downregulates myelin-associated glycoprotein, which also contributes to the dysmyelinating effect of the retinoid.

Cell cycle control of Notch signaling and the functional regionalization of the neuroepithelium during vertebrate neurogenesis.

A critical feature of vertebrate neural precursors is the to-and-fro displacement of their nuclei as cell cycle progresses, thus giving rise to a pseudostratified epithelium. This nuclear behavior, referred to as interkinetic nuclear migration (INM), is translated into the disposition of the cell somas at different orthogonal levels depending on the cell cycle stage they are. The finding that important regulators of neurogenesis, such as the proneural and neurogenic genes, undergo cyclic changes of expression and function in coordination with the cell cycle and the INM, and that the neurogenic process correlates with a particular window of the cell cycle, in coincidence with the apical localization in the neuroepithelium of neural precursors, is a novel concept that facilitates our understanding of the neurogenic process in vertebrates. As such, recent data support the notion that the three-dimensional structure of the neuroepithelium is crucial for proper neuronal production. In this review, we describe current knowledge of the molecular mechanisms involved in the differential expression and function of the proneural and neurogenic gene products along the cell cycle, and we discuss important consequences for vertebrate neurogenesis derived from this observation.

Instability of Notch1 and Delta1 mRNAs and reduced Notch activity in vertebrate neuroepithelial cells undergoing S-phase.

Vertebrate neurogenesis is controlled through lateral inhibitory signals triggered by the Notch receptor and its ligand Delta. In the E4 chick embryo, the capacity of neural precursors to express the neurogenic genes Notch1 and Delta1 becomes reduced during S-phase, suggesting that their competence to trigger lateral inhibitory signals varies at different stages of the cell cycle. Here we show that the reduction of neurogenic gene expression during S-phase is extensive to later developmental stages and to other species; and it correlates with lower expression of lunatic Fringe and diminished capability to induce the expression of cHairy1/Hes1 and Hes5-1. We also show that the cell cycle-dependence of Notch1 and Delta1 expression is due to a remarkable decrease of mRNA stability during S-phase. These results provide evidence that the capacity of vertebrate neural precursors to express neurogenic genes and trigger lateral inhibitory signals is functionally coordinated with the cell cycle.

Occupancy and function of the -150 sterol regulatory element and -65 E-box in nutritional regulation of the fatty acid synthase gene in living animals.

Upstream regulatory factor (USF) and sterol regulatory element binding protein (SREBP) play key roles in the transcriptional regulation of the fatty acid synthase (FAS) gene by feeding and insulin. Due to the dual binding specificity of SREBP, as well as the presence of multiple consensus sites for these transcription factors in the FAS promoter, their physiologically relevant functional binding sites have been controversial. Here, in order to determine the occupancy of the putative USF and SREBP binding sites, we examined their protein-DNA interactions in living animals by using formaldehyde cross-linking and immunoprecipitation of chromatin and tested the function of these elements by employing mice transgenic for a reporter gene driven by various 5' deletions as well as site-specific mutations of the FAS promoter. We show that the -332 and -65 E-boxes are bound by USF in both fasted and refed mice, while the -150 SRE is bound by SREBP-1 only in refed mice. We also found that mutation of either the -150 SRE or the -65 E-box abolishes the feeding-induced activation of the FAS promoter in transgenic mice. Furthermore, in vivo occupancy of the FAS promoter by SREBP in the fed state can be prevented by mutation not only of the -150 SRE but, unexpectedly, of the -65 E-box as well. We conclude that the FAS promoter is activated during refeeding via the induced binding of SREBP to the -150 SRE and that USF binding to the -65 E-box is also required for SREBP binding and activation of the FAS promoter.

Silencing of the mouse H-rev107 gene encoding a class II tumor suppressor by CpG methylation.

H-rev107 is a tumor suppressor originally isolated in revertants of H-ras-transformed cell lines. The gene is ubiquitously expressed in normal tissues but down-regulated in primary carcinomas or in many cell lines derived from tumors, including WEHI 7.1 lymphoma cells. Here, we show that unlike in H-rev107-expressing cells or tissues the 5'-end of H-rev107 containing a CpG-rich region of 421 bp is highly methylated in WEHI 7.1 lymphoma cells, correlating with silencing of this gene. Repression of H-rev107 transcription in these cells could be relieved by chemically induced hypomethylation with 5-aza-dC. In addition, upon in vitro methylation, expression of the luciferase reporter gene driven by the H-rev107 promoter decreased by 80% in WEHI 7.1 and 293 cells. Furthermore, co-transfection of the methyl-CpG binding proteins, MeCP2 and MBD2, inhibited H-rev107 promoter activity up to 70% in SL2 cells when the promoter was methylated. By chromatin immunoprecipitation assays, we observed in vivo binding of MeCP2 and MBD2 to the 5'-end of H-rev107 in WEHI 7.1 cells, which was reduced to undetectable levels upon 5-aza-dC treatment, concluding that MeCP2 and MBD2 might be involved in silencing the methylated H-rev107 gene in lymphoma cells and probably certain tumors.

Murine H-rev107 gene encoding a class II tumor suppressor: gene organization and identification of an Sp1/Sp3-binding GC-box required for its transcription.

H-rev107, which belongs to class II tumor suppressor genes, is ubiquitously expressed in normal cells, but is downregulated in many carcinomas and tumor cell lines. Sequence analysis showed that the murine H-rev107 gene is composed of five exons and four introns. Transfections revealed that 7.6 kb of the H-rev107 promoter directed a high level expression of the reporter gene. There were no significant differences in promoter activity when various 5'-deletion promoter constructs from -7.6 kb to -113 bp were employed. By further deletion and mutation analysis, we found that the region between -83 and -75 containing a GC-box was essential for promoter activity in NIH3T3 or REF52 fibroblasts expressing H-rev107 at moderate to high levels. Gelshifts demonstrated in vitro binding of Sp1 and Sp3 to this GC-box. Cotransfection of Sp1 and Sp3 functionally stimulated promoter activity in SL2 cells. By chromatin immunoprecipitation assays, we observed in vivo binding of Sp1 and Sp3 to the proximal promoter region in NIH3T3 cells and liver, concluding that the transcription of the H-rev107 gene is dependent on Sp1/Sp3-binding to the -83/-75 GC-box.

Suppression of fatty acid synthase promoter by polyunsaturated fatty acids.

Dietary polyunsaturated fat is known to suppress expression of fatty acid synthase (FAS), a central enzyme in de novo lipogenesis. The sterol regulatory element-binding protein (SREBP) has recently been shown to be involved in this suppression. We previously reported that the first 2.1 kb of the FAS promoter are sufficient for transcriptional induction by a high carbohydrate diet as well as suppression by polyunsaturated fat in transgenic mice. Here, we first examined the DNA sequences responsible for SREBP-mediated suppression of FAS promoter activity by polyunsaturated fatty acids (PUFA) in vivo. Feeding polyunsaturated fat prevented both the low-level activation of the -278 FAS promoter which contains the -150 sterol response element (SRE), as well as the maximal activation of the longer -444 FAS promoter. We observed that ectopic expression of the activated form of SREBP in liver prevented PUFA-mediated suppression of both the endogenous FAS and FAS promoter-reporter transgene expression. We also found that the promoter region required for PUFA suppression in vivo is located between -278 to -131, where SREBP functions. Using HepG2 cells, we further examined the specific FAS promoter elements required for PUFA suppression. We found that the -150 SRE, as well as the -65 E-Box, contribute to PUFA suppression of the FAS promoter, at least in vitro.