Role of microRNA-709 in murine liver

Indiana University-Purdue University Indianapolis (IUPUI)MicroRNAs are small RNA molecules that regulate expression of genes involved in development, cell differentiation, proliferation and death. It has been estimated that in eukaryotes, approximately 0.5 to 1% of predicted genes encode a microRNA, which in humans, regulate at least 30% of genes at an average of 200 genes per miRNA. Some microRNAs are tissue-specific, while others are ubiquitously expressed. In liver, a few microRNAs have been identified that regulate specialized functions. The best known is miR-122, the most abundant liver-specific miRNA, which regulates cholesterol biosynthesis and other genes of fatty acid metabolism; it also regulates the cell cycle through inhibition of cyclin G1. To discover other miRNAs with relevant function in liver, we characterized miRNA profiles in normal tissue and identified miR-709. Our data indicates this is a highly abundant hepatic miRNA and is dysregulated in an animal model of type 2 diabetes. To understand its biological role, miR-709 gene targets were identified by analyzing the transcriptome of primary hepatocytes transfected with a miR-709 mimic. The genes identified fell within four main categories: cytoskeleton binding, extracellular matrix attachment, endosomal recycling and fatty acid metabolism. Thus, similar to miR-122, miR-709 downregulates genes from multiple pathways. This would be predicted, given the abundance of the miRNA and the fact that the estimated number of genes targeted by a miRNA is in the hundreds. In the case of miR-709, these suggested a coordinated response during cell proliferation, when cytoskeleton remodeling requires substantial changes in gene expression. Consistently, miR-709 was found significantly upregulated in an animal model of hepatocellular carcinoma. Likewise, in a mouse model of liver regeneration, mature miR-709 was increased. To study the consequences of depleting miR-709 in quiescent and proliferating cells, primary hepatocytes and hepatoma cells were cultured with antagomiRs (anti-miRs). The presence of anti-miR-709 caused cell death in proliferating cells. Quiescent primary hepatocytes responded by upregulating miR-709 and its host gene, Rfx1. These studies show that miR-709 targets genes relevant to cystokeleton structural genes. Thus, miR-709 and Rfx1 may be needed to facilitate cytoskeleton reorganization, a process that occurs after liver injury and repopulation, or during tumorigenesis

Acetylation of Replication Protein A (RPA) Improves its DNA Binding Property

poster abstractGenome maintenance is critical for cellular survival and growth. Replication Protein A (RPA), a single-strand DNA (ssDNA) binding protein, is vital for various aspects of genome maintenance such as replication, recombination, repair and checkpoint activation. RPA binding to ssDNA protects it from degradation by cellular nucleases, prevents secondary structure formation and from illegitimate recombination. Within the cell, RPA is subject to many post-translational modifications including phosphorylation, SUMOylation and ribosylation. These modifications regulate the activity of RPA with DNA and other binding partners. RPA has been reported to be also modified by acetylation. We found that human RPA (hRPA) can be in vitro acetylated by p300, an acetyl transferase (AT). To study the effect of this modification on its ssDNA binding function, we made use of electro-mobility gel shift assay (EMSA) and bio-layer interferometry (BLI) technology. Using various length oligos, we tested the binding property of unmodified and acetylated RPA. Our results showed that acetylation of RPA increased its binding affinity compared to unmodified RPA. Interestingly, the acetylated form was also able to bind more stably to shorter length oligos compared to the unmodified form. This suggests that the acetylation of RPA improves its ssDNA binding function. This alteration in its enzymatic activity would have significant implications in maintenance of genome fidelity since improved DNA binding function of RPA will protect the genome from both endogenous and exogenous stresses. Additionally, using mass spectrometry analysis we have identified the lysine residues that get modified by the acetyl group both in vitro and in vivo. We are currently studying the factors that trigger this post-translational modification in the cell

Lack of liver glycogen causes hepatic insulin resistance and steatosis in mice

Disruption of the Gys2 gene encoding the liver isoform of glycogen synthase generates a mouse strain (LGSKO) that almost completely lacks hepatic glycogen, has impaired glucose disposal, and is pre-disposed to entering the fasted state. This study investigated how the lack of liver glycogen increases fat accumulation and the development of liver insulin resistance. Insulin signaling in LGSKO mice was reduced in liver, but not muscle, suggesting an organ-specific defect. Phosphorylation of components of the hepatic insulin-signaling pathway, namely IRS1, Akt, and GSK3, was decreased in LGSKO mice. Moreover, insulin stimulation of their phosphorylation was significantly suppressed, both temporally and in an insulin dose response. Phosphorylation of the insulin-regulated transcription factor FoxO1 was somewhat reduced and insulin treatment did not elicit normal translocation of FoxO1 out of the nucleus. Fat overaccumulated in LGSKO livers, showing an aberrant distribution in the acinus, an increase not explained by a reduction in hepatic triglyceride export. Rather, when administered orally to fasted mice, glucose was directed toward hepatic lipogenesis as judged by the activity, protein levels, and expression of several fatty acid synthesis genes, namely, acetyl-CoA carboxylase, fatty acid synthase, SREBP1c, chREBP, glucokinase, and pyruvate kinase. Furthermore, using cultured primary hepatocytes, we found that lipogenesis was increased by 40% in LGSKO cells compared with controls. Of note, the hepatic insulin resistance was not associated with increased levels of pro-inflammatory markers. Our results suggest that loss of liver glycogen synthesis diverts glucose toward fat synthesis, correlating with impaired hepatic insulin signaling and glucose disposal

Gene targets of mouse miR-709: regulation of distinct pools

MicroRNA (miRNA) are short non-coding RNA molecules that regulate multiple cellular processes, including development, cell differentiation, proliferation and death. Nevertheless, little is known on whether miRNA control the same gene networks in different tissues. miR-709 is an abundant miRNA expressed ubiquitously. Through transcriptome analysis, we have identified targets of miR-709 in hepatocytes. miR-709 represses genes implicated in cytoskeleton organization, extracellular matrix attachment, and fatty acid metabolism. Remarkably, none of the previously identified targets in non-hepatic tissues are silenced by miR-709 in hepatocytes, even though several of these genes are abundantly expressed in liver. In addition, miR-709 is upregulated in hepatocellular carcinoma, suggesting it participates in the genetic reprogramming that takes place during cell division, when cytoskeleton remodeling requires substantial changes in gene expression. In summary, the present study shows that miR-709 does not repress the same pool of genes in separate cell types. These results underscore the need for validating gene targets in every tissue a miRNA is expressed

Role of microRNA-709 in murine liver

Indiana University-Purdue University Indianapolis (IUPUI)MicroRNAs are small RNA molecules that regulate expression of genes involved in development, cell differentiation, proliferation and death. It has been estimated that in eukaryotes, approximately 0.5 to 1% of predicted genes encode a microRNA, which in humans, regulate at least 30% of genes at an average of 200 genes per miRNA. Some microRNAs are tissue-specific, while others are ubiquitously expressed. In liver, a few microRNAs have been identified that regulate specialized functions. The best known is miR-122, the most abundant liver-specific miRNA, which regulates cholesterol biosynthesis and other genes of fatty acid metabolism; it also regulates the cell cycle through inhibition of cyclin G1. To discover other miRNAs with relevant function in liver, we characterized miRNA profiles in normal tissue and identified miR-709. Our data indicates this is a highly abundant hepatic miRNA and is dysregulated in an animal model of type 2 diabetes. To understand its biological role, miR-709 gene targets were identified by analyzing the transcriptome of primary hepatocytes transfected with a miR-709 mimic. The genes identified fell within four main categories: cytoskeleton binding, extracellular matrix attachment, endosomal recycling and fatty acid metabolism. Thus, similar to miR-122, miR-709 downregulates genes from multiple pathways. This would be predicted, given the abundance of the miRNA and the fact that the estimated number of genes targeted by a miRNA is in the hundreds. In the case of miR-709, these suggested a coordinated response during cell proliferation, when cytoskeleton remodeling requires substantial changes in gene expression. Consistently, miR-709 was found significantly upregulated in an animal model of hepatocellular carcinoma. Likewise, in a mouse model of liver regeneration, mature miR-709 was increased. To study the consequences of depleting miR-709 in quiescent and proliferating cells, primary hepatocytes and hepatoma cells were cultured with antagomiRs (anti-miRs). The presence of anti-miR-709 caused cell death in proliferating cells. Quiescent primary hepatocytes responded by upregulating miR-709 and its host gene, Rfx1. These studies show that miR-709 targets genes relevant to cystokeleton structural genes. Thus, miR-709 and Rfx1 may be needed to facilitate cytoskeleton reorganization, a process that occurs after liver injury and repopulation, or during tumorigenesis

Sphaeromyxa cornuti n. sp., a New Species of Myxosporean Infecting the Gallbladder of the Moorish Idol, Zanclus cornutus (Linnaeus, 1758) from Lakshadweep Waters

Purpose: The present study describes a new species of myxosporean, Sphaeromyxa cornuti n. sp. infecting the gallbladder of the Moorish idol, Zanclus cornutus (Linnaeus 1758) collected from Lakshadweep waters of the Arabian Sea. Methods: Fish were collected using traps and cages. The morphology of mature spores recovered from the gallbladder of Z. cornutus was studied under Nomarski Differential Interference Contrast (DIC) optics. The molecular and phylogenetic analyses were based on SSU rDNA. Results: Sphaeromyxa cornuti n. sp. is characterized by arcuate myxospores with tapering extremities and round ends in valvular, and slightly sigmoid in sutural views (19.2–24.7 µm × 4.1–5.7 µm). The two polar capsules are unequally elongate-ovoid in shape and positioned at opposite ends of the spore (6.2–9.7 µm × 1.7–2.6 µm). Each encloses an irregularly folded, ribbon-like polar tubule, which is oriented parallel to polar capsule axis. In molecular and phylogenetic analyses, the present myxosporean revealed significant differences with related forms and clustered together with S. hellandi within the ‘incurvata’ group of the Sphaeromyxa clade with high nodal support. Conclusions: Morphological, morphometric, molecular and phylogenetic differences between our material and previously described species of Sphaeromyxa, along with host and geographic variations indicate that the present myxosporean is unique and the name Sphaeromyxa cornuti n. sp. is proposed. This forms the first report of a myxosporean parasite-infecting Z. cornutus

Morphological and molecular characterization of Ceratomyxa xanthopteri n. sp. (Myxosporea: Ceratomyxidae) from the marine ornamental fish Acanthurus xanthopterus Valenciennes 1835 (Acanthuridae) off Vizhinjam coast, Kerala

A new species of Ceratomyxa infecting the gallbladder of the marine ornamental fish Acanthurus xanthopterus collected from the Vizhinjam coast of Kerala is described. The parasite exhibited a prevalence of 100%. Mature spores recovered from the gallbladder were slightly crescentic with rounded lateral extremities and possessed convex anterior and slightly concave to straight posterior margins. Spore valves two, equal, joined by a straight and prominent suture. Myxospores measured 5.5 ± 0.6 μm in length and 15.9 ± 2.3 μm in thickness. Polar capsules two, equal, spherical, positioned anteriorly on either sides of the suture, 2.3 ± 0.2 μm long and 2.2 ± 0.2 μm wide. Polar filament with four to five coils, 21.2 ± 0.6 μm when extruded. Posterior angle 173.6 ± 5.2°. Early sporogonic stages and monosporic, disporic, and multisporic plasmodial stages were spherical to irregular in shape, with or without filopodia. Histopathologic analysis revealed that spores and developing stages were attached to the gallbladder wall as well as found free in the lumen. Morphologic and morphometric comparison of the present parasite with known species of Ceratomyxa indicated significant differences. In molecular and phylogenetic analyses, the present myxosporean revealed high divergence with related forms and occupied an independent position within the Ceratomyxa clade with high nodal support. Considering the morphological, morphometric, molecular, and phylogenetic dissimilarities with the previously described species of Ceratomyxa and the differences in host and geographic locations, the present species of myxosporean is treated as new and is named Ceratomyxa xanthopteri n. sp

Impact of silencing hepatic SREBP-1 on insulin signaling.

Sterol Regulatory Element Binding Protein-1 (SREBP-1) is a conserved transcription factor of the basic helix-loop-helix leucine zipper family (bHLH-Zip) that plays a central role in regulating expression of genes of carbohydrate and fatty acid metabolism in the liver. SREBP-1 activity is essential for the control of insulin-induced anabolic processes during the fed state. In addition, SREBP-1 regulates expression of key molecules in the insulin signaling pathway, including insulin receptor substrate 2 (IRS2) and a subunit of the phosphatidylinositol 3-kinase (PI3K) complex, PIK3R3, suggesting that feedback mechanisms exist between SREBP-1 and this pathway. Nevertheless, the overall contribution of SREBP-1 activity to maintain insulin signal transduction is unknown. Furthermore, Akt is a known activator of mTORC1, a sensor of energy availability that plays a fundamental role in metabolism, cellular growth and survival. We have silenced SREBP-1 and explored the impact on insulin signaling and mTOR in mice under fed, fasted and refed conditions. No alterations in circulating levels of insulin were observed. The studies revealed that depletion of SREBP-1 had no impact on IRS1Y612, AktS473, and downstream effectors GSK3αS21 and FoxO1S256 during the fed state. Nevertheless, reduced levels of these molecules were observed under fasting conditions. These effects were not associated with changes in phosphorylation of mTOR. Overall, our data indicate that the contribution of SREBP-1 to maintain insulin signal transduction in liver is modest

SREBP-1 expression increases insulin signaling in primary hepatocytes.

<p><b>(A)</b> Mouse primary hepatocytes were cultured in DMEM containing 5 mM glucose, 10% FBS and 100 IU/ml penicillin/100 μg/ml streptomycin, 100 nM dexamethasone. Cells were transduced with an adenovirus expressing SREBP-1c or a control vector (Null) at the multiplicity of infection (MOI) indicated on the top. Cells were harvested 48 hours later. IRS2 levels decreased while ACACA/B increased in hepatocytes treated with SREBP-1c, as expected. <b>(B)</b> Insulin signaling in primary hepatocytes transduced with an adenovirus expressing the mature form of human SREBP-1c or a control vector (Null) at multiplicity of infection (MOI) 20, 40, or 100. Data are representative of 2 separate experiments.</p