Mark Ibberson

Endogenous RNAs Modulate MicroRNA Sorting to Exosomes and Transfer to Acceptor Cells

MicroRNA (miRNA) transfer via exosomes may mediate cell-to-cell communication. Interestingly, specific miRNAs are enriched in exosomes in a cell-type-dependent fashion. However, the mechanisms whereby miRNAs are sorted to exosomes and the significance of miRNA transfer to acceptor cells are unclear. We used macrophages and endothelial cells (ECs) as a model of heterotypic cell communication in order to investigate both processes. RNA profiling of macrophages and their exosomes shows that miRNA sorting to exosomes is modulated by cell-activation-dependent changes of miRNA target levels in the producer cells. Genetically perturbing the expression of individual miRNAs or their targeted transcripts promotes bidirectional miRNA relocation from the cell cytoplasm/P bodies (sites of miRNA activity) to multivesicular bodies (sites of exosome biogenesis) and controls miRNA sorting to exosomes. Furthermore, the use of Dicer-deficient cells and reporter lentiviral vectors (LVs) for miRNA activity shows that exosomal miRNAs are transferred from macrophages to ECs to detectably repress targeted sequences.

Gluco-incretins control insulin secretion at multiple levels as revealed in mice lacking GLP-1 and GIP receptors

The role of the gluco-incretin hormones GIP and GLP-1 in the control of beta cell function was studied by analyzing mice with inactivation of each of these hormone receptor genes, or both. Our results demonstrate that glucose intolerance was additively increased during oral glucose absorption when both receptors were inactivated. After intraperitoneal injections, glucose intolerance was more severe in double- as compared to single-receptor KO mice, and euglycemic clamps revealed normal insulin sensitivity, suggesting a defect in insulin secretion. When assessed in vivo or in perfused pancreas, insulin secretion showed a lack of first phase in Glp-1R(-/-) but not in Gipr(-/-) mice. In perifusion experiments, however, first-phase insulin secretion was present in both types of islets. In double-KO islets, kinetics of insulin secretion was normal, but its amplitude was reduced by about 50% because of a defect distal to plasma membrane depolarization. Thus, gluco-incretin hormones control insulin secretion (a) by an acute insulinotropic effect on beta cells after oral glucose absorption (b) through the regulation, by GLP-1, of in vivo first-phase insulin secretion, probably by an action on extra-islet glucose sensors, and (c) by preserving the function of the secretory pathway, as evidenced by a beta cell autonomous secretion defect when both receptors are inactivated.

GLUT2 is a high affinity glucosamine transporter

When expressed in Xenopus oocytes, GLUT1, 2 and 4 transport glucosamine with V max values that are three‐ to four‐fold lower than for glucose. The K ms for glucosamine and glucose of GLUT1 and GLUT4 were similar. In contrast, GLUT2 had a much higher apparent affinity for glucosamine than for glucose (K m=0.8±0.1 mM vs. ∼17–20 mM). Glucosamine transport by GLUT2 was confirmed in mammalian cells and, using hepatocytes from control or GLUT2‐null mice, HgCl2‐inhibitable glucosamine uptake by liver was shown to be exclusively through GLUT2. These data have implications for glucosamine effects on impaired glucose metabolism and for structure–function studies of transporter sugar binding sites.

Identification of a mammalian H+-myo-inositol symporter expressed predominantly in the brain

Inositol and its phosphorylated derivatives play a major role in brain function, either as osmolytes, second messengers or regulators of vesicle endo‐ and exocytosis. Here we describe the identification and functional characterization of a novel H+‐myo‐ inositol co‐transporter, HMIT, expressed predominantly in the brain. HMIT cDNA encodes a 618 amino acid polypeptide with 12 predicted transmembrane domains. Functional expression of HMIT in Xenopus oocytes showed that transport activity was specific for myo‐inositol and related stereoisomers with a Michaelis–Menten constant of ∼100 μM, and that transport activity was strongly stimulated by decreasing pH. Electrophysiological measurements revealed that transport was electrogenic with a maximal transport activity reached at pH 5.0. In rat brain membrane preparations, HMIT appeared as a 75–90 kDa protein that could be converted to a 67 kDa band upon enzymatic deglycosylation. Immunofluorescence microscopy analysis showed HMIT expression in glial cells and some neurons. These data provide the first characterization of a mammalian H+‐coupled myo‐ inositol transporter. Predominant central expression of HMIT suggests that it has a key role in the control of myo‐inositol brain metabolism.

Genome-wide profiling of the cardiac transcriptome after myocardial infarction identifies novel heart-specific long non-coding RNAs

Aim Heart disease is recognized as a consequence of dysregulation of cardiac gene regulatory networks. Previously, unappreciated components of such networks are the long non-coding RNAs (lncRNAs). Their roles in the heart remain to be elucidated. Thus, this study aimed to systematically characterize the cardiac long non-coding transcriptome post-myocardial infarction and to elucidate their potential roles in cardiac homoeostasis. Methods and results We annotated the mouse transcriptome after myocardial infarction via RNA sequencing and ab initio transcript reconstruction, and integrated genome-wide approaches to associate specific lncRNAs with developmental processes and physiological parameters. Expression of specific lncRNAs strongly correlated with defined parameters of cardiac dimensions and function. Using chromatin maps to infer lncRNA function, we identified many with potential roles in cardiogenesis and pathological remodelling. The vast majority was associated with active cardiac-specific enhancers. Importantly, oligonucleotide-mediated knockdown implicated novel lncRNAs in controlling expression of key regulatory proteins involved in cardiogenesis. Finally, we identified hundreds of human orthologues and demonstrate that particular candidates were differentially modulated in human heart disease. Conclusion These findings reveal hundreds of novel heart-specific lncRNAs with unique regulatory and functional characteristics relevant to maladaptive remodelling, cardiac function and possibly cardiac regeneration. This new class of molecules represents potential therapeutic targets for cardiac disease. Furthermore, their exquisite correlation with cardiac physiology renders them attractive candidate biomarkers to be used in the clinic.

Immunolocalization of GLUTX1 in the Testis and to Specific Brain Areas and Vasopressin- Containing Neurons

GLUTX1 or GLUT8 is a newly characterized glucose transporter isoform that is expressed at high levels in the testis and brain and at lower levels in several other tissues. Its expression was mapped in the testis and brain by using specific antibodies. In the testis, immunoreactivity was expressed in differentiating spermatocytes of type 1 stage but undetectable in mature spermatozoa. In the brain, GLUTX1 distribution was selective and localized to a variety of structures, mainly archi- and paleocortex. It was found in hippocampal and dentate gyrus neurons as well as amygdala and primary olfactory cortex. In these neurons, its location was close to the plasma membrane of cell bodies and sometimes in proximal dendrites. High GLUTX1 levels were detected in the hypothalamus, supraoptic nucleus, median eminence, and the posterior pituitary. Neurons of these areas synthesize and secrete vasopressin and oxytocin. As shown by double immunofluorescence microscopy and immunogold labeling, GLUTX1 was expressed only ...

Peroxisomal and microsomal lipid pathways associated with resistance to hepatic steatosis and reduced pro-inflammatory state

Accumulation of fat in the liver increases the risk to develop fibrosis and cirrhosis and is associated with development of the metabolic syndrome. Here, to identify genes or gene pathways that may underlie the genetic susceptibility to fat accumulation in liver, we studied A/J and C57Bl/6 mice that are resistant and sensitive to diet-induced hepatosteatosis and obesity, respectively. We performed comparative transcriptomic and lipidomic analysis of the livers of both strains of mice fed a high fat diet for 2, 10, and 30 days. We found that resistance to steatosis in A/J mice was associated with the following: (i) a coordinated up-regulation of 10 genes controlling peroxisome biogenesis and β-oxidation; (ii) an increased expression of the elongase Elovl5 and desaturases Fads1 and Fads2. In agreement with these observations, peroxisomal β-oxidation was increased in livers of A/J mice, and lipidomic analysis showed increased concentrations of long chain fatty acid-containing triglycerides, arachidonic acid-containing lysophosphatidylcholine, and 2-arachidonylglycerol, a cannabinoid receptor agonist. We found that the anti-inflammatory CB2 receptor was the main hepatic cannabinoid receptor, which was highly expressed in Kupffer cells. We further found that A/J mice had a lower pro-inflammatory state as determined by lower plasma levels and IL-1β and granulocyte-CSF and reduced hepatic expression of their mRNAs, which were found only in Kupffer cells. This suggests that increased 2-arachidonylglycerol production may limit Kupffer cell activity. Collectively, our data suggest that genetic variations in the expression of peroxisomal β-oxidation genes and of genes controlling the production of an anti-inflammatory lipid may underlie the differential susceptibility to diet-induced hepatic steatosis and pro-inflammatory state.

Network-guided analysis of genes with altered somatic copy number and gene expression reveals pathways commonly perturbed in metastatic melanoma.

Cancer genomes frequently contain somatic copy number alterations (SCNA) that can significantly perturb the expression level of affected genes and thus disrupt pathways controlling normal growth. In melanoma, many studies have focussed on the copy number and gene expression levels of the BRAF, PTEN and MITF genes, but little has been done to identify new genes using these parameters at the genome-wide scale. Using karyotyping, SNP and CGH arrays, and RNA-seq, we have identified SCNA affecting gene expression (‘SCNA-genes’) in seven human metastatic melanoma cell lines. We showed that the combination of these techniques is useful to identify candidate genes potentially involved in tumorigenesis. Since few of these alterations were recurrent across our samples, we used a protein network-guided approach to determine whether any pathways were enriched in SCNA-genes in one or more samples. From this unbiased genome-wide analysis, we identified 28 significantly enriched pathway modules. Comparison with two large, independent melanoma SCNA datasets showed less than 10% overlap at the individual gene level, but network-guided analysis revealed 66% shared pathways, including all but three of the pathways identified in our data. Frequently altered pathways included WNT, cadherin signalling, angiogenesis and melanogenesis. Additionally, our results emphasize the potential of the EPHA3 and FRS2 gene products, involved in angiogenesis and migration, as possible therapeutic targets in melanoma. Our study demonstrates the utility of network-guided approaches, for both large and small datasets, to identify pathways recurrently perturbed in cancer.

LKB1 and AMPK differentially regulate pancreatic β-cell identity

Fully differentiated pancreatic β cells are essential for normal glucose homeostasis in mammals. Dedifferentiation of these cells has been suggested to occur in type 2 diabetes, impairing insulin production. Since chronic fuel excess (“glucotoxicity”) is implicated in this process, we sought here to identify the potential roles in β‐cell identity of the tumor suppressor liver kinase B1 (LKB1/STK11) and the downstream fuel‐sensitive kinase, AMP‐activated protein kinase (AMPK). Highly β‐cell‐restricted deletion of each kinase in mice, using an Ins1‐controlled Cre, was therefore followed by physiological, morphometric, and massive parallel sequencing analysis. Loss of LKB1 strikingly (2.0‐12‐fold, E<0.01) increased the expression of subsets of hepatic (Alb, Iyd, Elovl2) and neuronal (Nptx2, Dlgap2, Cartpt, Pdyn) genes, enhancing glutamate signaling. These changes were partially recapitulated by the loss of AMPK, which also up‐regulated β‐cell “disallowed” genes (Slc16a1, Ldha, Mgst1, Pdgfra) 1.8‐ to 3.4‐fold (E<0.01). Correspondingly, targeted promoters were enriched for neuronal (Zfp206; P= 1.3×10‐33) and hypoxia‐regulated (HIF1; P= 2.5×10‐16) transcription factors. In summary, LKB1 and AMPK, through only partly overlapping mechanisms, maintain β‐cell identity by suppressing alternate pathways leading to neuronal, hepatic, and other characteristics. Selective targeting of these enzymes may provide a new approach to maintaining β‐cell function in some forms of diabetes.—Kone, M., Pullen, T. J., Sun, G., Ibberson, M., Martinez‐Sanchez, A., Sayers, S., Nguyen‐Tu, M.‐S., Kantor, C., Swisa, A., Dor, Y., Gorman, T., Ferrer, J., Thorens, B., Reimann, F., Gribble, F., McGinty, J. A., Chen, L., French, P. M., Birzele, F., Hildebrandt, T., Uphues, I., Rutter, G. A., LKB1 and AMPK differentially regulate pancreatic β‐cell identity. FASEB J. 28, 4972–4985 (2014). www.fasebj.org

Discovery and functional characterization of cardiovascular long noncoding RNAs

Recent advances in sequencing and genomic technologies have resulted in the discovery of thousands of previously unannotated long noncoding RNAs (lncRNAs). However, their function in the cardiovascular system remains elusive. Here we review and discuss considerations for cardiovascular lncRNA discovery, annotation and functional characterization. Although we primarily focus on the heart, the proposed pipeline should foster functional and mechanistic exploration of these transcripts in various cardiovascular pathologies. Moreover, these insights could ultimately lead to novel therapeutic approaches targeting lncRNAs for the amelioration of cardiovascular diseases including heart failure.