24 results found
Nguyen-Tu M-S, Martinez-Sanchez A, Leclerc I, et al., 2020, Adipocyte-specific deletion of Tcf7l2 induces dysregulated lipid metabolism and impairs glucose tolerance in mice, DIABETOLOGIA, Vol: 64, Pages: 129-141, ISSN: 0012-186X
Rutter GA, Georgiadou E, Martinez-Sanchez A, et al., 2020, Metabolic and functional specialisations of the pancreatic beta cell: gene disallowance, mitochondrial metabolism and intercellular connectivity, DIABETOLOGIA, Vol: 63, Pages: 1990-1998, ISSN: 0012-186X
Martinez-Sanchez A, Lazzarano S, Sharma E, et al., 2020, High-Throughput Identification of MiR-145 Targets in Human Articular Chondrocytes, LIFE-BASEL, Vol: 10
Georgiadou E, Haythorne E, Dickerson MT, et al., 2020, The pore-forming subunit MCU of the mitochondrial Ca2+ uniporter is required for normal glucose-stimulated insulin secretion in vitro and in vivo in mice, Diabetologia, Vol: 63, Pages: 1368-1381, ISSN: 0012-186X
Aims/hypothesisMitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca2+ uptake into pancreatic beta cell mitochondria potentiates or antagonises this process is still a matter of debate. Although the mitochondrial Ca2+ importer (MCU) complex is thought to represent the main route for Ca2+ transport across the inner mitochondrial membrane, its role in beta cells has not previously been examined in vivo.MethodsHere, we inactivated the pore-forming subunit of the MCU, encoded by Mcu, selectively in mouse beta cells using Ins1Cre-mediated recombination. Whole or dissociated pancreatic islets were isolated and used for live beta cell fluorescence imaging of cytosolic or mitochondrial Ca2+ concentration and ATP production in response to increasing glucose concentrations. Electrophysiological recordings were also performed on whole islets. Serum and blood samples were collected to examine oral and i.p. glucose tolerance.ResultsGlucose-stimulated mitochondrial Ca2+ accumulation (p< 0.05), ATP production (p< 0.05) and insulin secretion (p< 0.01) were strongly inhibited in beta cell-specific Mcu-null (βMcu-KO) animals, in vitro, as compared with wild-type (WT) mice. Interestingly, cytosolic Ca2+ concentrations increased (p< 0.001), whereas mitochondrial membrane depolarisation improved in βMcu-KO animals. βMcu-KO mice displayed impaired in vivo insulin secretion at 5 min (p< 0.001) but not 15 min post-i.p. injection of glucose, whilst the opposite phenomenon was observed following an oral gavage at 5 min. Unexpectedly, glucose tolerance was improved (p< 0.05) in young βMcu-KO (<12 weeks), but not in older animals vs WT mice.Conclusions/interpretationMCU is crucial for mitochondrial Ca2+ uptake in pancreatic beta cells and is required for normal GSIS. The apparent compensatory mechanisms that maintain glucose tolerance in βMcu-KO mice remain
Clough TJ, Baxan N, Coakley EJ, et al., 2020, Synthesis and in vivo behaviour of an exendin-4-based MRI probe capable of beta-cell-dependent contrast enhancement in the pancreas, Dalton Transactions: an international journal of inorganic chemistry, Vol: 49, Pages: 4732-4740, ISSN: 1477-9226
Global rates of diabetes mellitus are increasing, and treatment of the disease consumes a growing proportion of healthcare spending across the world. Pancreatic β-cells, responsible for insulin production, decline in mass in type 1 and, to a more limited degree, in type 2 diabetes. However, the extent and rate of loss in both diseases differs between patients resulting in the need for the development of novel diagnostic tools, which could quantitatively assess changes in mass of β-cells over time and potentially lead to earlier diagnosis and improved treatments. Exendin-4, a potent analogue of glucagon-like-peptide 1 (GLP-1), binds to the receptor GLP-1R, whose expression is enriched in β-cells. GLP-1R has thus been used in the past as a means of targeting probes for a wide variety of imaging modalities to the endocrine pancreas. However, exendin-4 conjugates designed specifically for MRI contrast agents are an under-explored area. In the present work, the synthesis and characterization of an exendin-4-dota(ga)-Gd(III) complex, GdEx, is reported, along with its in vivo behaviour in healthy and in β-cell-depleted C57BL/6J mice. Compared to the ubiquitous probe, [Gd(dota)]−, GdEx shows selective uptake by the pancreas with a marked decrease in accumulation observed after the loss of β-cells elicited by deleting the microRNA processing enzyme, DICER. These results open up pathways towards the development of other targeted MRI contrast agents based on similar chemistry methodology.
Cheung R, Pizza G, Rolando DM, et al., 2019, miR-125b Is Regulated by Glucose via AMPK and Impairs beta-Cell Function, 79th Scientific Sessions of the American-Diabetes-Association (ADA), Publisher: AMER DIABETES ASSOC, ISSN: 0012-1797
Martinez-Sanchez A, Nguyen-Tu M-S, Cebola I, et al., 2018, MiR-184 expression is regulated by AMPK in pancreatic islets., FASEB Journal, Vol: 32, Pages: 2587-2600, ISSN: 0892-6638
AMPK is a critical energy sensor and target for widely used antidiabetic drugs. In β-cells, elevated glucose concentrations lower AMPK activity, and the ablation of both catalytic subunits (βAMPKdKO mice) impairs insulin secretion in vivo and β-cell identity. MicroRNAs (miRNAs) are small RNAs that silence gene expression that are essential for pancreatic β-cell function and identity and altered in diabetes. Here, we have explored the miRNAs acting downstream of AMPK in mouse and human β-cells. We identified 14 down-regulated and 9 up-regulated miRNAs in βAMPKdKO vs. control islets. Gene ontology analysis of targeted transcripts revealed enrichment in pathways important for β-cell function and identity. The most down-regulated miRNA was miR-184 (miR-184-3p), an important regulator of β-cell function and compensatory expansion that is controlled by glucose and reduced in diabetes. We demonstrate that AMPK is a potent regulator and an important mediator of the negative effects of glucose on miR-184 expression. Additionally, we reveal sexual dimorphism in miR-184 expression in mouse and human islets. Collectively, these data demonstrate that glucose-mediated changes in AMPK activity are central for the regulation of miR-184 and other miRNAs in islets and provide a link between energy status and gene expression in β-cells.-Martinez-Sanchez, A., Nguyen-Tu, M.-S., Cebola, I., Yavari, A., Marchetti, P., Piemonti, L., de Koning, E., Shapiro, A. M. J., Johnson, P., Sakamoto, K., Smith, D. M., Leclerc, I., Ashrafian, H., Ferrer, J., Rutter, G. A. MiR-184 expression is regulated by AMPK in pancreatic islets.
Martinez-Sanchez A, Nguyen-Tu M-S, Leclerc I, et al., 2018, Manipulation and Measurement of AMPK Activity in Pancreatic Islets., Methods Mol Biol, Vol: 1732, Pages: 413-431
The role of the energy sensor AMPK-activated protein kinase (AMPK) in the insulin-secreting β-cell remains unclear and a subject of intense research. With this chapter, we aim to provide a detailed description of the methods that our group routinely applies to the study of AMPK function in mouse and human pancreatic islets. Thus, we provide detailed protocols to isolate and/or culture mouse and human islets, to modulate and measure AMPK activity in isolated islets, and to evaluate its impact on islet function.
Martinez-Sanchez A, Shapiro J, Marchetti P, et al., 2017, AMP-Activated Protein Kinase (AMPK) Regulates beta-Cell MicroRNA Expression, 77th Scientific Sessions of the American-Diabetes-Association, Publisher: AMER DIABETES ASSOC, Pages: A29-A29, ISSN: 0012-1797
Martinez-Sanchez A, Rutter GA, Latreille M, 2017, MiRNAs in β-Cell development, identity, and disease., Frontiers in Genetics, Vol: 7, ISSN: 1664-8021
Pancreatic β-cells regulate glucose metabolism by secreting insulin, which in turn stimulates the utilization or storage of the sugar by peripheral tissues. Insulin insufficiency and a prolonged period of insulin resistance are usually the core components of type 2 diabetes (T2D). Although, decreased insulin levels in T2D have long been attributed to a decrease in β-cell function and/or mass, this model has recently been refined with the recognition that a loss of β-cell "identity" and dedifferentiation also contribute to the decline in insulin production. MicroRNAs (miRNAs) are key regulatory molecules that display tissue-specific expression patterns and maintain the differentiated state of somatic cells. During the past few years, great strides have been made in understanding how miRNA circuits impact β-cell identity. Here, we review current knowledge on the role of miRNAs in regulating the acquisition of the β-cell fate during development and in maintaining mature β-cell identity and function during stress situations such as obesity, pregnancy, aging, or diabetes. We also discuss how miRNA function could be harnessed to improve our ability to generate β-cells for replacement therapy for T2D.
Yavari A, Stocker CJ, Ghaffari S, et al., 2016, Chronic Activation of γ2 AMPK Induces Obesity and Reduces β Cell Function., Cell Metabolism, Vol: 23, Pages: 821-836, ISSN: 1932-7420
Despite significant advances in our understanding of the biology determining systemic energy homeostasis, the treatment of obesity remains a medical challenge. Activation of AMP-activated protein kinase (AMPK) has been proposed as an attractive strategy for the treatment of obesity and its complications. AMPK is a conserved, ubiquitously expressed, heterotrimeric serine/threonine kinase whose short-term activation has multiple beneficial metabolic effects. Whether these translate into long-term benefits for obesity and its complications is unknown. Here, we observe that mice with chronic AMPK activation, resulting from mutation of the AMPK γ2 subunit, exhibit ghrelin signaling-dependent hyperphagia, obesity, and impaired pancreatic islet insulin secretion. Humans bearing the homologous mutation manifest a congruent phenotype. Our studies highlight that long-term AMPK activation throughout all tissues can have adverse metabolic consequences, with implications for pharmacological strategies seeking to chronically activate AMPK systemically to treat metabolic disease.
Martinez-Sanchez A, Pullen TJ, Chabosseau PL, et al., 2016, Disallowance of acyl-CoA thioesterase 7 (Acot7) in beta cells is required for normal insulin secretion and glucose tolerance, DIABETIC MEDICINE, Vol: 33, Pages: 38-38, ISSN: 0742-3071
Rutter GA, 2016, Disallowance of Acot7 in b-Cells IsRequired for Normal GlucoseTolerance and Insulin Secretion, Diabetes
Acot7, encoding acyl-CoA thioesterase-7, is one of ∼60 genes expressed ubiquitously across tissues but relatively silenced, or “disallowed”, in pancreatic β-cells. The capacity of ACOT7 to hydrolyse long-chain acyl-CoA esters suggests potential roles in β-oxidation, lipid biosynthesis, signal transduction or insulin exocytosis. Here, we explored the physiological relevance of β-cell-specific Acot7 silencing by re-expressing ACOT7 in these cells. ACOT7 overexpression in clonal MIN6 and INS1(832/13) β-cells impaired insulin secretion in response to glucose plus fatty acids. Furthermore, examined in a panel of transgenic mouse lines, we demonstrate that overexpression of mitochondrial ACOT7 selectively in the adult β-cell reduced glucose tolerance dose-dependently and impaired glucose-stimulated insulin secretion. By contrast, depolarisation-induced secretion was unaffected, arguing against a direct action on the exocytotic machinery. Acyl-CoA levels, ATP/ADP increases, membrane depolarization and Ca2+ fluxes were all markedly reduced in transgenic mouse islets, whereas glucose-induced O2-consumption was unchanged. Whilst glucose-induced increases in ATP/ADP ratio were similarly lowered after ACOT7 over-expression in INS1(832/13) cells, changes in mitochondrial membrane potential (ΔΨ) were unaffected, consistent with an action of Acot7 to increase cellular ATP consumption. Since Acot7 mRNA levels are increased in human islets in type 2 diabetes, inhibition of the enzyme might provide a novel therapeutic strategy.
Seidl CI, Martinez-Sanchez A, Murphy CL, 2016, Derepression of MicroRNA-138 Contributes to Loss of the Human Articular Chondrocyte Phenotype, ARTHRITIS & RHEUMATOLOGY, Vol: 68, Pages: 398-409, ISSN: 2326-5191
Martinez-Sanchez A, Nguyen-Tu M-S, Rutter GA, 2015, DICER Inactivation Identifies Pancreatic β-Cell "Disallowed" Genes Targeted by MicroRNAs., Mol Endocrinol, Vol: 29, Pages: 1067-1079
Pancreatic β-cells are the body's sole source of circulating insulin and essential for the maintenance of blood glucose homeostasis. Levels of up to 66 "disallowed" genes, which are strongly expressed and play housekeeping roles in most other mammalian tissues, are unusually low in β-cells. The molecular mechanisms involved in repressing these genes are largely unknown. Here, we explore the role in gene disallowance of microRNAs (miRNAs), a type of small noncoding RNAs that silence gene expression at the posttranscriptional level and are essential for β-cell development and function. To selectively deplete miRNAs from adult β-cells, the miRNA-processing enzyme DICER was inactivated by deletion of the RNase III domain with a tamoxifen-inducible Pdx1CreER transgene. In this model, β-cell dysfunction was apparent 2 weeks after recombination and preceded a decrease in insulin content and loss of β-cell mass. Of the 14 disallowed genes studied, quantitative RT-quantitative real-time PCR revealed that 6 genes (Fcgrt, Igfbp4, Maf, Oat, Pdgfra, and Slc16a1) were up-regulated (1.4- to 2.1-fold, P < .05) at this early stage. Expression of luciferase constructs bearing the 3'-untranslated regions of the corresponding mRNAs in wild-type or DICER-null β-cells demonstrated that Fcgrt, Oat, and Pdgfra are miRNA direct targets. We thus reveal a role for miRNAs in the regulation of disallowed genes in β-cells and provide evidence for a novel means through which noncoding RNAs control the functional identity of these cells independently of actions on β-cell mass.
Rutter GA, Pullen TJ, Hodson DJ, et al., 2015, Pancreatic beta-cell identity, glucose sensing and the control of insulin secretion, BIOCHEMICAL JOURNAL, Vol: 466, Pages: 203-218, ISSN: 0264-6021
Kone M, Sun G, Ibberson M, et al., 2014, LKB1 and AMPK differentially regulate pancreatic beta-cell identity, Faseb Journal, Vol: 28, Pages: 4972-4985, ISSN: 1530-6860
Fully differentiated pancreatic b cellsare essential for normal glucose homeostasis in mammals.Dedifferentiation of these cells has been suggestedto occur in type 2 diabetes, impairing insulinproduction. Since chronic fuel excess (“glucotoxicity”)is implicated in this process, we sought here to identifythe potential roles in b-cell identity of the tumor suppressorliver kinase B1 (LKB1/STK11) and the downstreamfuel-sensitive kinase, AMP-activated proteinkinase (AMPK). Highly b-cell-restricted deletion ofeach kinase in mice, using an Ins1-controlled Cre, wastherefore followed by physiological, morphometric,and massive parallel sequencing analysis. Loss of LKB1strikingly (2.0–12-fold, E<0.01) increased the expressionof subsets of hepatic (Alb, Iyd, Elovl2) and neuronal(Nptx2, Dlgap2, Cartpt, Pdyn) genes, enhancing glutamatesignaling. These changes were partially recapitulatedby the loss of AMPK, which also up-regulated b-cell“disallowed” genes (Slc16a1, Ldha, Mgst1, Pdgfra) 1.8- to3.4-fold (E<0.01). Correspondingly, targeted promoterswere enriched for neuronal (Zfp206; P51.3310233)and hypoxia-regulated (HIF1; P52.5310216) transcriptionfactors. In summary, LKB1 and AMPK, through onlypartly overlapping mechanisms, maintain b-cell identityby suppressing alternate pathways leading to neuronal,hepatic, and other characteristics. Selective targetingof these enzymes may provide a new approach tomaintaining b-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 differentiallyregulate pancreatic b-cell identity.
Martinez-Sanchez A, Pullen TJ, Rutter GA, 2014, Repression of Acot7 in beta-Cells Is Required for the Potentiation of GSIS by Fatty Acids, Publisher: AMER DIABETES ASSOC, Pages: A552-A552, ISSN: 0012-1797
Martinez-Sanchez A, Murphy CL, 2013, miR-1247 Functions by Targeting Cartilage Transcription Factor SOX9, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 288, Pages: 30802-30814, ISSN: 0021-9258
Martinez-Sanchez A, Murphy C, 2013, MicroRNA Target Identification—Experimental Approaches, Biology, Vol: 2, Pages: 189-205, ISSN: 2079-7737
MicroRNAs (miRNAs) are small non-coding RNA molecules of 21–23 nucleotides that control gene expression at the post-transcriptional level. They have been shown to play a vital role in a wide variety of biological processes and dysregulated expression of miRNAs is observed in many pathologies. Understanding the mechanism of action and identifying functionally important mRNA targets of a specific miRNA are essential to unravelling its biological function and to assist miRNA-based drug development. This review summarizes the current understanding of the mechanistic aspects of miRNA-mediated gene repression and focuses on the different approaches for miRNA target identification that have been proposed in recent years.
Martinez-Sanchez A, Dudek KA, Murphy CL, 2012, Regulation of human chondrocyte function through direct inhibition of cartilage master regulator SOX9 by microRNA-145 (miRNA-145)., J Biol Chem, Vol: 287, Pages: 916-924
Articular cartilage enables weight bearing and near friction-free movement in the joints. Critical to its function is the production of a specialized, mechanocompetent extracellular matrix controlled by master regulator transcription factor SOX9. Mutations in SOX9 cause campomelic dysplasia, a haploinsufficiency disorder resulting in severe skeletal defects and dwarfism. Although much is understood about how SOX9 regulates cartilage matrix synthesis and hence joint function, how this master regulator is itself regulated remains largely unknown. Here we identify a specific microRNA, miR-145, as a direct regulator of SOX9 in normal healthy human articular chondrocytes. We show that miR-145 directly represses SOX9 expression in human cells through a unique binding site in its 3'-UTR not conserved in mice. Modulation of miR-145 induced profound changes in the human chondrocyte phenotype. Specifically, increased miR-145 levels cause greatly reduced expression of critical cartilage extracellular matrix genes (COL2A1 and aggrecan) and tissue-specific microRNAs (miR-675 and miR-140) and increased levels of the hypertrophic markers RUNX2 and MMP13, characteristic of changes occurring in osteoarthritis. We propose miR-145 as an important regulator of human chondrocyte function and a new target for cartilage repair.
Dudek K, Lafont J, Martinez-Sanchez A, et al., 2010, Type II Collagen Expression is Regulated by Tissue-specific miR-675 in Human Articular Chondrocytes, The Journal of Biological Chemistry
MiRNAs have recently been shown to be essential for normal cartilage development in the mouse. However the role of specific miRNAs in cartilage function is unknown. Using rarely available healthy human chondrocytes (obtained from 8 to 50 year olds) we detected a most highly abundant primary miRNA H19 whose expression was heavily dependent on cartilage master regulator SOX9. Across a range of murine tissues expression of both H19 and H19-derived miR-675 mirrored that of cartilage-specific SOX9. MiR-675 was shown to upregulate essential cartilage matrix component COL2A1, and overexpression of miR-675 rescued COL2A1 levels in H19-depleted or SOX9-depleted cells. We thus provide evidence that SOX9 positively regulates COL2A1 in human articular chondrocytes via a previously unreported miR-675-dependent mechanism. This represents a novel pathway regulating cartilage matrix production and identifies miR-675 as a promising new target for cartilage repair.
Cuesta R, Martínez-Sanchez A, Gebauer F, 2009, miR-181a Regulates Cap-Dependent Translation of p27kip1mRNA in Myeloid Cells, MOLECULAR AND CELLULAR BIOLOGY
p27kip1 (p27) is a cell cycle inhibitor and tumor suppressor whose expression is tightly regulated in the cell.Translational control of p27 mRNA has emerged as a prominent mechanism to regulate p27 expression duringdifferentiation, quiescence, and cancer progression. The microRNAs miR-221 and miR-222 repress p27 expressionin various cancer cells, and this repression promotes tumor cell proliferation. In addition, thepresence of an internal ribosome entry site in the 5 untranslated region (UTR) of p27 mRNA has beenreported. Here, we show that p27 mRNA is translated via a cap-dependent mechanism in HeLa and HL60 cellsand that the previously reported IRES activity can be attributed to cryptic promoters in the sequencecorresponding to the p27 5 UTR. Furthermore, cap-dependent translation of p27 mRNA is repressed bymiR-181a in undifferentiated HL60 cells. Repression by miR-181a is relieved during differentiation of HL60into monocyte-like cells, allowing the accumulation of p27, which is necessary to fully block cell cycle progressionand reach terminal differentiation. These results identify miR-181a as a regulator of p27 mRNA translationduring myeloid cell differentiation.
Nguyen-Tu M-S, Martinez-Sanchez A, Leclerc I, et al., Reduced expression of TCF7L2 in adipocyte impairs glucose tolerance associated with decreased insulin secretion, incretins levels and lipid metabolism dysregulation in male mice
<jats:title>Abstract</jats:title><jats:p>Transcription factor 7-like 2 (TCF7L2) is a downstream effector of the Wnt/beta-catenin signalling pathway and its expression is critical for adipocyte development. The precise role of TCF7L2 in glucose and lipid metabolism in adult adipocytes remains to be defined. Here, we aim to investigate how changes in TCF7L2 expression in mature adipocytes affect glucose homeostasis. <jats:italic>Tcf7l2</jats:italic> was selectively ablated from mature adipocytes in C57BL/6J mice using an adiponectin promoter-driven <jats:italic>Cre</jats:italic> recombinase to recombine alleles floxed at exon 1 of the <jats:italic>Tcf7l2</jats:italic> gene. Mice lacking <jats:italic>Tcf7l2</jats:italic> in mature adipocytes displayed normal body weight. Male mice exhibited normal glucose homeostasis at eight weeks of age. Male heterozygote knockout mice (aTCF7L2het) exhibited impaired glucose tolerance (AUC increased 1.14 ± 0.04 -fold, p=0.03), as assessed by intraperitoneal glucose tolerance test, and changes in fat mass at 16 weeks (increased by 1.4 ± 0.09-fold, p=0.007). Homozygote knockout mice exhibited impaired oral glucose tolerance at 16 weeks of age (AUC increased 2.15 ± 0.15-fold, p=0.0001). Islets of Langerhans exhibited impaired glucose-stimulated insulin secretion <jats:italic>in vitro</jats:italic> (decreased 0.54 ± 0.13-fold aTCF7L2KO vs control, p=0.02), but no changes in <jats:italic>in vivo</jats:italic> glucose-stimulated insulin secretion. Female mice in which one or two alleles of the <jats:italic>Tcf7l2</jats:italic> gene was knocked out in adipocytes displayed no changes in glucose tolerance, insulin sensitivity or insulin secretion. Plasma levels of glucagon-like peptide-1 and gastric inhibitory polypeptide were lowered in knockout mice (decreased 0.57 ± 0.03-fold and 0.41 ± 0.12-fold
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