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A Syntenic Cross Species Aneuploidy Genetic Screen Links RCAN1 Expression to ?-Cell Mitochondrial Dysfunction in Type 2 Diabetes.

ABSTRACT: Type 2 diabetes (T2D) is a complex metabolic disease associated with obesity, insulin resistance and hypoinsulinemia due to pancreatic ?-cell dysfunction. Reduced mitochondrial function is thought to be central to ?-cell dysfunction. Mitochondrial dysfunction and reduced insulin secretion are also observed in ?-cells of humans with the most common human genetic disorder, Down syndrome (DS, Trisomy 21). To identify regions of chromosome 21 that may be associated with perturbed glucose homeostasis we profiled the glycaemic status of different DS mouse models. The Ts65Dn and Dp16 DS mouse lines were hyperglycemic, while Tc1 and Ts1Rhr mice were not, providing us with a region of chromosome 21 containing genes that cause hyperglycemia. We then examined whether any of these genes were upregulated in a set of ~5,000 gene expression changes we had identified in a large gene expression analysis of human T2D ?-cells. This approach produced a single gene, RCAN1, as a candidate gene linking hyperglycemia and functional changes in T2D ?-cells. Further investigations demonstrated that RCAN1 methylation is reduced in human T2D islets at multiple sites, correlating with increased expression. RCAN1 protein expression was also increased in db/db mouse islets and in human and mouse islets exposed to high glucose. Mice overexpressing RCAN1 had reduced in vivo glucose-stimulated insulin secretion and their ?-cells displayed mitochondrial dysfunction including hyperpolarised membrane potential, reduced oxidative phosphorylation and low ATP production. This lack of ?-cell ATP had functional consequences by negatively affecting both glucose-stimulated membrane depolarisation and ATP-dependent insulin granule exocytosis. Thus, from amongst the myriad of gene expression changes occurring in T2D ?-cells where we had little knowledge of which changes cause ?-cell dysfunction, we applied a trisomy 21 screening approach which linked RCAN1 to ?-cell mitochondrial dysfunction in T2D.

SUBMITTER: Peiris H

PROVIDER: S-EPMC4873152 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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A Syntenic Cross Species Aneuploidy Genetic Screen Links RCAN1 Expression to β-Cell Mitochondrial Dysfunction in Type 2 Diabetes.

Peiris Heshan H Duffield Michael D MD Fadista Joao J Jessup Claire F CF Kashmir Vinder V Genders Amanda J AJ McGee Sean L SL Martin Alyce M AM Saiedi Madiha M Morton Nicholas N Carter Roderick R Cousin Michael A MA Kokotos Alexandros C AC Oskolkov Nikolay N Volkov Petr P Hough Tertius A TA Fisher Elizabeth M C EM Tybulewicz Victor L J VL Busciglio Jorge J Coskun Pinar E PE Becker Ann A Belichenko Pavel V PV Mobley William C WC Ryan Michael T MT Chan Jeng Yie JY Laybutt D Ross DR Coates P Toby PT Yang Sijun S Ling Charlotte C Groop Leif L Pritchard Melanie A MA Keating Damien J DJ

PLoS genetics 20160519 5

Type 2 diabetes (T2D) is a complex metabolic disease associated with obesity, insulin resistance and hypoinsulinemia due to pancreatic β-cell dysfunction. Reduced mitochondrial function is thought to be central to β-cell dysfunction. Mitochondrial dysfunction and reduced insulin secretion are also observed in β-cells of humans with the most common human genetic disorder, Down syndrome (DS, Trisomy 21). To identify regions of chromosome 21 that may be associated with perturbed glucose homeostasis ...[more]

PMID: 27195491

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Project description:PurposeCell death is an essential process in normal development and homeostasis. In eyes, corneal epithelial injury leads to the death of cells in underlying stroma, an event believed to initiate corneal wound healing. The molecular basis of wound induced corneal stromal cell death is not understood in detail. Studies of others have indicated that ceramide may play significant role in stromal cell death following LASIK surgery. We have undertaken the present study to investigate the mechanism of death induced by C6 ceramide in cultures of human corneal stromal (HCSF) fibroblasts.MethodsCultures of HCSF were established from freshly excised corneas. Cell death was induced in low passage (p<4) cultures of HCSF by treating the cells with C6 ceramide or C6 dihydroceramide as a control. Cell death was assessed by Live/Dead cell staining with calcein AM and ethidium homodimer-1 as well as Annexin V staining, caspase activation and TUNEL staining Mitochondrial dysfunction was assessed by Mito Sox Red, JC-1 and cytochrome C release Gene expression was examined by qPCR and western blotting.ResultsOur data demonstrate ceramide caused mitochondrial dysfunction as evident from reduced MTT staining, cyto c release from mitochondria, enhanced generation of ROS, and loss in mitochondrial membrane potential (??m). Cell death was evident from Live -Dead Cell staining and the inability to reestablish cultures from detached cells. Ceramide induced the expression of the harikari gene(HRK) and up-regulated JNK phosphorylation. In ceramide treated cells HRK was translocated to mitochondria, where it was found to interact with mitochondrial protein p32. The data also demonstrated HRK, p32 and BAD interaction. Ceramide-induced expression of HRK, mitochondrial dysfunction and cell death were reduced by HRK knockdown with HRK siRNA.ConclusionOur data document that ceramide is capable of inducing death of corneal stromal fibroblasts through the induction of HRK mediated mitochondria dysfunction.

Dataset Information

A Syntenic Cross Species Aneuploidy Genetic Screen Links RCAN1 Expression to ?-Cell Mitochondrial Dysfunction in Type 2 Diabetes.

Publications

A Syntenic Cross Species Aneuploidy Genetic Screen Links RCAN1 Expression to β-Cell Mitochondrial Dysfunction in Type 2 Diabetes.

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