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Research Resource: A Dual Proteomic Approach Identifies Regulated Islet Proteins During ?-Cell Mass Expansion In Vivo.

ABSTRACT: Diabetes is characterized by insulin insufficiency due to a relative paucity of functional ?-cell mass. Thus, strategies for increasing ?-cell mass in situ are sought-after for therapeutic purposes. Pregnancy is a physiological state capable of inducing robust ?-cell mass expansion, however, the mechanisms driving this expansion are not fully understood. Thus, the aim of this study was to characterize pregnancy-induced changes in the islet proteome at the peak of ?-cell proliferation in mice. Islets from pregnant and nonpregnant littermates were compared via 2 proteomic strategies. In vivo pulsed stable isotope labeling of amino acids in cell culture was used to monitor de novo protein synthesis during the first 14.5 days of pregnancy. In parallel, protein abundance was determined using ex vivo dimethyl labelling at gestational day 14.5. Comparison of the 2 datasets revealed 170 islet proteins to be up regulated as a response to pregnancy. These included several proteins, not previously associated with pregnancy-induced islet expansion, such as CLIC1, STMN1, MCM6, PPIB, NEDD4, and HLTF. Confirming the validity of our approach, we also identified proteins encoded by genes known to be associated with pregnancy-induced islet expansion, such as CHGB, IGFBP5, MATN2, EHHADH, IVD, and BMP1. Bioinformatic analyses demonstrated enrichment and activation of the biological functions: "protein synthesis" and "proliferation," and predicted the transcription factors HNF4?, MYC, MYCN, E2F1, NFE2L2, and HNF1? as upstream regulators of the observed expressional changes. As the first characterization of the islet-proteome during pregnancy, this study provides novel insight into the mechanisms involved in promoting pregnancy-induced ?-cell mass expansion and function.

SUBMITTER: Horn S

PROVIDER: S-EPMC5414659 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Research Resource: A Dual Proteomic Approach Identifies Regulated Islet Proteins During β-Cell Mass Expansion In Vivo.

Horn Signe S Kirkegaard Jeannette S JS Hoelper Soraya S Seymour Philip A PA Rescan Claude C Nielsen Jens H JH Madsen Ole D OD Jensen Jan N JN Krüger Marcus M Grønborg Mads M Ahnfelt-Rønne Jonas J

Molecular endocrinology (Baltimore, Md.) 20151209 1

Diabetes is characterized by insulin insufficiency due to a relative paucity of functional β-cell mass. Thus, strategies for increasing β-cell mass in situ are sought-after for therapeutic purposes. Pregnancy is a physiological state capable of inducing robust β-cell mass expansion, however, the mechanisms driving this expansion are not fully understood. Thus, the aim of this study was to characterize pregnancy-induced changes in the islet proteome at the peak of β-cell proliferation in mice. Is ...[more]

PMID: 26649805

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Project description:GH and GH receptors are expressed throughout life, and GH elicits a diverse range of responses, including growth and altered metabolism. It is therefore important to understand the full spectrum of GH signaling pathways and cellular responses. We applied mass spectrometry-based phosphoproteomics combined with stable isotope labeling with amino acids in cell culture to identify proteins rapidly phosphorylated in response to GH in 3T3-F442A preadipocytes. We identified 132 phosphosites in 95 proteins that exhibited rapid (5 or 15 min) GH-dependent statistically significant increases in phosphorylation by more than or equal to 50% and 96 phosphosites in 46 proteins that were down-regulated by GH by more than or equal to 30%. Several of the GH-stimulated phosphorylation sites were known (e.g. regulatory Thr/Tyr in Erks 1 and 2, Tyr in signal transducers and activators of transcription (Stat) 5a and 5b, Ser939 in tuberous sclerosis protein (TSC) 2 or tuberin). The remaining 126 GH-stimulated sites were not previously associated with GH. Kyoto Encyclopedia of Genes and Genomes pathway analysis of GH-stimulated sites indicated enrichment in proteins associated with the insulin and mammalian target of rapamycin (mTOR) pathways, regulation of the actin cytoskeleton, and focal adhesions. Akt/protein kinase A consensus sites (RXRXXS/T) were the most commonly phosphorylated consensus sites. Immunoblotting confirmed GH-stimulated phosphorylation of all seven novel GH-dependent sites tested [regulatory sites in proline-rich Akt substrate, 40 kDA (PRAS40), regulatory associated protein of mTOR, ATP-citrate lyase, Na(+)/H(+) exchanger-1, N-myc downstream regulated gene 1, and Shc]). The immunoblot results suggest that many, if not most, of the GH-stimulated phosphosites identified in this large-scale quantitative phosphoproteomics analysis, including sites in multiple proteins in the Akt/ mTOR complex 1 pathway, are phosphorylated in response to GH. Their identification significantly broadens our thinking of GH-regulated cell functions.

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Research Resource: A Dual Proteomic Approach Identifies Regulated Islet Proteins During ?-Cell Mass Expansion In Vivo.

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Research Resource: A Dual Proteomic Approach Identifies Regulated Islet Proteins During β-Cell Mass Expansion In Vivo.

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