β cell lines ins-1  (Chunbo Co Ltd)

Journal: PLoS Biology

Article Title: Pancreatic β cell microRNA-26a alleviates type 2 diabetes by improving peripheral insulin sensitivity and preserving β cell function

doi: 10.1371/journal.pbio.3000603

Figure Lengend Snippet: (A and B) Islets were isolated from either RIP TG or WT littermates fed an HFD for 16 weeks and cocultured with MPHs. (A) Levels of miR-26a in islets (left panel) and MPHs (right panel). (B) AKT phosphorylation in MPHs stimulated with insulin. (C and D) Exosomes were isolated from islets of RIP TG or WT littermates fed an HFD for 16 weeks and then transferred to MPHs. (C) Levels of miR-26a in islet exosomes (left panel) and MPHs (right panel). (D) AKT phosphorylation in MPHs stimulated with insulin. (E) Exosomes secreted from Min6 cells were labeled with PKH26 and then added to MPHs. (F–I) Exosomes were collected from media of Min6 cells transfected with miR-26a mimics (miR-26a) or NCs and then transferred to MPHs. (F) Levels of miR-26a in Min6-derived exosomes (left panel) and MPHs (right panel). (G) Levels of miR-26a in MPHs treated with exosomes from miR-26a–overexpressing Min6 cells that were treated with the exosome inhibitor GW4869 or controls. (H) AKT phosphorylation in MPHs stimulated with insulin. (I) Levels of miR-26a targets in MPHs. Results are representative of 3 replicated independent experiments (A–I), and ImageJ quantification of the pAKT/AKT ratio is shown (B, D, and H). The data underlying this figure may be found in and . Data are shown as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.005, Student t test. Acsl3/4 , acyl-CoA synthetase long chain family member 3/4; AKT, AKT serine/threonine kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; INS, insulin; Min6 cells, murine β cells; MPHs, murine primary hepatocytes; NC, negative control; pAKT, phosphorylated AKT; Pkcθ , protein kinase C theta; RIP, rat insulin promoter; TG, transgenic; WT, wild type.

Article Snippet: The β cell lines Min6 and INS-1 were kindly provided by Professor Chunbo Teng (Northeast Forestry University, Harbin, China).

Techniques: Isolation, Phospho-proteomics, Labeling, Transfection, Derivative Assay, Negative Control, Transgenic Assay

Journal: PLoS Biology

Article Title: Pancreatic β cell microRNA-26a alleviates type 2 diabetes by improving peripheral insulin sensitivity and preserving β cell function

doi: 10.1371/journal.pbio.3000603

Figure Lengend Snippet: (A) Static insulin secretion performed with islets from mice fed an HFD for 12 weeks at indicated glucose, arginine, and KCl concentrations ( n = 3). (B) Time course of GSIS in islets from mice fed an HFD for 14 weeks ( n = 4). (C–E) Proteomic analysis on islets of either RIP TG or WT littermate controls fed an HFD for 2 days. (C) Venn diagram comparing islet proteins. The number of proteins that are differentially expressed are shown. RIP TG mice showed an increase of 204 proteins and a decrease of 104 proteins. (D) KEGG pathway analysis on islet proteins deregulated in RIP TG mice. (E) Heat map of protein levels of genes involved in focal adhesion. (F) The protein levels of FLNA and CAV1 in the islets were verified by western blotting. (G and H) Representative IF imaging of F-actin in isolated islets treated with glucose (scale bar, 20 μm) (G). Quantification of F-actin intensity are shown (H) ( n = 9 islets from 3 independent pancreatic samples per genotype). (I–L) Min6 cells were transfected with either miR-26a mimics (miR-26a) or NCs, followed by H 2 O 2 treatment ( n = 3). (I) Representative IF imaging of F-actin (scale bar, 20 μm). (J) Ratio of G-actin and F-actin fluorescence intensity. (K) Protein levels of F-actin and G-actin. (L) The activation kinetics of focal adhesion. The data underlying this figure may be found in and . Data are shown as mean ± SD. * P < 0.05, Student t test. ACTN4, actinin alpha 4; AKT, AKT serine/threonine kinase; CAV1, caveolin 1; CDC42, cell division cycle 42; COL6A, collagen type VI alpha; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; FLNA, filamin alpha; F-actin, filamentous actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSIS, glucose-stimulated insulin secretion; G-actin, globular actin; HFD, high-fat diet; IF, immunofluorescence; KEGG, Kyoto Encyclopedia of Genes and Genomes; LAMB2, laminin subunit beta 2; LAMC1, laminin subunit gamma 1; Min6 cells, murine β cells; MIP, maximum intensity projection; MYLK, myosin light chain kinase; NC, negative control; pAKT, phosphorylated AKT; p-ERK, phosphorylated ERK; p-FAK, phosphorylated FAK; PPAR, peroxisome proliferator activated receptor; RIP, rat insulin promoter; TG, transgenic; SOS, SOS Ras/Rac guanine nucleotide exchange factor; VCL, vinculin; WT, wild type.

Article Snippet: The β cell lines Min6 and INS-1 were kindly provided by Professor Chunbo Teng (Northeast Forestry University, Harbin, China).

Techniques: Western Blot, Imaging, Isolation, Transfection, Fluorescence, Activation Assay, Immunofluorescence, Negative Control, Transgenic Assay

Journal: PLoS Biology

Article Title: Pancreatic β cell microRNA-26a alleviates type 2 diabetes by improving peripheral insulin sensitivity and preserving β cell function

doi: 10.1371/journal.pbio.3000603

Figure Lengend Snippet: (A) Representative IF staining for insulin and glucagon in pancreatic islets (scale bar, 200 μm) ( n = 4). (B) Representative HE-stained pancreas (scale bar, 200 μm) ( n = 4). (C) Representative IHC staining for insulin in pancreatic islets (scale bar, 100 μm) ( n = 4). (D) β cell mass ( n = 4). (E) Islet area relative to pancreas area ( n = 8–10). (F) Percentage of large islets ( n = 5–8). (G) mRNA levels of the markers for β cell hyperplasia ( n = 3). (H) Quantification of IF staining for insulin and PCNA in pancreas ( n = 4). (I) mRNA levels of the markers for β cell replication ( n = 3). (J) The top 50 enriched genes in the islet of WT mouse identified by Ago2 RNA immunoprecipitation sequencing are presented in a heat map ( n = 3). Red and blue depict higher and lower gene enrichment, respectively. Color intensity indicates magnitude of enrichment differences. (K) Relative luciferase activity in 293T cells transfected with reporter constructs containing the 3′ UTR of target genes and co-transfected with either miR-26a mimics (miR-26a) or NCs. (L) Expression of miR-26a target genes in islets of RIP TG and WT littermates fed an HFD for 16 weeks ( n = 3–4). The data underlying this figure may be found in . Data are shown as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.005, Student t test. Adamts19, ADAM metallopeptidase with thrombospondin type 1 motif 19; Ago2, argonaute RISC catalytic component 2; AKT, AKT serine/threonine kinase; Amph, amphiphysin; Ap5m1, adaptor related protein complex 5 subunit Mu 1; Atad2b, ATPase family AAA domain containing 2B; Baz2b, bromodomain adjacent to zinc finger domain 2B; Brap, BRCA1 associated protein; Cacna1c, calcium voltage-gated channel subunit alpha1 C; Carmil1, capping protein regulator and myosin 1 linker 1; Ccnd2 , cyclin D2; Cep350, centrosomal protein 350; Chordc1, cysteine and histidine rich domain containing 1; Col11a1, collagen type XI alpha 1 chain; Col22a1, collagen type XXII alpha 1 chain; Crebrf, CREB3 regulatory factor; Ctgf, connective tissue growth factor; Dab2, DAB adaptor protein 2; Dapk1, death associated protein kinase 1; DKO, double knockout; E2f7, E2F transcription factor 7; Epha7, EPH receptor A7; Esr1, estrogen receptor 1; Ext1, exostosin glycosyltransferase 1; Foxm1 , forkhead box M 1; HE, hematoxylin–eosin; HFD, high-fat diet; Hnrnpul1, heterogeneous nuclear ribonucleoprotein U like 1; IF, immunofluorescence; Igf1r , insulin like growth factor 1 receptor; IHC, immunohistochemistry; Insr , insulin receptor; Irs2 , insulin receptor substrate 2; Lman1, lecti mannose binding 1; Lrat, lecithin retinol acyltransferase; Mcc, MCC regulator of WNT signaling pathway; Megf9, multiple EGF like domains 9; Memo1, mediator of cell motility 1; Mki67 , marker of proliferation Ki-67; Mtpn, myotrophin; Mut, mutant; NC, negative control; Nek10, NIMA related kinase 10; Neto1, neuropilin and tolloid like 1; Nras, neuroblastoma RAS viral oncogene; Nwd2, NACHT and WD repeat domain containing 2; Onecut2, one cut homeobox 2; Pcdh9, protocadherin 9; PCNA, proliferative cell nuclear antigen; Pdx1 , pancreatic and duodenal homeobox 1; Pfkfb2, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2; Phf21a, PHD finger protein 21A; Pja2, praja ring finger ubiquitin ligase 2; Plcb1, phospholipase C beta 1; Prkag2, protein kinase AMP-activated non-catalytic subunit gamma 2; Prkg1, protein kinase CGMP-dependent 1; Pxylp1, 2-phosphoxylose phosphatase 1; Rb1, RB transcriptional corepressor 1; Rcn1, reticulocalbin 1; Ret, Ret proto-oncogene; Rhoq, Ras homolog family member Q; RIP, rat insulin promoter; Ryk, receptor like tyrosine kinase; Scml4, scm polycomb group protein like 4; Slc26a4, solute carrier family 26 member 4; Sox5, SRY-box transcription factor 5; Spock2, SPARC/Osteonectin, CWCV and Kazal like domains proteoglycan 2; Tab2, TGF-beta activated kinase 1 binding protein 2; Tacc2, transforming acidic coiled-coil containing protein 2; Tet2, tet methylcytosine dioxygenase 2; TG, transgenic; Tnks2, tankyrase 2; Trps1, transcriptional repressor GATA binding 1; Tsc22d2, TSC22 domain family member 2; Ube4b, ubiquitination factor E4B; Usp9x, ubiquitin specific peptidase 9 X-linked; WT, wild type.

Article Snippet: The β cell lines Min6 and INS-1 were kindly provided by Professor Chunbo Teng (Northeast Forestry University, Harbin, China).

Techniques: Staining, Immunohistochemistry, RNA Immunoprecipitation, Sequencing, Luciferase, Activity Assay, Transfection, Construct, Expressing, Double Knockout, Immunofluorescence, Binding Assay, Marker, Mutagenesis, Negative Control, Ubiquitin Proteomics, Transgenic Assay