title: Proteome of H-411E (liver) cells exposed to insulin and tumor necrosis factor-alpha: analysis of proteins involved in insulin resistance.
abstract: Insulin resistance may be modeled in H-411E liver cells in tissue culture with the use of the cytokine tumor necrosis factor-alpha (TNF-alpha) and insulin. This tissue-culture model nicely mimics IR in human type 2 diabetes mellitus. After incubation of liver cells in tissue culture with INS alone, TNF-alpha alone, and TNF-alpha plus insulin, as well as a control sample, liver-cell extracts were separated on 2D polyacrylamide-gel electrophoresis on the basis of isoelectric point and molecular weight. We analyzed the gel images with the use of PD Quest software (Bio-Rad Laboratories, Hercules, Calif) to identify differentially expressed protein spots (ie, up or down with insulin vs down or up with TNF-alpha plus insulin). In separate experiments, phosphorus-32 incorporation/autoradiography and phosphoprotein staining were used to characterize treatment-induced phosphorylations. Affected protein spots were identified with the use of peptide fingerprinting and matrix-assisted laser desorption ionization time of flight mass spectrometry. The first series of experiments identified 6 differentially expressed proteins: eukaryotic translation initiation factor-3, subunit 2, regulator of G-protein signaling-5, superoxide dismutase, protein disulfide isomerase A6, proteasome subunit-alpha type 3, and regucalcin. In addition, we observed changes in the phosphorylation of protein disulfide isomerase A6. A second series of experiments identified 7 additional proteins with significantly altered differential expression: cell-division protein kinase-4, kinogen heavy chain, carbonic anhydrase-7, E 3 ubiquitin protein ligase, URE-B1; Rab GDP dissociation inhibitor-beta, Rab GDP dissociation inhibitor-beta2, and MAWDBP. It can be seen that differentially expressed proteins, affected by treatment with insulin or with TNF-alpha plus insulin, include regulators of translation, protein degradation, cellular Ca ++ , G-proteins, and free-radical production. Although one cannot detail the mechanism or mechanisms of TNF-alpha induced IR from this data alone, it is easy to relate all of these proteins to a role in insulin signal transduction and, hence, insulin resistance.
authors.affiliation: Research and Medical Service, Veterans Affairs Medical Center, Memphis, Tennessee 38104, USA.
authors.forename: Solomon S
authors.initials: SS
authors.lastname: Solomon
authors.forename: Nicholas
authors.initials: N
authors.lastname: Buss
authors.forename: James
authors.initials: J
authors.lastname: Shull
authors.forename: Susanne
authors.initials: S
authors.lastname: Monnier
authors.forename: Gipsy
authors.initials: G
authors.lastname: Majumdar
authors.forename: Jian
authors.initials: J
authors.lastname: Wu
authors.forename: Ivan C
authors.initials: IC
authors.lastname: Gerling
mesh_terms: D000818:Animals; D003720:Densitometry; D003924:Diabetes Mellitus, Type 2 / Q000150:complications; D015180:Electrophoresis, Gel, Two-Dimensional; D015870:Gene Expression / Q000187:drug effects; D007328:Insulin / Q000494:pharmacology*; D007333:Insulin Resistance / Q000235:genetics; D008099:Liver / Q000737:chemistry* / Q000187:drug effects; D008114:Liver Neoplasms, Experimental; D008954:Models, Biological; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D011506:Proteins / Q000032:analysis* / Q000235:genetics; D020543:Proteome / Q000032:analysis* / Q000235:genetics; D051381:Rats; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D007328:Insulin; D010750:Phosphoproteins; D011506:Proteins; D020543:Proteome; D014409:Tumor Necrosis Factor-alpha