Faculty Collaboration Database

Proteome of H-411E (liver) cells exposed to insulin and tumor necrosis factor-alpha: analysis of proteins involved in insulin resistance. J Lab Clin Med 2005 May;145(5):275-83

Date

05/20/2005

Pubmed ID

15902099

DOI

10.1016/j.lab.2005.02.013

Scopus ID

2-s2.0-19344372177 (requires institutional sign-in at Scopus site)   33 Citations

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.

Author List

Solomon SS, Buss N, Shull J, Monnier S, Majumdar G, Wu J, Gerling IC

Author

Susanne M. Cabrera MD Associate Professor in the Pediatrics department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Densitometry
Diabetes Mellitus, Type 2
Electrophoresis, Gel, Two-Dimensional
Gene Expression
Insulin
Insulin Resistance
Liver
Liver Neoplasms, Experimental
Models, Biological
Phosphoproteins
Phosphorylation
Proteins
Proteome
Rats
Signal Transduction
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Tumor Cells, Cultured
Tumor Necrosis Factor-alpha