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Overexpression of Akt1 upregulates glycogen synthase activity and phosphorylation of mTOR in IRS‐1 knockdown HepG2 cells
Author(s) -
Varma Shailly,
Khandelwal Ramji L.
Publication year - 2007
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.21525
Subject(s) - gene knockdown , protein kinase b , pi3k/akt/mtor pathway , downregulation and upregulation , phosphorylation , insulin receptor , insulin , biology , insulin receptor substrate , microbiology and biotechnology , medicine , endocrinology , chemistry , signal transduction , cell culture , biochemistry , insulin resistance , genetics , gene
Insulin receptor substrate (IRS) proteins are important docking proteins in mediating the insulin signaling cascade. We have investigated the effect of short interfering RNA (siRNA) mediated knockdown of IRS‐1 on insulin signaling cascade in primary human hepatocellular carcinoma HepG2 cell line and HepG2 cells overexpressing Akt1/PKB‐α (HepG2‐CA‐Akt/PKB). IRS‐1 knockdown in both cell lines resulted in reduction of insulin stimulated Akt1 phosphorylation at Ser 473. In parental HepG2 cells, IRS‐1 knockdown resulted in reduction (ca. 50%) in the basal level of phosphorylated mTOR (Ser 2448) irrespective of insulin treatment. In contrast, HepG2‐CA‐Akt/PKB cells showed an upregulation in the basal level of phosphorylated mTOR (Ser 2448) (ca. 40%). Insulin mediated phosphorylation of mTOR was reduced. IRS‐1 knockdown also reduced the cell proliferation of parental HepG2 cells by ca. 30% in the presence/absence of insulin, whereas in HepG2‐CA‐Akt/PKB the cell proliferation was reduced by 15% and treatment of insulin further reduced it to ca. 50% (vs. control). IRS‐1 knockdown also reduced the glycogen synthase (GS) activity in parental HepG2 cells, however, it was upregulated in HepG2‐CA‐Akt/PKB cells. These results suggest that knockdown of IRS‐1 abolished basal as well as insulin mediated phosphorylation/activity of proteins involved in cell proliferation or glycogen metabolism in the parental Hep2 cells. IRS‐1 knockdown in cells overexpressing constitutively active Akt1/PKB‐α either did not change or upregulated the basal levels of phosphorylated/active proteins. However, insulin mediated response was either not altered or downregulated in these cells. J. Cell. Biochem. 103: 1424–1437, 2008. © 2007 Wiley‐Liss, Inc.