Restored insulin-sensitivity in IRS-1–deficient mice treated by adenovirus-mediated gene therapy
Kohjiro Ueki, … , Ryozo Nagai, Takashi Kadowaki
Kohjiro Ueki, … , Ryozo Nagai, Takashi Kadowaki
Published May 15, 2000
Citation Information: J Clin Invest. 2000;105(10):1437-1445. https://doi.org/10.1172/JCI7656.
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Article

Restored insulin-sensitivity in IRS-1–deficient mice treated by adenovirus-mediated gene therapy

Abstract

Insulin resistance is commonly observed both in overt diabetes and in individuals prone to, but not yet manifesting, diabetes. Hence the maintenance or restoration of insulin sensitivity may prevent the onset of this disease. We previously showed that homozygous disruption of insulin receptor substrate-1 (IRS-1) in mice resulted in insulin resistance but not diabetes. Here, we have explored the mechanism of systemic insulin resistance in these mice and used adenovirus-mediated gene therapy to restore their insulin sensitivity. Mice expressing the IRS-1transgene showed almost normal insulin sensitivity. Expression of an IRS-1 mutant (IRS-1Δp85) lacking the binding site for the p85 subunit of phosphatidylinositol 3-kinase (PI3K) also restored insulin sensitivity, although PI3K is known to play a crucial role in insulin’s metabolic responses. Protein kinase B (PKB) activity in liver was decreased in null mice compared with the wild-type and the null mice expressing IRS-1 or IRS-1Δp85. In primary hepatocytes isolated from null mice, expression of IRS-1 enhanced both PI3K and PKB activities, but expression of IRS-1Δp85 enhanced only PKB. These data suggest that PKB in liver plays a pivotal role in systemic glucose homeostasis and that PKB activation might be sufficient for reducing insulin resistance even without full activation of PI3K.

Authors

Kohjiro Ueki, Toshimasa Yamauchi, Hiroyuki Tamemoto, Kazuyuki Tobe, Ritsuko Yamamoto-Honda, Yasushi Kaburagi, Yasuo Akanuma, Yoshio Yazaki, Sininchi Aizawa, Ryozo Nagai, Takashi Kadowaki

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Figure 5

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PKB activity in IRS-1–deficient primary hepatocytes and liver. (a) The e...
PKB activity in IRS-1–deficient primary hepatocytes and liver. (a) The effect of IRS-1wt or IRS-1Δp85 on PKB phosphorylation and activity in IRS-1–deficient primary hepatocytes. After being treated without or with 100 nM insulin for 5 minutes, the cell lysates were subjected to SDS-PAGE, followed by Western blot analysis (IB) with anti-PKB antibodies (αPKB-CT) (upper panel), or subjected to immunoprecipitation (IP) with αPKB-CT followed by the immune complex kinase assay as described in Methods (lower panel). The results are expressed as the ratio to the value of LacZ without insulin, and each bar represents the mean ± SD of more than three independent experiments. AP < 0.05 LacZ insulin (+) versus IRS-1wt insulin (+). BP < 0.05 LacZ insulin (+) versus IRS-1Δp85 insulin (+). (b) The effect of IRS-1wt or IRS-1Δp85 on PKB activity in IRS-1–deficient mouse liver. After being treated without or with insulin for 5 minutes, the liver lysates were immunoprecipitated with αPKB-CT and then subjected to PKB kinase assay. The results are expressed as the ratio to the value of LacZ without insulin, and each bar represents the mean ± SD of more than three independent experiments. AP < 0.01 LacZ insulin (+) versus wild-type insulin (+). BP < 0.05 LacZ insulin (+) versus IRS-1wt insulin (+). CP < 0.05 LacZ insulin (+) versus IRS-1Δp85 insulin (+). (c) The effect of PI3K inhibition on PKB activation on IRS-1 deficient primary hepatocytes. The indicated proteins were introduced into IRS-1–deficient primary hepatocytes by adenovirus-mediated gene transfer. Cells were incubated with or without 50 nM wortmannin for 30 minutes and were treated with 100 nM insulin for 5 minutes. The cell lysates were subjected to immunoprecipitation with 4G10 or αPKB-CT, followed by PI3K assay (upper panel) and PKB immune complex kinase assay (lower panel), respectively. In the lower panel, the results are expressed as the ratio to the value of LacZ without insulin, and each bar represents the mean of two experiments. wor, wortmannin.