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 1

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The effect of IRS-1 reconstitution by adenovirus-mediated gene transfer ...
The effect of IRS-1 reconstitution by adenovirus-mediated gene transfer in the IRS-1–deficient mice. The IRS-1–deficient mice were treated with the adenoviruses as described in Methods. (a) Specific reconstitution of IRS-1 protein in the knockout mouse liver. The lysates of each tissue (liver, soleus muscle [muscle], and epididymal fat pad [fat]) from the indicated mouse (wild-type mouse [wild], null mouse treated with LacZ adenovirus [null LacZ], and null mouse treated with wild-type IRS-1 [null IRS-1wt]) were prepared as described in Methods. The lysates were immunoprecipitated (IP) with the antibodies against the COOH-terminal region of IRS-1 (αIRS-1-CT) and subjected to SDS-PAGE (7% gel), followed by Western blotting (IB) with the same antibodies. (b) Tyrosine-phosphorylated proteins in liver in response to insulin. Mice were anesthetized, and 1 μg/g body weight of insulin was injected through the inferior vena cava. After 5 minutes, the liver was removed and lysed with the tissue homogenization buffer as described in Methods. The lysates were immunoprecipitated with anti-phosphotyrosine antibodies (4G10) and subjected to SDS-PAGE (7% gel), followed by Western blotting with the same antibodies. PY, anti-phosphotyrosine antibodies. (c) ITT on mice treated with the indicated adenoviruses. ITT was performed on the indicated group of mice (eight mice in each group). Each bar represents ± SD. AP < 0.02 wild-type or null IRS-1wt versus null or null LacZ. BP < 0.01 wild-type or null IRS-1wt versus null or null LacZ. CP < 0.01 wild-type or null IRS-1wt versus null or null LacZ. (d) Insulin-induced MAP kinase activity in the liver infected with the indicated adenoviruses. After being treated without or with insulin for 5 minutes, the liver lysates were immunoprecipitated with anti-MAP kinase antibodies (αC92) and subjected to the immune complex kinase assay as described in Methods. The results are expressed as the ratio to the value of wild-type without insulin. Each bar represents the mean ± SD of more than three independent experiments. (e) Insulin-induced PI3K activity associated with the tyrosine-phosphorylated proteins in the liver infected with the indicated adenoviruses. After being treated without or with insulin for 5 minutes, the liver lysates were immunoprecipitated with anti-phosphotyrosine antibodies and then subjected to PI3K assay as described in Methods. The lower panel shows the representative results (lanes 1–3; wild-type, lanes 4–6; null LacZ, lanes 7–9; null treated with IRS-1wt, lanes 1, 4, and 7; insulin [–], lanes 2, 3, 5, 6, 8, and 9; insulin [+]). In the upper panel, the results are expressed as the ratio to the value of wild-type without insulin, and each bar represents the mean ± SD of more than three independent experiments. AP < 0.05 wild-type insulin (+) versus null LacZ insulin (+). BP < 0.05 null IRS-1wt insulin (+) versus null LacZ insulin (+).