Subcellular Localization of Insulin Receptor Substrate Family Proteins Associated With Phosphatidylinositol 3-Kinase Activity and Alterations in Lipolysis in Primary Mouse Adipocytes From IRS-1 Null Mice
- Youki Tsuji1,
- Yasushi Kaburagi1,
- Yasuo Terauchi1,
- Shinobu Satoh2,
- Naoto Kubota1,
- Hiroyuki Tamemoto1,
- Fredric B. Kraemer3,
- Hisahiko Sekihara2,
- Shinichi Aizawa4,
- Yasuo Akanuma5,
- Kazuyuki Tobe1,
- Satoshi Kimura1 and
- Takashi Kadowaki1
- 1Department of Metabolic Diseases, Graduate School of Medicine, University of Tokyo, Tokyo
- 2Third Department of Internal Medicine, Yokohama City University, Yokohama, Japan
- 3Stanford University School of Medicine, Stanford, California
- 4Laboratory of Morphogenesis IMEG, Kumamoto University School of Medicine, Kumamoto
- 5Institute for Diabetes Care and Research, Asahi Life Foundation, Tokyo, Japan
Abstract
To clarify the roles of insulin receptor substrate (IRS) family proteins in phosphatidylinositol (PI) 3-kinase activation and insulin actions in adipocytes, we investigated the intracellular localization of IRS family proteins and PI 3-kinase activation in response to insulin by fractionation of mouse adipocytes from wild-type and IRS-1 null mice. In adipocytes from wild-type mice, tyrosine-phosphorylated IRS-1 and IRS-2, which were found to associate with PI 3-kinase in response to insulin, were detected in the plasma membrane (PM) and low-density microsome (LDM) fractions. By contrast, tyrosine-phosphorylated IRS-3 (pp60), which was found to associate with PI 3-kinase, was predominantly localized in the PM fraction. In adipocytes from IRS-1–null mice, insulin-stimulated PI 3-kinase activity in anti-phosphotyrosine (αPY) immunoprecipitates in the LDM fraction was almost exclusively mediated via IRS-2 and was reduced to 25%; however, insulin-stimulated PI 3-kinase activity in the PM fraction was primarily mediated via IRS-3 and was reduced to 60%. To determine the potential functional impact of the distinct subcellular localization of IRSs and associating PI 3-kinase activity on adipocyte-specific metabolic actions, we examined lipolysis in IRS-1 null mice. The level of isoproterenol-induced lipolysis was increased 5.1-fold in adipocytes from IRS-1 null mice as compared with wild-type mice. Moreover, hormone-sensitive lipase (HSL) protein was increased 4.3-fold in adipocytes from IRS-1–null mice compared with wild-type mice, and HSL mRNA expression was also increased. The antilipolytic effect of insulin in IRS-1 null adipocytes, however, was comparable to that in wild-type mice. Thus, discordance between these two insulin actions as well as the transcriptional and translational effect (HSL mRNA and protein regulation) and the PM effect (antilipolysis) of insulin may be explained by distinct roles of both PI 3-kinase activity associated with IRS-1/IRS-2 and PI 3-kinase activity associated with IRS-3 in insulin actions related to their subcellular localization.
Footnotes
-
Address correspondence and reprint requests to Takashi Kadowaki, Department of Metabolic Diseases, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: kadowaki-3im{at}h.u-tokyo.ac.jp.
Received for publication 16 September 2000 and accepted in revised form 14 March 2001.
αIRS, anti-IRS antibody; αPY, anti-phosphotyrosine; BSA, bovine serum albumin; CYTO, cytosol; GST, glutathione S-transferase; HSL, hormone-sensitive lipase; IRS, insulin receptor substrate; KRBH, Krebs-Ringer bicarbonate–HEPES; HDM, high-density microsome; LDM, low-density microsome; NEFA, nonesterified fatty acid; PCR, polymerase chain reaction; PDE3B, phosphodiesterase 3B; PH, pleckstrin homology; PI, phosphatidylinositol; PM, plasma membrane; PMSF, phenylmethylsulfonyl fluoride; PTB, phosphotyrosine-binding; SH2, Src homology-2; TOTAL, total lysates.
