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. 2004 Aug 24;101(34):12670-5.
doi: 10.1073/pnas.0402053101. Epub 2004 Aug 16.

Insulin resistance in skeletal muscles of caveolin-3-null mice

Jin Oshikawa  1 , Koji Otsu, Yoshiyuki Toya, Takashi Tsunematsu, Raleigh Hankins, Jun-ichi Kawabe, Susumu Minamisawa, Satoshi Umemura, Yasuko Hagiwara, Yoshihiro Ishikawa
Affiliations

Insulin resistance in skeletal muscles of caveolin-3-null mice

Jin Oshikawa et al. Proc Natl Acad Sci U S A. .

Abstract

Type 2 diabetes is preceded by the development of insulin resistance, in which the action of insulin is impaired, largely in skeletal muscles. Caveolin-3 (Cav3) is a muscle-specific subtype of caveolin, an example of a scaffolding protein found within membranes. Cav is also known as growth signal inhibitor, although it was recently demonstrated that the genetic disruption of Cav3 did not augment growth in mice. We found, however, that the lack of Cav3 led to the development of insulin resistance, as exemplified by decreased glucose uptake in skeletal muscles, impaired glucose tolerance test performance, and increases in serum lipids. Such impairments were markedly augmented in the presence of streptozotocin, a pancreatic beta cell toxin, suggesting that the mice were susceptible to severe diabetes in the presence of an additional risk factor. Insulin-stimulated activation of insulin receptors and downstream molecules, such as IRS-1 and Akt, was attenuated in the skeletal muscles of Cav3 null mice, but not in the liver, without affecting protein expression or subcellular localization. Genetic transfer of Cav3 by needle injection restored insulin signaling in skeletal muscles. Our findings suggest that Cav3 is an enhancer of insulin signaling in skeletal muscles but does not act as a scaffolding molecule for insulin receptors.

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Figures

Fig. 1.
Fig. 1.
Decreased insulin signaling in skeletal muscle tissues of Cav3KO. Two minutes after insulin injection, tissues were harvested to determine insulin signaling in WT (WT) and Cav3KO (KO). (A) Phosphorylation of IRβ in skeletal muscles was quantitated by immunoblotting with a phosphotyrosine antibody (IB: P-Tyr) after immunoprecipitation with an IRβ antibody (IP: IRβ) in the absence [insulin(-)] or presence [insulin(+)] of insulin stimulation. *, P < 0.05 relative to WT; n = 8. (Inset) A representative photo. (B) Phosphorylation of IRS-1 was quantitated by immunoblotting with a phosphotyrosine antibody after immunoprecipitation with an IRS-1 antibody (IP: IRS1) in the absence or presence of insulin stimulation. *, P < 0.05 relative to WT, n = 8. (Inset) A representative photo. (C) PI3Kp85, coimmunoprecipitated with IRS-1, was quantitated by immunoblotting with a PI3Kp85 antibody (IB: PI3Kp85) after immunoprecipitation with an IRS-1 antibody (IP: IRS1) in the absence or presence of insulin stimulation. *, P < 0.05 relative to WT, n = 8. (Inset) A representative photo. (D) Phosphorylation of Akt1/PKB was quantitated by immunoblotting with an antiphospho-serine473-Akt1/PKB antibody (IB: pAkt) in the absence or presence of insulin stimulation. *, P < 0.05 relative to WT, n = 4. (Inset) A representative photo. (E) Phosphorylation of IRβ in the liver was quantitated by immunoblotting with a phosphotyrosine antibody (IB: P-Tyr) after immunoprecipitation with an IRβ antibody (IP: IRβ) in the absence or presence of insulin stimulation. (Inset) A representative photo. (F) Expression of Cav1, Cav2, and Cav3 in skeletal muscles was compared between WT and Cav3KO by immunoblotting.
Fig. 2.
Fig. 2.
Unaltered expression and subcellular localization of IRβ and its related molecules in Cav3KO. (A) Protein expression of IRβ, IRS-1, and P13Kp85 in soleus muscle tissues of WT (WT, open bars) and Cav3KO (KO, filled bars). Means ± SEM are shown. n = 8. (Insets) Representative photos. (B) mRNA expression of IRβ in soleus muscle tissues of WT and Cav3KO. GAPDH was used as loading control. Means ± SEM are shown. n = 8. (Inset) A representative photo. (C) Coimmunoprecipitation assays of Cav3 and IRβ. The caveolar fractions from soleus muscle tissues were used for coimmunoprecipitation assays (IP) by using IRβ and Cav3 antibodies, followed by immunodetection (IB) of Cav3 (Upper) and IRβ (Lower), respectively. Arrows indicate Cav3 (Upper) and IRβ (Lower). Nonimmune mouse and rabbit IgGs were used as control. (D and E) Subcellular localization of IRβ (D) or PTP1B (E) in soleus muscle tissues of WT (WT) and Cav3KO (KO). After sucrose gradient centrifugation, IRβ or PTP1B was detected by immunoblotting (arrows).
Fig. 3.
Fig. 3.
Impaired insulin signaling in Cav3KO. (A) Glucose uptake in skeletal muscles. 2-Deoxy-d-[1,2-3H]glucose (2-DG) uptake into soleus muscles was measured for 30 min. The muscle samples from WT (open bars, WT) and Cav3KO (filled bars, KO) were incubated in the absence [insulin(-)] or presence [insulin(+)] of insulin (14 nM), followed by measurements of 2-DG uptake. Means ± SEM are shown. *, P < 0.05 relative to WT; n = 4. (B) Blood glucose levels in glucose tolerance test. Glucose (2 g/kg) was administered i.p., and blood glucose levels were determined 0, 30, 60, 90, and 120 min after injection. Open circles, WT; filled circles, Cav3KO. Arrow indicates the time of glucose injection. *, P < 0.05 relative to WT; n = 8. (C) Blood insulin levels after glucose injection. Insulin concentrations were similarly determined at 0, 15, and 30 min after glucose injection. n = 8. (D) Blood glucose levels in glucose-tolerance test in STZ-treated mice. STZ (100 mg/kg) was administered to WT (open circles) and Cav3KO (filled circles), and the glucose tolerance test was performed after 2 weeks. *, P < 0.05 relative to WT, n = 4.
Fig. 4.
Fig. 4.
Increased serum lipids in Cav3KO. Serum was obtained from overnight-fasted mice and measured for triglycerides (A), free fatty acids (B), and total cholesterol (C). Open bars, WT (WT); filled bars, Cav3KO (KO). Means ± SEM are shown. *, P < 0.05 relative to WT, n = 8.
Fig. 5.
Fig. 5.
Increased insulin signaling in skeletal muscle tissues by Cav3 gene transfer. Adenovirus harboring either Cav3 or GFP as control was injected into soleus muscles of Cav3KO. Insulin was given intravenously 48 h after injection, and soleus muscles were harvested for immunoprecipitation and immunoblotting assays. (A) Phosphorylation of IRβ (P-IR) was quantitated by phosphotyrosine antibody after immunoprecipitation in the absence [insulin(-)] or presence [insulin(+)] of insulin stimulation between control (CT) and Cav3 injection (Cav3). Means ± SEM are shown. *, P < 0.05 relative to WT; n = 6. (Insets) A representative photo of the expression of Cav3 in soleus muscles (Upper) and of the phosphorylation of IRβ in the presence or absence of insulin (Lower). (B) Phosphorylation of IRS-1 was quantitated by phosphotyrosine antibody after immunoprecipitation in the absence or presence of insulin stimulation between control and Cav3 injection. *, P < 0.05 relative to WT; n = 6. (Inset) A representative photo of the phosphorylation of IRS-1 (P-IRS-1) in the presence or absence of insulin.
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