Increased p85/55/50 Expression and Decreased Phosphotidylinositol 3-Kinase Activity in Insulin-Resistant Human Skeletal Muscle
Article Figures & Tables
Figures
- FIG. 1.
PI 3-kinase regulatory subunits in human muscle. A: A representative Western blot analysis of the basal muscle homogenates with a pan anti-p85 antibody and antiactin antibody. B: The ratio subunit to actin in lean, obese (Ob), and type 2 diabetic subjects for individual subunits as well as for combined subunits. *P < 0.01, **P < 0.001 vs. lean.
- FIG. 2.
Phosphorylation of IRS-1. A: A representative Western blot for pSer307 IRS-1 and pY IRS-1. Areas representing insulin-stimulated levels of pSer307 IRS-1/total IRS-1 (B) and pY-IRS-1/total IRS-1 (C) in lean, obese (Ob), and type 2 diabetic subjects are shown. *P < 0.001 vs. lean basal; **P < 0.001 vs. lean insulin stimulated.
- FIG. 3.
Interaction of p85 and p110 with IRS-1. A: Western blot analysis of IRS-1 bound p85 and p110 in the IRS-1 immunoprecipitates is shown. The signal intensities showing insulin-stimulated binding of p110 to IRS-1 (B) and p85 to IRS-1 (C) are shown. *P < 0.05, **P < 0.001 vs. lean. Ob, obese.
- FIG. 4.
PI 3-kinase activity. A: A representative autoradiogram showing PI 3-phosphate (PI-3P) formed by the kinase activity in the anti-IRS-1 immunoprecipitates. B: Quantitative comparison of insulin-stimulated PI 3-kinase activity in human muscle from lean, obese (Ob), and type 2 diabetic subjects. *P < 0.05, **P < 0.01 vs. lean. CPM, counts per minute.
- FIG. 5.
Insulin-stimulated activation of PKC-ζ/λ. Insulin-stimulated PKC activity was measured in the immune agarose beads obtained after immunoprecipitation of PKC-ζ/λ from the lysates with anti–COOH-terminal PKC-ζ antiserum. *P < 0.01 vs. lean basal; **P < 0.005 vs. lean insulin stimulated. CPM, counts per minute; Ob, obese.
- FIG. 6.
Insulin-stimulated phosphorylation of Akt in human muscle. Comparison of insulin-stimulated phosphorylation of Thr (308)-Akt (A and B) and Ser (473)-Akt (C and D) in human muscle from lean, obese (Ob), and type 2 diabetic subjects.
- FIG. 7.
Insulin-stimulated PKC-θ activity and phosphorylation of JNK-1 and IKK-β. PKC-θ activities in the lysates from muscle obtained before and after insulin infusion (A) and the cytosol and membrane fractions (B). *P < 0.01 vs. lean basal. Representative Western blots for phospho-JNK (p-JNK) and total JNK (C) and phospho-IKK-β (pIKKβ) and total IKK-β signals (E). Results from densitometric scans for pJNK (D) and pIKK-β signals (F). *P < 0.05, **P < 0.01 vs. lean basal. Ob, obese.
- FIG. 8.
Insulin-stimulated phosphorylation of ERK-1 and p38 kinase. Representative Western blots for phospho-ERK (A) and phospho-p38 (C). B and D: The results after densitometric quantitation. *P < 0.05 vs. lean basal (B) or P = 0.05 vs. lean basal (D). **P < 0.05 vs. lean basal; §P < 0.01 vs. lean insulin stimulated.
Tables
- TABLE 1
Subject characteristics
Nondiabetic Obese Type 2 diabetic n 8 8 7 Age (years) 47.25 ± 4.8 43.5 ± 4.0 48.0 ± 5.2 Weight (kg) 75.2 ± 5.4 96.7 ± 4.0 110.0 ± 5.4 BMI (kg/m2) 24.4 ± 1.2 33.3 ± 1.06* 37.1 ± 2.64* Fasting plasma glucose (mmol/l) 5.09 ± 0.12 5.1 ± 0.1 11.3 ± 1.5* Glucose infusion rate (mg · kg−1 · min−1) 9.9 ± 0.93 7.0 ± 0.37† 2.26 ± 0.76* Basal insulin (pmol/l) 66.7 ± 10 125.36 ± 26‡ 315 ± 78‡ Insulin after 300-min clamping (pmol/l) 1,070 ± 47 1,290 ± 96 1,090 ± 74 Basal plasma free fatty acids (μmol/l) 346 ± 36 400 ± 30§ 750 ± 120‡ Free fatty acids after insulin clamping (μmol/l) 19 ± 3.8 16 ± 3.5 270 ± 100 Triacylglycerol (mg/dl) 88.75 ± 6.8 128.6 ± 21‖ 390 ± 127† Data are means ± SE. All P values were determined by comparing with nondiabetic subjects.
- *
* P < 0.005;
- †
† P < 0.05;
- ‡
‡ P < 0.02;
- §
§ NS;
- ‖
‖ P = 0.05.
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