Improved Insulin Sensitivity and Resistance to Weight Gain in Mice Null for the Ahsg Gene
Article Figures & Tables
Figures
- FIG. 1.
IR autophosphorylation and TK activity. In vitro IR autophosphorylation (A) and IRTK activity (B) were determined from IRs partially purified from liver membrane fraction on WGA. A: A representative autoradiograph of in vitro IR β-subunit autophosphorylation (PY) (basal or in the presence of 1, 10, or 100 nmol/l insulin) (upper panel). Four separate experiments were performed, with IRs purified individually from livers of weight-matched, 8- to 10-week-old male WT and KO mice; n = 4 per genotype. Western blot of IR β-subunit confirmed equal loading of IR (lower panel), and the result of the combined data of four separate experiments is shown (bar diagram). To assess the status of in vivo basal and insulin-induced IR autophosphorylation, saline or insulin (0.1, 1, or 10 μmol/l) was injected through the portal vein of weight-matched, 8- to 10-week-old male WT and KO mice. IR was immunoprecipitated from liver (C) or muscle (D) homogenates with anti-IR β-subunit antibody and immunoblotted with anti-phosphotyrosine antibody. Samples were normalized for loading by assaying total level of IR β-subunit. The quantified data (ratio of IR autophosphorylation to total level of IR β-subunit) are shown as bar diagrams (mean ± SE; n = 4 mice per genotype). * P < 0.05, **P < 0.01, ***P < 0.001, WT vs. KO.
- FIG. 2.
Insulin signal transduction. A: Liver homogenates from saline- or insulin-injected (0.1, 1, or 10 μmol/l) weight-matched, 8- to 10-week-old male WT and KO mice were resolved on SDS-PAGE, transferred, and detected by chemiluminescence with antibodies against phospho-MAPK or phospho-Akt. Membranes were stripped and blotted for ERK2 and Akt1, respectively, to normalize for sample loading. A representative blot (from four to five separate experiments) for each protein is depicted. B: Hindlimb muscle homogenates were resolved on SDS-PAGE, transferred, and detected by chemiluminescence with antibodies against phospho-MAPK or phospho-Akt. Membranes were stripped and blotted for ERK2 and Akt1, respectively, to normalize for sample loading. A representative blot (from four to five separate experiments) for each protein is depicted.
- FIG. 3.
Glucose and insulin tolerance tests in KO and WT mice. After an overnight fast, an oral glucose load (1 mg/g) (A and B) or i.p. glucose load (1.5 mg/g) (C and D) was given to 10-week-old fetuin KO and WT mice. Insulin tolerance tests were done on fed (random) mice using an i.p. injection of 0.75 units/kg (E) or 0.15 units/kg (F) regular human insulin. Blood glucose (A, B, C, E, F) or plasma insulin (D) was measured as described in research design and methods. Results shown are from either male or female mice, as similar findings were observed in both male and female mice. Data are means ± SE. *P < 0.05, **P < 0.01, *** P < 0.001, WT vs. KO.
- FIG. 4.
Euglycemic-hyperinsulinemic clamp studies in conscious KO and WT mice. Glucose infusion rate (A) and 2-DOG uptake in white adipose tissue, soleus, and gastrocnemius muscles (B) were determined using the euglycemic-hyperinsulinemic clamp technique in fasted 12- to 16-week-old male mice. Tissue glycogen content (C) was assayed at the end of the euglycemic-hyperinsulinemic clamp. Results are means ± SE for five animals per genotype. *P < 0.05, WT vs. KO.
- FIG. 5.
Plasma insulin and HOMA in WT and KO mice fed LF or HF diets. After an overnight fast, HF- or LF-fed (9 weeks) fetuin KO and WT mice were given an i.p. glucose tolerance test (1.5 mg glucose/g), and blood glucose and plasma insulin concentrations were measured. Results are means ± SE. A: *P < 0.05, WTHF vs. KOHF. B: *P < 0.05, WTHF vs. WTLF, KOHF, or KOLF.
Tables
- TABLE 1
Body weight, blood glucose, and plasma measurements in 10-week-old KO and WT mice
Wild type Knockout P Body weight (g) 17.9 ± 0.8 (11) 13.9 ± 0.9 (10) <0.01 Fasting blood glucose (mg/dl) 79.8 ± 5.9 (9) 98.3 ± 7.7 (10) 0.245 Fed blood glucose (mg/dl) 120.2 ± 6.4 (11) 107.0 ± 10.0 (10) 0.272 Fasting insulin (ng/ml) 0.177 ± 0.01 (11) 0.191 ± 0.01 (10) 0.337 Fed insulin (ng/ml) 0.213 ± 0.02 (7) 0.187 ± 0.02 (8) 0.448 Fasting FFAs (mEq/l) 0.827 ± 0.06 (11) 0.599 ± .03 (11) <0.001 Fasting triglycerides (mg/dl) 59.52 ± 1.71 (12) 42.31 ± 4.33 (11) <0.001 Fasting leptin (ng/ml) 1.33 ± 0.07 (12) 1.63 ± 0.46 (12) 0.068 Data are means ± SE (number of animals). Body weight, glucose, and insulin were measured in male mice; FFAs, triglycerides, and leptin were measured in female mice.
- TABLE 2
Body weight parameters, blood glucose, plasma insulin in WT and KO mice fed LF or HF diets
WTHF WTLF KOHF KOLF P Genotype Diet n 11 11 11 10 Body weight (g) 28.1 ± 0.9a 24.3 ± 0.9b 24.4 ± 1.0b 22.5 ± 0.9b <0.01 <0.005 Weight/length (g/cm) 2.85 ± 0.09a 2.55 ± 0.07b 2.5 ± 0.1b 2.39 ± 0.08b <0.01 <0.05 Liver weight (g) 1.07 ± 0.04 1.1 ± 0.04 1.00 ± 0.04 1.07 ± 0.03 NS NS Total fat weight (g) 5.8 ± 2.1a 3.4 ± 1.9b 3.7 ± 2.1b 2.62 ± 1.1b <0.01 <0.005 Total fat (%) 20.1 ± 0.6a 13.5 ± 0.6b 14.4 ± 0.6b 11.4 ± 0.4b <0.01 <0.005 Food intake (kcal) 6,045 ± 180a 3,505 ± 36b 5,652 ± 499a 3,713 ± 229b NS <0.01 Fasting glucose (mg/dl) 93.64 ± 7.1 83.64 ± 7.2 84.91 ± 3.9 80.8 ± 4.8 NS NS Fasting insulin (ng/ml) 0.36 ± 0.03a (n = 8) 0.32 ± 0.01 (n = 9) 0.28 ± 0.01b (n = 9) 0.30 ± 0.01 (n = 8) <0.05 NS Data are means ± SE. Fetuin KO and WT mice (females, 10 weeks old) were fed HF or LF diets ad libitum for 9 weeks and were monitored weekly for food intake. End-of-study body weight parameters and total caloric intake are shown. Values were dissimilar superscript letters (a or b) are significantly different from each other based on genotype or diet.
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