Proceedings of The Physiological Society
University of Cambridge (2008) Proc Physiol Soc 11, PC35
Ontogeny of insulin signalling pathways in ovine fetal skeletal muscle during late gestation
J. K. Jellyman1, R. L. Cripps2, M. S. Martin-Gronert2, D. A. Giussani1, S. E. Ozanne2, A. J. Forhead1, A. L. Fowden1
1. Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom. 2. Clinical Biochemistry, University of Cambridge, Cambridge, United Kingdom.
In man and other animals, the incidence of adult insulin resistance is increased when fetal growth is poor, which suggests that insulin sensitivity is determined, in part, prenatally1. In many species, an increase in fetal glucocorticoid concentrations is responsible for prepartum tissue maturation2. Fetal glucocorticoid levels are also raised earlier in gestation by conditions known to impair fetal growth and adult insulin sensitivity3. Little is known about the ontogeny and developmental control of the insulin signalling pathways in utero. This study determined i) the ontogenic changes in these pathways during late gestation and ii) the glucocorticoid dependence of these changes in sheep. After maternal and fetal euthanasia (200 mg/kg, Na pentobarbitone), hind limb skeletal muscle was collected from 4 groups of fetal sheep: 1) untreated controls (n=22) at 110, 120, 130 and 140 days (d) of gestation (term ~145d, n≥ 4 per group), 2) adrenalectomised (AX) at 115d and age-matched controls delivered at 145d (n=6 per group), 3) catheterised and infused with cortisol (2-3 mg/kg/day in saline) or saline for 5 days before delivery at 130d (n=6 per group), 4) maternal dexamethasone (2x12 mg in saline im) or saline treatment before delivery at 127d (n=6 per group). All surgical procedures were carried out under halothane anaesthesia (2%, in O2:N2O). After protein extraction and standardization, abundance of the insulin receptor (IR)-b subunit, insulin-like growth factor type I receptor (IGF-1R), protein kinase C zeta (PKC-ζ) and the insulin-sensitive glucose transporter (GLUT4) were measured by SDS-PAGE and Western Blotting using ovine validated antibodies4 (Santa Cruz, CA or Abcam Ltd, UK). Results are mean (± SE) arbitrary units. Statistical significance was assessed by Student’s t-Test or ANOVA plus Tukey Test, as appropriate. From 110d to 120d, there were increases in muscle protein abundance of IGF-1R (10.10±1.30 to 19.15±1.00, P<0.05) and PKC-ζ (5.57±0.20 to 21.25±3.41, P<0.05) but not IRβ in (17.90±3.30 to 27.65±4.69, P>0.05). At 140d, muscle protein abundance of IR-b (13.65±2.58), IGF-1R b (4.47±0.95) and PKC-ζ (7.04±0.76) were significantly less (P<0.05) than the values at 120d but not 110d. Muscle protein GLUT4 abundance was significantly lower at 130 and 140d (18.34±2.75 and 20.63±3.04, respectively) than at 110 and 120d (72.13±6.37 and 73.28±7.32 respectively; P<0.05). Fetal adrenalectomy, intrafetal cortisol and maternal dexamethasone treatment did not significantly alter muscle abundance of any of the proteins, except PKC-ζ, which was higher in AX (10.58±1.24) than control fetuses (7.04±0.76; P<0.05). These data show that ontogenic changes in the insulin-signalling pathway occur in fetal skeletal muscle towards term but that these are unlikely to be glucocorticoid dependent.
Where applicable, experiments conform with Society ethical requirements