DOWN-REGULATION OF AMP-ACTIVATED PROTEIN KINASE IN FETAL MUSCLE OF OBESE, OVERNOURISHED PREGNANT SHEEP

Abstract

Estimates suggest that 18-35% of pregnant women in the USA, are clinically obese. This pregnancy-associated increase in overweight and obese women is accompanied by a 5-fold increase in gestational diabetes mellitus. Babies of obese mothers are macrosomic even in the absence of gestational diabetes. For ethical reasons it is impossible to evaluate the pathophysiological effects on specific signaling pathways in human fetal tissues that result from the adverse fetal metabolic environment due to maternal obesity. The objective of this study was to assess any alteration of AMP-activated protein kinase (AMPK) and insulin signalling in skeletal muscle of fetuses of obese ewes on a high caloric intake. METHODS: Non pregnant ewes were randomly assigned to a control (Con, 100% of NRC recommendations, n=7) or obesogenic (OB, 150% of NRC, n=7) diet from 60 days before to 75 days after conception at which time ewes were euthanized with an overdose of sodium pentobarbital. The fetal longissimus dorsi (Ld) muscle was collected at necropsy. Phosphorylated and unphosphorylated forms of key signaling proteins were measured by western analysis with appropriate antibodies. Insulin was measured by RIA. RESULTS: Body weight and crown rump length of fetuses of OB ewes were ∼30% greater (P<0.05) than fetuses from Con ewes (374 ± 10 g and 24.6 ± 0.2 cm vs. 268 ± 12 g and 22.1 ± 0.2 cm, respectively, P<0.05). While the weights of most fetal tissues from OB ewes were greater (P<0.05) than fetuses of CON ewes, only pancreatic weight was heavier as a percentage of fetal weight in fetuses from ewes on the OB versus the Con diet (P<0.05). At necropsy, fetal plasma glucose was 25.35 ± 2.10 mg/dl and 43.63 ± 5.58 mg/dl and plasma insulin was 1.14 ± 0.35 IU/ml and 7.29 ± 1.31 IU/ml in fetuses of Con and OB mothers respectively (p<0.05). Phosphorylated AMPK at Thr 172 (the active form) was down-regulated by 14.9 ± 4.2 % (p<0.05) in the fetal muscle of OB ewes. Phosphorylation of acetyl-CoA carboxylase (ACC), a down-stream target of AMPK was also reduced by 18.9 ± 7.1% (p<0.05) in OB compared to Con fetal muscle. The expression of PPARgamma, a marker of adipogenesis, was higher by 18.2 ± 3.4% (p<0.05) in the fetal muscle from OB compared to Con pregnancies. No alteration in the content of total insulin receptor in fetal muscle was observed but the phosphorylation of insulin receptor at Tyr 1146 was higher (85.4 ± 17.7%, p<0.01) in OB fetal muscle. Despite increased activation of the insulin receptor, down-stream insulin signaling was down-regulated in fetal muscle of OB ewes. The phosphorylation of Akt at Ser 473, a key mediator of insulin signaling, was 41.2 ± 6.7% lower in OB fetal muscle. mTOR phosphorylation at Ser 2448, a site mediated by Akt, was reduced for 29.1 ± 8.1%, as was 4E-BP1 (28.9 ± 5.9%, p<0.05). Phosphorylation of tuberous sclerosis 2 (TSC2) at Thr 1462, a mediator between Akt and mTOR, was also reduced for 27.5 ± 7.3% (p<0.05). CONCLUSIONS: These data clearly show that OB fetal muscle developed insulin resistance. Because AMPK phosphorylates insulin receptor substrate-1 (IRS-1) at Ser 789 which enhances the function of IRS-1, we propose that the low AMPK activity in OB fetal muscle reduced the insulin sensitivity by down-regulation of IRS-1 function, which could be at least partially responsible for the observed insulin resistance in OB fetal muscle. USDA 2006-55618-16914 and NIH INBRE 1P20RR16474 (platform)