INSULIN SIGNALING AND AMP-ACTIVATED PROTEIN KINASE ARE DOWN-REGULATED IN SKELETAL MUSCLE OF OVERNOURISHED, OBESE PREGNANT SHEEP (2007)

In the USA, estimates suggest that 18–35% of pregnant women are clinically obese. More importantly, a shift towards higher gestational weight gain appears evident resulting from the excessive nutrient intake during gestation in affluent countries. This pregnancy-associated increase in overweight and obese women is accompanied by a 5-fold increase in gestational diabetes mellitus. The objective of this study was to evaluate the effects of maternal obesity and over-nutrition on insulin signaling and AMP-activated protein kinase (AMPK) activity in skeletal muscle using a pregnant sheep model. 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 maternal semitendenosus (St) muscle was sampled for analyses. Phosphorylated and unphosphorylated forms of key signaling proteins were measured by western analysis with appropriate antibodies. Intramuscular triglyceride (IMTG) content was measured following extraction and saponification in 4% ethanolic KOH as free glycerol and expressed as the percentage of muscle weight. Sections were stained for intramyocellular fat by oil-red O. Insulin was measured by RIA. RESULTS: Ewes on the OB diet increased their body weight by ∼30% from diet initiation to mating (P<0.05) and increased an additional ∼13% in body weight from mating to necropsy on day 75 of gestation (P<0.05). In contrast, control fed ewes maintained similar body weights from diet initiation to necropsy. At necropsy, maternal plasma glucose was 52.1 ± 3.4 mg/dl and 70.2 ± 6.3 mg/dl and plasma insulin was 4.8 ± 1.6 IU/ml and 25.0 ± 4.0 IU/ml in Con and OB mothers respectively. However, insulin signaling was down-regulated in skeletal muscle of OB sheep in the presence of a circulating high insulin concentration. Phosphorylation of Akt at Ser 473 was reduced by 45.0 ± 5.4% (p<0.05), mammalian target of rapamycin (mTOR) at Ser 2448 was reduced by 39.9 ± 3.3% (p<0.05), and phospho-eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) at Thr 37/46 was reduced by 37.7 ± 8.5% (p<0.05) in OB sheep compared to Con sheep. The phosphorylation of AMPK at Thr 172 was reduced by 14.9 ± 4.2% (p<0.05) and acetyl-CoA carboxylase (ACC) at Ser79 was reduced by16.3 ± 1.7% (p<0.05) by the OB diet, as was phosphorylation of insulin receptor substrate-1 (IRS-1) at Ser 789 by 20.3 ± 5.6% (p<0.05). The expression of peroxisome proliferators-activated receptor-gamma (PPARgamma) and the content of intramuscular fat was increased by 61.9 ± 9.5% (p<0.05) in OB muscle compared to Con muscle. Finally, histological staining showed an increase of fat within the skeletal muscle fibers – a situation that is associated with increased insulin resistance. CONCLUSIONS: These data demonstrate that in the presence of the high energy intake and maternal obesity, maternal skeletal muscle of OB sheep showed a higher triglyceride and fat content and developed insulin resistance. Obese pregnant sheep provide a good model for studying pathophysiological changes in gestational diabetes in obese women. NIH INBRE 1P20RR16474 and USDA 2006-55618-16914 (platform)