Total cellular protein was extracted using Pierce RIPA Buffer (Thermo Scientific) supplemented with PhosSTOP Protease Inhibitor Cocktail Tablets (Roche Life Science). For the placenta, frozen tissues were thawed for several minutes in pre-chilled RIPA buffer and homogenized with a Polytron (Kinematica). Western blotting was performed by using AnykD Mini-PROTEAN TGX Gels (Biorad, Hercules, CA, USA) and nylon/nitrocellulose membranes (Biorad). Primary and secondary antibodies used were: rabbit anti-IRS1 (1:500, sc-559, Santa Cruz Biotechnology), rabbit anti-GLUT4 (1:1,000, 07-1404, Millipore), mouse anti-β-Actin (1:5,000, Sigma-Aldrich), goat anti-Mouse (sc-2005, Santa Cruz Biotechnology), and/or anti-Rabbit (sc-2004, Santa Cruz Biotechnology), human anti-HRP (1:2,000, Santa Cruz Biotechnology). HRP activity was detected by using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Densitometric analysis and protein quantification of the western blots was performed by using Image Lab Software (version 5.2.1). Blots were stripped briefly in 0.2 M sodium hydroxide and reprobed for mouse anti-β-Actin (Sigma-Aldrich) as a loading control.