During gestation, heterozygous C57BLKS/J-Lepr(db/+) mice develop spontaneous gestational diabetes mellitus (GDM), and the newborn fetuses are macrosomic compared with offspring from wild-type (+/+) mothers. To investigate the effects of the leptin receptor mutation on maternal metabolism and fetal growth during pregnancy, we studied +/+, db/+, and db/+ transgenic mice that overexpress the human GLUT4 gene two- to three-fold (db/+TG6). During pregnancy, fasting plasma glucose and hepatic glucose production were twofold greater in db/+ than +/+ mice, despite similar insulin levels. In skeletal muscle, insulin-stimulated tyrosine phosphorylation was decreased in pregnant +/+ mice, and even more so in db/+ mice: insulin receptor beta (IR-beta), +/+ 34%, db/+ 57% decrease, P<0.05; insulin receptor substrate 1 (IRS-1), +/+ 44%, db/+ 61% decrease, P<0.05; and phosphoinositol (PI) 3-kinase (p85alpha), +/+ 33%, db/+ 65% decrease, P<0.05. Overexpression of GLUT4 in db/+TG6 mice markedly improved glucose-stimulated insulin secretion, by 250%, and increased IRbeta, IRS-1, and p85alpha phosphorylation twofold, despite no change in concentration of these proteins. Plasma leptin concentration increased 40-fold during pregnancy, from 2.2+/-0.5 to 92+/-11 ng/ml and 3.6+/-0.1 to 178+/-34 ng/ml in +/+ and db/+ mice, respectively (P<0.01), but was increased to only 23+/-3 ng/ml in pregnant db/+TG6 mice (P<0.001). Maternal fat mass and energy intake were greater in db/+ mice, and fat mass was reduced by GLUT4 overexpression, independent of food intake. Fetal body weight was increased by 8.1 and 7.9% in db/+ and db/+TG6 mothers, respectively (P<0.05), regardless of fetal genotype, whereas fetuses from db/+TG8 mothers (four- to fivefold overexpression) weighed significantly less compared with pups from +/+ or db/+ mothers (P<0.05). These results suggest that the single mutant db allele effects susceptibility to GDM through abnormalities in insulin receptor signaling, defective insulin secretion, and greater nutrient availability. GLUT4 overexpression markedly improves insulin-signaling in GDM, resulting in increased insulin secretion and improved glycemic control. However, maternal hyperglycemia appears not to be the sole cause of fetal macrosomia. These data suggest that GDM is associated with defects in insulin receptor signaling in maternal skeletal muscle, and this may be an important factor provoking maternal and fetal perinatal complications.