The ability of insulin to suppress gluconeogenesis in type II diabetes mellitus is impaired; however, the cellular mechanisms for this insulin resistance remain poorly understood. To address this question, we generated transgenic (TG) mice overexpressing the phosphoenolpyruvate carboxykinase (PEPCK) gene under control of its own promoter. TG mice had increased basal hepatic glucose production (HGP), but normal levels of plasma free fatty acids (FFAs) and whole-body glucose disposal during a hyperinsulinemic-euglycemic clamp compared with wild-type controls. The steadystate levels of PEPCK and glucose-6-phosphatase mRNAs were elevated in livers of TG mice and were resistant to down-regulation by insulin. Conversely, GLUT2 and glucokinase mRNA levels were appropriately regulated by insulin, suggesting that insulin resistance is selective to gluconeogenic gene expression. Insulin-stimulated phosphorylation of the insulin receptor, insulin receptor substrate (IRS)-1, and associated phosphatidylinositol 3-kinase were normal in TG mice, whereas IRS-2 protein and phosphorylation were down-regulated compared with control mice. These results establish that a modest (2-fold) increase in PEPCK gene expression in vivo is sufficient to increase HGP without affecting FFA concentrations. Furthermore, these results demonstrate that PEPCK overexpression results in a metabolic pattern that increases glucose-6-phosphatase mRNA and results in a selective decrease in IRS-2 protein, decreased phosphatidylinositol 3-kinase activity, and reduced ability of insulin to suppress gluconeogenic gene expression. However, acute suppression of HGP and glycolytic gene expression remained intact, suggesting that FFA and/or IRS-1 signaling, in addition to reduced IRS-2, plays an important role in downstream insulin signal transduction pathways involved in control of gluconeogenesis and progression to type II diabetes mellitus.Type II diabetes mellitus is a complex metabolic disease with an environmental and genetic component. Hyperglycemia develops for reasons that are not completely understood; however, a prominent defect is the inability of insulin to inhibit hepatic glucose production (HGP).1 Increased gluconeogenesis is believed to be the major cause of increased HGP and fasting hyperglycemia in type II diabetes mellitus patients (1). Gluconeogenesis is a highly regulated process, catalyzed by several enzymes subject to regulation by insulin. The first site of regulation, the conversion of oxalacetate to phosphoenolpyruvate, is catalyzed by the enzyme phosphoenolpyruvate carboxykinase (PEPCK). PEPCK is considered a key rate-controlling enzyme in the pathway of gluconeogenesis from pyruvate, lactate, and alanine (2). Normally, insulin rapidly and substantially inhibits PEPCK gene transcription and gluconeogenesis in liver and in rat hepatoma cells. However, in several animal models of obesity and type II diabetes, gluconeogenesis and PEPCK mRNA are increased by 2-3-fold over non-diabetic animals, despite circulating insulin levels that may be ...

Nanoparticles consisting of single molecules of DNA condensed with polyethylene glycol-substituted lysine 30-mers efficiently transfect lung epithelium following intrapulmonary administration. Nanoparticles formulated with lysine polymers having different counterions at the time of DNA mixing have distinct geometric shapes: trifluoroacetate or acetate counterions produce ellipsoids or rods, respectively. Based on intracytoplasmic microinjection studies, nanoparticle ellipsoids having a minimum diameter less than the 25 nm nuclear membrane pore efficiently transfect nondividing cells. This 25 nm size restriction corresponds to a 5.8 kbp plasmid when compacted into spheroids, whereas the 8-11 nm diameter of rod-like particles is smaller than the nuclear pore diameter. In mice, up to 50% of lung cells are transfected after dosing with a rod-like compacted 6.9 kbp lacZ expression plasmid, and correction of the CFTR chloride channel was observed in humans following intranasal administration of a rod-like compacted 8.3 kbp plasmid. To further investigate the potential size and shape limitations of DNA nanoparticles for in vivo lung delivery, reporter gene activity of ellipsoidal and rod-like compacted luciferase plasmids ranging in size between 5.3 and 20.2 kbp was investigated. Equivalent molar reporter gene activities were observed for each formulation, indicating that microinjection size limitations do not apply to the in vivo gene transfer setting. Gene Therapy (2006) The structural features of gene transfer complexes can be optimized to facilitate efficient gene transfer in vivo. We have developed nanoparticles consisting of single molecules of plasmid DNA condensed with polyethylene glycol (PEG)-substituted lysine peptides.1 The volume, shape and size dimensions of these DNA nanoparticles depend on several parameters, including plasmid size and the lysine counterion present at the time of DNA mixing.2 For example, lysine polymers containing trifluoroacetate and acetate counterions result in the formation of ellipsoidal and rod-like nanoparticles, with the volume of these complexes closely predicted by the partial specific volumes of the constituent components. 1,2For ellipsoidal nanoparticles, microinjection studies indicated a size limitation for nuclear access and transgene expression, with nanoparticles having a minor diameter approaching 25 nm having decreased efficiency. This 25 nm size restriction corresponds to a 5.8 kbp plasmid when compacted into spheroids. In contrast, the diameter of rod-like nanoparticles ranges between 8 and 11 nm for a series of plasmid sizes, whereas the length of the rod is linearly proportional to the plasmid molecular weight. Because the nuclear membrane pore diameter is approximately 25 nm, these data suggested a size dimension that may limit nuclear transit in these microinjection studies. For intrapulmonary delivery of DNA nanoparticles in mice, efficient gene transfer has been observed for larger plasmids using rod-like formulations. For example, up to 50% of lung cells ar...

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.

mice. These results demonstrate that C/EBP␤ deletion decreases plasma FFA levels and increases insulin signal transduction specifically in skeletal muscle, and both contribute to increased whole-body insulin sensitivity.

Gestational diabetes mellitus (GDM) results from an interaction between susceptibility genes and the diabetogenic effects of pregnancy. During pregnancy, mice heterozygous for the lepin receptor (db/+) gain more weight, are glucose intolerant, and produce macrosomic fetuses compared with wild-type (+/+) mothers, suggesting that an alteration in leptin action may play a role in GDM and fetal overgrowth. To investigate whether leptin administration or pair-feeding can reduce adiposity and thereby prevent GDM and neonatal overgrowth, we examined energy balance, glucose and insulin tolerance, and fetal growth in pregnant db/+ and +/+ mice treated with recombinant human leptin-IgG during late pregnancy. Leptin reduced food intake and adiposity in pregnant db/+ mice to levels similar to pregnant +/+ mice and significantly reduced maternal weight gain. Maternal glucose levels were markedly lower during glucose and insulin challenge tests in leptin-treated db/+ mice relative to db/+ and pair-fed controls. Despite reduced energy intake and improved glucose tolerance, leptin administration did not reduce fetal overgrowth in offspring from db/+ mothers. Fetal and placental leptin levels were 1.3- to 1.5-fold higher in offspring from db/+ mothers and remained unchanged with leptin administration, whereas leptin treatment in +/+ mothers or pair-feeding decreased placental leptin concentration and reduced fetal birth weight. Our results provide evidence that leptin administration during late gestation can reduce adiposity and improve glucose tolerance in the db/+ mouse model of spontaneous GDM. However, fetal and placenta leptin levels are higher in db/+ mothers and are subject to reduced negative feedback in response to leptin treatment. These data suggest that alterations in placenta leptin may contribute to the regulation of fetal growth independently of maternal glucose levels.