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The children adjusted their EIs in response to different preloads, and the younger children did so more effectively than did the older children. Poor short-term energy compensation may constitute a behavioral marker for positive energy balance.

The children adjusted their EIs in response to different preloads, and the younger children did so more effectively than did the older children. Poor short-term energy compensation may constitute a behavioral marker for positive energy balance.

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Aims/hypothesis: Peroxisome proliferator-activated receptor γ is an important regulator of adiposity in mouse and man, and common variation in the PPARG gene has been associated with birthweight, adult obesity, insulin sensitivity and type 2 diabetes. We hypothesised that these variants may be associated with childhood obesity. Methods: Height and weight were recorded for 2454 prepubertal children aged between 4 and 10 years, who were then genotyped for three common variants of the PPARG locus: C-681G, Pro12Ala and C1431T. Results: No single variant of PPARG was significantly associated with height, weight or BMI. However, when modelling the variants together we detected an opposing interaction between the −681G and the Ala12 variants in height and weight, but not BMI (p=0.018, 0.013 and 0.119 respectively). The data were consistent with the Ala12 carriers being deficient in energy storage/utilisation, leading to reduced growth. In contrast, the −681G variant, which has been associated with increased adult height, was associated with accelerated growth. The two variants were in strong linkage disequilibrium. However, rare individuals bearing the isolated variants demonstrated the greatest variation from the mean, the most contrasting genotypes being associated with a variation of 7 kg in weight and 6 cm in height, standardised to 7.4-year-olds (p=0.006 and p=0.02 espectively). Conclusions/interpretation: This study demonstrates that quantitative trait analysis of energy balance/ growth and the PPARG locus is complex and requires the use of multiple genetic markers.

The pivotal role of phosphatidylinositol 3-kinase (PI 3-kinase) in signal transduction has been well established in recent years. Receptor-regulated forms of PI 3-kinase are thought to phosphorylate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) at the 3-position of the inositol ring to give the putative lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ). Cellular levels of PtdIns(3,4,5)P 3 are currently measured by time-consuming procedures involving radiolabeling with high levels of 32 PO 4 , extraction, and multiple chromatography steps. To avoid these lengthy and hazardous procedures, many laboratories prefer to assay PI 3-kinase activity in cell extracts and/or appropriate immunoprecipitates. Such approaches are not readily applied to measurements of PtdIns(3,4,5)P 3 in extracts of animal tissues. Moreover, they can be misleading since the association of PI 3-kinases in molecular complexes is not necessarily correlated with the enzyme's activity state. Direct measurements of PtdIns(3,4,5)P 3 would also be desirable since its concentration may be subject to additional control mechanisms such as activation or inhibition of the phosphatases responsible for PtdIns(3,4,5)P 3 metabolism. We now report a simple, reproducible isotope dilution assay which detects PtdIns(3,4,5)P 3 at subpicomole sensitivity, suitable for measurements of both basal and stimulated levels of PtdIns(3,4,5)P 3 obtained from samples containing approximately 1 mg of cellular protein. Total lipid extracts, containing PtdIns(3,4,5)P 3 , are first subjected to alkaline hydrolysis which results in the release of the polar head group Ins(1,3,4,5)P 4 . The latter is measured by its ability to displace [ 32 P]Ins(1,3,4,5)P 4 from a highly specific binding protein present in cerebellar membrane preparations. We show that this assay solely detects PtdIns(3,4,5)P 3 and does not suffer from interference by other compounds generated after alkaline hydrolysis of total cellular lipids. Measurements on a wide range of cells, including rat-1 fibroblasts, 1321N1 astrocytoma cells, HEK 293 cells, and rat adipocytes, show wortmannin-sensitive increased levels of PtdIns(3,4,5)P 3 upon stimulation with appropriate agonists. The enhanced utility of this procedure is further demonstrated by measurements of PtdIns(3,4,5)P 3 levels in tissue derived from whole animals. Specifically, we show that stimulation with insulin increases PtdIns(3,4,5)P 3 levels in rat skeletal muscle in vivo with a time course which parallels the activation of protein kinase B in the same samples.PI 3-kinases 1 represent a family of enzymes which phosphorylate phosphoinositides on the 3-position of the inositol ring (1). The role of PI 3-kinases in mitogenic signaling, membrane ruffling, trafficking, cell motility, inflammatory and immune cell responses, activation of neutrophils, and the metabolic effects of insulin (2-8) has been well established using biochemical, pharmacological, and genetic approaches. At least two major classes of PI 3-kinas...

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