indistinguishable in the two groups (Figure 1b). The body weights of wild-type and ob-norm animals were similar after 20 weeks on a high-fat diet (52.660.4 g in wild-type versus 52.161.8 g in ob- norm, p = 0.79). As expected, the plasma leptin levels of animals in these two groups were very different (Figure 1f). Wild-type animals became markedly hyperleptinemic as they became obese (37.465.6 ng/mL at 18 weeks of age and 15466 ng/mL at 26 weeks of age), whereas the plasma leptin levels of ob-norm animals on a high-fat diet were the same as low-fat diet, chow fed controls (e.g., 7.062.3 ng/mL on a low-fat diet versus 8.162.3 ng/mL on a high-fat diet at 26 weeks of age, p = 0.75). The nearly identical body weight trajectory of wild-type and ob- norm animals on a high-fat diet was somewhat unexpected: unlike wild-type animals, the weight gain of ob-norm mice is not restrained by increases in plasma leptin. We measured weekly food intake in both cohorts after exposure to a high-fat diet, and while the ob-norm animals did eat more food than wild-type animals at a subset of time points (Figure 1e; p,0.01), this slightly elevated food intake did not translate into increased body weight or adiposity (Figure 1c). These data show that wild-type and ob-norm animals display a very similar progression of diet-induced obesity in response to a high-fat diet, despite the fact that they maintain a ,20-fold difference in plasma leptin levels. Diet-induced obesity causes insulin resistance, whereas leptin improves glucose homeostasis independent of its effects on body weight[33,34,35,36]. We therefore compared glucose metabolism in the four groups of animals in this experiment. Both wild-type and ob-norm animals on a high-fat diet developed hyperglycemia relative to low-fat diet controls (Figure 2a; p,0.001), and there was no significant difference in fasting blood glucose between cohorts on the same diet (p.0.15). We measured plasma insulin Leptin Resistance levels in all four groups at 18 and 26 weeks of age (Figure 2b). Both wild-type and ob-norm animals on a high-fat diet were hyperinsulinemic relative to low-fat diet controls (p,0.05), confirming that their hyperglycemia was a consequence of insulin resistance. By contrast, there was no significant difference in plasma insulin between cohorts on the same diet (p.0.24). These data indicate that the differences in blood glucose and insulin levels were determined by diet, not plasma leptin levels. We performed glucose tolerance tests to measure the ability of each cohort to clear a bolus of sugar from the blood (Figure 2c). For mice on a low-fat diet, plasma glucose levels were indistinguishable between wild-type and ob-norm cohorts throughout the test (p.0.5). This confirms that leptin replacement at physiological levels was sufficient to normalize glucose homeostasis in the lean ob-norm animals. By contrast, both cohorts exposed to a high-fat diet showed delayed glucose clearance (Figure 2c; p,0.01). The magnitude of this impairment was greater in ob-norm animals relative to wild-type controls at 60 and 120 minutes (Figure 2c; p,0.01). Thus, while the high-fat fed wild-type and ob-norm animals have similar fasting hyperglycemia and hyperinsulinemia, the ob-norm animals have an additional defect in glucose metabolism that is revealed by glucose challenge suggesting that their relative leptin deficiency may further impair glucose metabolism of DIO mice. Leptin-deficient ob/ob mice are hypoactive and have reduced energy expenditure. We therefore performed respirometry and activity measurements to compare the four groups of animals in this experiment (Figure 2). Activity was quantified by measuring beam breaks in three dimensions in animals that had been acclimated in metabolic cages. For the groups on a low-fat diet, there was no significant difference in average beam breaks Figure 2. Glucose metabolism and energy expenditure. A. Plasma glucose concentrations in ad libitum fed wild-type and ob-norm mice that were maintained on a high-fat diet (open bars) or low-fat diet (filled bars). B. Plasma insulin concentrations of wild-type and ob-norm mice at 18 and 26 weeks of age that were maintained on a high-fat diet (open bars) or low-fat diet (filled bars). C. Plasma glucose concentrations in response to a glucose injection in wild-type (black) and ob-norm (red) mice. Triangles represent mice on a low-fat diet and squares represent mice on a high-fat diet. * indicates p,0.05. D. Average beam breaks per minute for wild-type and ob-norm mice maintained on a low-fat (filled bar) or high-fat (open bar) diet. E. Average oxygen consumption for wild-type and ob-norm mice in the light phase (open bars) and dark phase (closed bars). doi:10.1371/journal.pone.0011376.g002 PLoS ONE | www.plosone.org 3 June 2010 | Volume 5 | Issue 6 | e11376