between the wild-type and ob-norm groups (Figure 2d). For animals on a high-fat diet, there was a trend toward decreased activity in the ob-norm group, but this did not reach significance (35.964.3 breaks/min in wild-type versus 27.762.6 breaks/min in the ob/ob norm, p = 0.12). Similarly, we found that there was no significant difference in oxygen consumption, during either the light or dark phases, between wild-type and ob-norm cohorts on the same diet (Figure 2e). Having established that wild-type and ob-norm animals have a similar body weight and physiology when maintained on the same diet, other than a modest impairment of glucose metabolism among ob-norm animals on a high fat diet, we next tested whether long-term exposure to a high- or low-fat diet affected the leptin sensitivity of these two groups. We used two assays for this purpose. First, in order to measure functional sensitivity to leptin, we tested whether a short-term leptin infusion could reduce food intake and body weight in each of the four cohorts. We did this by replacing the mini-osmotic pumps in all animals with pumps that delivered leptin at either the same rate (‘‘vehicle’’) or at a rate 450 ng/h higher. This results in a 4-fold increase in the leptin infusion rate in ob-norm animals (150 ng/h versus 600 ng/h leptin) and has previously been shown to result in a 4-fold increase in plasma leptin in lean wild-type animals[37]. We then measured food intake and body weight for 12 days, at which point the pumps were replaced with pumps dispensing leptin or PBS at the baseline rate. As expected, wild-type mice maintained on a low-fat diet remained sensitive to exogenous leptin, reducing their food intake (15.160.6 kcal/d for vehicle versus 12.560.5 kcal/d for leptin, p,0.05) and showed a progressive reduction of body weight throughout the 12-day infusion (Figure 3). By contrast, wild-type mice that had been maintained on a high-fat diet for 20 weeks Leptin Resistance showed no reduction in food intake or body weight in response to the 450 ng/h leptin infusion (Figure 3). These results are consistent with previous reports showing that prolonged diet- induced obesity induces robust leptin resistance in mice[10]. ob-norm mice that had been maintained on a low-fat diet were sensitive to leptin, and showed a similar reduction of food intake as their wild-type counterparts, (15.261.4 kcal/d for vehicle versus 12.161.1 kcal/d for leptin, p = 0.08) and losing approximately 10 percent of their body mass over the course of the 12-day infusion (22.861.1% for control versus 211.661.0% for leptin, p,0.05). In contrast, and unlike wild-type controls on a high fat diet, ob- norm mice that had been exposed to a high-fat diet remained highly sensitive to exogenous leptin. These animals showed a significantly reduced food intake (18.361.5 kcal/d for vehicle versus 13.860.8 kcal/d for leptin, p,0.05) and body weight (0.660.6% for vehicle versus 27.361.8% for leptin, p,0.05) in response to the leptin infusion. Because wild-type and ob-norm animals differed only in their plasma leptin levels, this result confirms that hyperleptinemia is functionally required for the development of leptin resistance after long-term exposure to a high-fat diet. In a second assay of leptin sensitivity, we next tested whether leptin could stimulate acute phosphorylation of STAT3 in neurons of the mediobasal hypothalamus among the four groups of mice[10,13,38]. Mice were given an intraperitoneal bolus of either vehicle (PBS) or leptin (2 mg/kg) and sacrificed 30 minutes later by cardiac perfusion. This dose of leptin has previously been shown to increase plasma leptin levels by 40-fold[10]. Brains were dissected, and STAT3 phosphorylation was quantified by immunohistochem- istry (Figure 4). Wild-type animals maintained on a low-fat diet showed a robust increase in the number of pSTAT3 positive cells in response to leptin (Figure 4a; 4.061.7 cells for vehicle versus 67615 Figure 3. Leptin sensitivity in a 12-day infusion. A. Body weight of wild-type animals that received a leptin (red) or PBS (black) infusion for 12- days (gray block). B. Body weight of ob-norm animals that received a leptin (red) or PBS (black) infusion for 12-days (gray block). C. Difference in weight loss between leptin and vehicle for each cohort over the 12-day leptin infusion. Values are expressed as percentage change in body weight. D. Average daily food intake during the 12-day infusion for animals in each cohort. doi:10.1371/journal.pone.0011376.g003 PLoS ONE | www.plosone.org 4 June 2010 | Volume 5 | Issue 6 | e11376