responsive neurons[23,24,25,26,27,28]. This model is supported by the fact that pharmacological or genetic modulation of fat metabolism in the hypothalamus has been shown to influence energy balance and leptin sensitivity[26,29,30]. Moreover, leptin resistance is known to develop most strongly in the arcuate nucleus of the hypothalamus, which relative to other regions of the brain has enhanced access to circulating nutrients[13]. In addition, it has been observed in some[10,31], but not all[32], experimental settings that mice fed a high-fat diet fat do not consume more calories than mice fed a low-fat diet; this implies that dietary fat itself, rather than increased energy intake, may be responsible for leptin resistance in these animals. In order to distinguish between these two possibilities, we separated the contributions of hyperleptinemia and dietary fat to the development of leptin resistance by (1) using osmotic infusion pumps to clamp the plasma leptin of ob/ob mice to the level found in lean wild-type animals over the long term, and then (2) measuring the leptin sensitivity of these animals after being placed on either a low- or high-fat diet. Results The development of central leptin resistance in C57Bl/6J mice requires exposure to a high-fat diet for 20 weeks[10,14]. To establish the possible contribution of hyperleptinemia versus a high fat diet itself to the development of leptin resistance, we developed an experimental protocol in which, beginning at weaning, the plasma leptin levels of ob/ob mice could be fixed to the level of lean wild-type mice (,5 ng/mL) for this duration (Figure 1a). We performed extensive dose response studies infusing leptin into ob/ ob mice via osmotic infusion pumps and found that wild type plasma leptin levels of approximately 5 ng/mL in ob/ob mice Leptin Resistance could be achieved by delivering leptin at 150 ng/h. This infusion rate could also be stably maintained for longer than six months by replacement of the pumps every 28 days. Pumps dispensing 150 ng/h leptin were implanted in male ob/ ob mice at four weeks of age (hereafter referred to as ‘‘ob-norm’’), and leptin treatment normalized the body weight of these animals within three weeks (Figure 1b). Identical pumps dispensing vehicle (PBS) were implanted in a control group of male wild-type mice. At six weeks of age, animals from each group were assigned to either a high-fat diet (60% of calories from fat) or a low-fat chow diet (13% of calories from fat) and maintained on this diet for 20 weeks with monthly pump replacements. The body weight of the ob-norm and wild-type mice on the low- fat diet was similar throughout the course of the experiment (Figure 1b). There was no significant difference in average weekly food intake between the two groups (Figure 1d; 13.560.3 kcal/d for ob-norm versus 13.160.2 kcal/d for wild-type, p = 0.26) and their body fat percentages were similar when measured at 14 and 18 weeks (Figure 1c). Plasma leptin levels were measured periodically and, there was no significant difference in leptin levels between the two cohorts on the low-fat diet (Figure 1f; p.0.22). Interestingly, a small age-dependent increase in plasma leptin was observed in wild- type mice on a low-fat diet, and this was also observed in the ob- norm animals (2.660.4 ng/mL versus 1.960.4 at 18 weeks of age, and 7.062.3 versus 6.160.9 ng/mL at 26 weeks of age for ob-norm and wild-type, respectively; p,0.01 for the comparison between 18 and 26 weeks within either cohort). Because the only source of leptin in the ob-norm mice was pumps that delivered a constant dose, this small age-dependant increase likely reflects age-dependant changes in leptin metabolism or excretion. Exposure to a high-fat diet resulted in obesity in both wild-type and ob-norm animals, and the time course of weight gain was Figure 1. Energy balance in wild-type and ob-normalized animals on low and high-fat diets. A. Schematic of experimental strategy. Wild- type and ob/ob animals were implanted with pumps dispensing PBS or leptin, respectively, at 4 weeks of age, and pumps were replaced every four weeks as indicated by red arrows. Leptin sensitivity was assayed at 26 weeks of age. B. Body weight of wild-type (black) and ob-norm (red) mice on a high-fat or low-fat diet. C. Body fat percentage of wild-type and ob-norm mice at 14 and 18 weeks of age that were maintained on a high-fat diet (open bars) or low-fat diet (filled bars). D. Daily food intake for wild-type (black) and ob-norm (red) mice on a low-fat diet. p.0.05 for wild-type vs. ob- norm at all time points. E. Daily food intake for wild-type (black) and ob-norm (red) mice on a high-fat diet. * indicates p,0.05. F. Plasma leptin 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). All error bars are +/2 SEM. doi:10.1371/journal.pone.0011376.g001 PLoS ONE | www.plosone.org 2 June 2010 | Volume 5 | Issue 6 | e11376