Leptin Resistance Figure 4. STAT3 phosphorylation in response to acute leptin injection. A. Staining for pSTAT3 in the mediobasal hypothalamus of mice on a low-fat diet given an intraperitoneal injection of either leptin or vehicle. B. Staining for pSTAT3 in the mediobasal hypothalamus of mice on a high-fat diet given an intraperitoneal injection of either leptin or vehicle. C. Quantitation of pSTAT3 positive cells in the arcuate nucleus for mice on a low-fat diet. D. Quantitation of pSTAT3 positive cells in the arcuate nucleus for mice on a high-fat diet. doi:10.1371/journal.pone.0011376.g004 cells for leptin, p,0.02). By contrast, wild-type mice maintained on a high-fat diet showed no increase in pSTAT3 positive cells in response to leptin (24.265.8 cells for vehicle versus 21.866.8 cells for leptin, p = 0.8), confirming that diet-induced obesity blunts the leptin-responsiveness of these first-order neurons. While wild-type mice on a high-fat diet were insensitive to exogenous leptin, these mice did have a modestly increased number of pSTAT3 positive cells at baseline compared to low-fat fed animals (Figure 4c, p,0.05). This supports prior suggestions that diet-induced obesity results in chronically elevated leptin signaling that cannot be further modulated by additional leptin[39,40,41]. We next tested ob-norm mice in the same assay. Ob-norm mice maintained on a low-fat diet showed increased STAT3 phosphor- ylation in response to leptin, and the magnitude of this increase was similar to wild-type controls (10.764.5 cells for vehicle versus 7161.7 cells for leptin, p,0.01). Unlike wild-type animals on a high fat diet, ob-norm mice that had been maintained on a high- fat diet also showed a robust increase in STAT3 phosphorylation in response to leptin (2.760.8 cells for vehicle versus 108621 cells for leptin, p,0.01). There was no indication that the leptin responsiveness of these high-fat diet fed, ob-norm mice was impaired in this assay relative to lean controls. These biochemical data are consistent with the physiological data from leptin infusion experiments and indicate that even long-term exposure to a high- fat diet is not sufficient to block the leptin-induced activation of JAK/STAT signaling in normoleptic animals. Instead, we conclude that hyperleptinemia is required for the development of leptin resistance in response to diet-induced obesity. Discussion The mechanisms responsible for the development of leptin resistance have been the focus of many studies. As leptin resistance results in obesity and other metabolic diseases, agents which can re-sensitize the obese to leptin would have great therapeutic value. However, the molecular events responsible for the development of leptin resistance remain poorly understood, in part because of the numerous physiological and nutritional differences between lean and diet-induced obese animals. In principle, any of these differences could contribute to impaired leptin sensitivity. In this study we asked whether one physiological parameter – elevated plasma leptin levels – is required for the development of leptin resistance in response to a high-fat diet. We focused on hyperleptinemia because negative feedback in response to excess signaling is a classical mechanism of hormone resistance (including insulin), and because there was already evidence supporting at least one molecular mechanism for negative feedback regulation of leptin (SOCS-3). These experiments were made possible by our having developed a protocol that could normalize the plasma leptin levels of ob/ob mice over the long-term using subcutaneous pumps. We found, consistent with previous studies, that leptin replacement in ob/ob animals is sufficient to normalize their body weight and other physiologic abnormalities, and furthermore that this normalization can be maintained for 6 months by replacing the pumps monthly. The data clearly show that the hyperleptinemia in mice fed a high fat diet is required for the development of leptin resistance. These results do not support the claim that a high-fat diet can block the response to leptin in ob/ob animals[42]. Recently, the peptide amylin has been shown to sensitize animals and humans to the effects of leptin[43,44]. An important challenge will be to further understand the cellular basis of leptin resistance and the mechanism by which amylin can improve it. In addition to its role in regulating energy balance in adult animals, leptin has trophic effects on hypothalamic neurons during early post-natal development[45]. We have shown that leptin treatment beginning at 4 weeks of age normalizes the food intake PLoS ONE | www.plosone.org 5 June 2010 | Volume 5 | Issue 6 | e11376