Two defects contribute to hypothalamic leptin resistance in mice with diet-induced obesity

Abstract

Obesity in humans and in rodents is usually associated with high circulating leptin levels and leptin resistance. To examine the molecular basis for leptin resistance, we determined the ability of leptin to induce hypothalamic STAT3 (signal transducer and activator of transcription) signaling in C57BL/6J mice fed either low-fat or high-fat diets. In mice fed the low-fat diet, leptin activated STAT3 signaling when administered via the intraperitoneal (ip) or the intracerebroventricular (icv) route, with the half-maximal dose being 30-fold less when given by the icv route. The high-fat diet increased body-weight gain and plasma leptin levels. After 4 weeks on the diet, hypothalamic STAT3 signaling after ip leptin administration was equivalent in both diet groups. In contrast, peripherally administered leptin was completely unable to activate hypothalamic STAT3 signaling, as measured by gel shift assay after 15 weeks of high-fat diet. Despite the absence of detectable signaling after peripheral leptin at 15 weeks, the mice fed the high-fat diet retained the capacity to respond to icv leptin, although the magnitude of STAT3 activation was substantially reduced. These results suggest that leptin resistance induced by a high-fat diet evolves during the course of the diet and has at least two independent causes: an apparent defect in access to sites of action in the hypothalamus that markedly limits the ability of peripheral leptin to activate hypothalamic STAT signaling, and an intracellular signaling defect in leptin-responsive hypothalamic neurons that lies upstream of STAT3 activation.

Figure 1

Gel shift assays of dose response for STAT3 activation after ip (open circles) and icv (filled circles) leptin administration, and time course of STAT3 activation after icv leptin administration in 8-week-old C57BL/6J mice on a standard chow diet. Each dose and timepoint represents four animals. (a) Dose response for STAT3 activation in hypothalami of ip- and icv-injected mice. For the icv injections, doses of leptin were 0 μg, 0.01 μg, 0.1 μg, 0.5 μg, and 5 μg in 3 μL phosphate-buffered saline. For the ip injections, doses of leptin were 0 μg, 0.1 μg, 5 μg, 10 μg, 50 μg, 100 μg, and 200 μg in 100 μL phosphate-buffered saline. Mice were sacrificed 30 minutes after injection. Data are presented as mean ± SEM. (b) Time course of STAT3 activation by leptin in hypothalami of icv-injected mice. Leptin (0.5 μg) was injected and animals were sacrificed 0, 5, 15, 30, and 60 minutes later. The gels were quantified by PhosphorImager^® analysis. Data are presented as mean ± SEM.

Figure 2

Physiological characteristics of male C57BL/6J mice fed high-fat (n = 22) and low-fat (n = 22) diets. Dietary treatment began when mice were 4 weeks old and lasted for 15 weeks. Filled circles represent a diet of 45% fat; open circles represent a diet of 10% fat. Data are presented as mean ± SEM. (a) Effects of diets on body weight. ^AFirst day of significant weight difference (P < 0.001) between mice fed 10% fat and those fed 45% fat. (b) Caloric intake on the two diets. ^AP < 0.05. (c) Effects of diets on serum leptin concentration. ^AP < 0.001.

Figure 3

Leptin-induced STAT3 DNA-binding activity in hypothalami of mice on diets of 45% fat (HFD) and 10% fat (LFD). Mice were starved overnight and then received an ip or icv leptin injection. They were sacrificed 30 minutes later, and nuclear extractions were used for EMSA (see Methods). Each lane represents one hypothalamus. All experiments were performed twice with similar results. The arrows indicate the migration of the STAT-DNA complexes. LFD values are normalized to 100%. (a and b) Treatment with ip leptin (100 μg) after 4 weeks on diets. (c and d) Treatment with ip leptin (100 μg) after 15 weeks on diets. ^AP < 0.001. (e and f) Treatment with icv leptin (0.5 μg) after 15 weeks on diets. ^AP < 0.03.

Figure 4

(a) Hypothalamic mRNA expression of SOCS-3, PIAS-3, STAT3, and ObRb (long isoform of leptin receptor) after 18 weeks on a high-fat (white bars; n = 8) or low-fat (black bars; n = 5) diet. (b) Expression in brain microvessels of short-form leptin receptor (ObRa) mRNA and mRNA of all leptin receptor isoforms (ObRall) in mice on low-fat (white bars) or high-fat (black bars) diets. Gene expression was measured by quantitative RT-PCR as described in Methods. Values for low-fat diet are normalized to 100%. Data are presented as mean ± SEM. Microvessel experiments were performed three times with similar results.