The aim of this study was to examine the basis for the leptin resistance that develops when C57BL/6J mice are placed on a high-fat diet. Mice were studied after 4 weeks and 15 weeks of low-fat or high-fat diet. Leptin was administered by either the ip or icv route, after which leptin signaling to activate STAT3 in the hypothalamus was assessed. To better understand the characteristics of leptin signaling to STAT3 in the hypothalamus, we first performed a dose-response study for STAT3 activation by gel shift assay after ip vs. icv leptin in mice fed a normal chow diet (Figure a). STAT3 activation was dose dependent with both routes of administration. The dose-response curve for icv leptin was shifted 30-fold to the left. The half-maximal dose of leptin was 0.5 μg after icv administration, and approximately 15 μg after ip administration. A dose of 30 μg icv leptin was no more effective than 5 μg icv (data not shown). The maximal response was obtained 15–30 minutes after 0.5 μg icv leptin (Figure b). This is similar to the maximal-response timepoint observed after intravascular injection that was reported by Vaisse et al. (10
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 (more ...)
Mice fed the high-fat diet had a measurable, significant weight increase after 4 days; after 15 weeks they weighed 24% more than those fed the low-fat diet (Figure a). Mice fed the high-fat diet also ingested more calories per day (Figure b). As expected, high-fat feeding and attendant obesity were associated with increased leptin levels (Figure c). The ability of leptin to induce STAT3 signaling was first studied after 4 weeks on the diets. After an overnight fast, mice were administered 100 μg recombinant mouse leptin by the ip route. Thirty minutes later, hypothalami were isolated, nuclear extracts were obtained, and STAT3 activation was assessed by EMSA. After 4 weeks on the diets, peripheral leptin treatment induced STAT3 DNA binding activity to an equivalent extent in hypothalami of mice fed low- and high-fat diets (Figure , a and b). In contrast, after 15 weeks on the diets, the same dose of ip leptin that was effective in mice fed the low-fat diet was unable to induce detectable STAT3 DNA binding activity in hypothalami of mice fed the high-fat diet (Figure , c and d). We obtained the same result in another independent experiment with six mice in each group (data not shown). Thus, prolonged high-fat feeding induced profound resistance to peripherally administered leptin as measured by STAT3 signaling in the hypothalamus. It is worth noting that even after 4 weeks on a high-fat diet, mice appear to be in positive energy balance, as suggested by early weight gain (Figure ), and they may therefore already have some degree of leptin resistance. We did not directly assess leptin resistance by evaluating the effects of leptin administration on food intake and body weight at 4 weeks in the current study. If leptin resistance exists at this point, our data suggest that the mechanism is different, or it is of much smaller magnitude than that seen after 15 weeks.
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 (more ...)
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 (more ...)
If the absence of leptin signaling after 15 weeks of high-fat diet was due to a defect in access of peripheral leptin to sites of action in the hypothalamus, then icv leptin administration should be able to overcome this defect. Consistent with this hypothesis, icv leptin given at a much lower dose than was given intraperitoneally (0.5 μg vs. 100 μg) clearly induced STAT3 activation in mice with 15 weeks of DIO (Figure e). Thus, one component of the leptin resistance in mice with 15 weeks of DIO can be attributed to a failure to deliver leptin to its sites of action in the hypothalamus, presumably by a defect in the blood-brain-barrier transport system. Importantly, however, whereas icv leptin can induce signaling in mice with DIO, the extent of this induction is 75% lower than that in mice fed the low-fat diet (Figure f). This finding was confirmed in an independent experiment with five mice in each group (data not shown). The results demonstrate that in addition to an apparent defect in leptin access to the hypothalamus, a signaling defect also exists upstream of STAT3 activation in leptin-responsive hypothalamic neurons in these DIO mice. To verify that the STAT3 DNA binding activity in the hypothalamus after leptin administration was dependent on the long form of the leptin receptor, we examined STAT3 DNA binding activity after administration of 100 μg ip leptin to db/db
mice, which lack the long form of leptin receptor (18
). As expected, we found no leptin-induced STAT3 DNA binding activity in hypothalami of db/db
mice, whereas controls showed a strong signal (data not shown).
These results suggest that at least two defects underlie the leptin resistance that develops during the course of high-fat feeding in C57BL/6J mice. A defect at the level of leptin access is inferred from the fact that icv administration of leptin at least partially overcomes the diminished signaling observed after peripheral administration in these mice. These findings are consistent with the data of Van Heek et al. (11
), who found that icv, but not ip leptin could diminish food intake and reduce body weight in a similar obesity model. Our data extends those physiologic findings to the level of signal transduction by demonstrating that leptin-induced STAT3 activation is undetectable after ip leptin, but is present after icv leptin administration, after 15 weeks of high-fat diet. The molecular mechanism for the apparent defect in leptin access or transport remains unknown at present. Leptin transport into the brain may involve the ObRa isoform (short form) of the leptin receptor, which is highly expressed within the blood-brain barrier (13
). In one study, expression of ObRa mRNA in cerebral microvessels of rats with DIO was increased (20
). We therefore examined the expression of ObRa mRNA in cerebral microvessels of mice with 15–18 weeks of DIO, and observed that no change in the expression of ObRa mRNA was evident (Figure b). In addition, we found no significant difference in expression of leptin receptor mRNA using primers designed to detect all known forms of ObR (ObRall, Figure b). Thus, the mechanism underlying what appears to be reduced leptin transport across the blood-brain barrier does not involve reduced expression of the mRNA encoding a putative leptin transporter within the blood-brain barrier. Further studies will be required to determine whether the protein expression or function of putative leptin transporters at the blood-brain barrier are impaired in DIO.
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 (more ...)
In addition to the defect in transport or availability of leptin, our data provide the first evidence that a second defect exists at the level of leptin signaling to activate STAT3 in DIO. Leptin activation of STAT3 requires the ObRb isoform (long form) of the leptin receptor, which associates with and activates Janus tyrosine kinase-2 (JAK2) in a ligand-dependent manner (21
). One potential mediator of reduced STAT3 activation in the hypothalamus of DIO mice is SOCS-3, a suppressor of cytokine signaling, whose expression we have shown previously to be acutely induced by leptin in neurons expressing ObRb in the hypothalami of normal rodents (14
). Forced expression of SOCS-3 in mammalian cells antagonizes proximal leptin signaling, probably by binding to and antagonizing JAK activity (26
). PIAS-3 (protein inhibitor of activated STAT) is a recently described antagonist of STAT3 DNA binding activity (27
), whose mRNA expression has not previously been examined in the hypothalamus of normal mice. Using quantitative RT-PCR, we examined the expression of SOCS-3 and PIAS-3 mRNA in hypothalami of mice fed low-fat or high-fat diets for 18 weeks. No significant differences were found (Figure a). In addition, we found no significant difference in hypothalamic STAT3 or ObRb mRNA expression between the two groups (Figure a). Thus, a molecular basis for the observed defect in leptin’s ability to activate STAT3 signaling remains to be determined.
Although C57BL/6J mice fed high-fat diets for 15 weeks fail to activate detectable hypothalamic STAT3 signaling in response to peripheral leptin, it is worth noting that these DIO mice are less obese than db/db
mice that lack all receptor-dependent STAT signaling. Additionally, unlike db/db
mice, DIO mice are fertile. Thus, despite our demonstration of severely impaired hypothalamic STAT3 activation after ip leptin, some important leptin signals are clearly reaching target neurons within the central nervous system in these mice. How does this conclusion relate to our inability to detect STAT3 activation either basally or after ip leptin administration in mice with 15 weeks of DIO? The leptin receptor ObRb isoform activates the JAK/STAT pathway, but is capable of activating the MAP kinase (23
) or PI-3 kinase pathway (28
) in certain mammalian cell lines as well. It is therefore possible that STAT3 pathways are selectively inhibited in key neural groups, but important signals via MAP kinase or PI-3 kinase pathways are effectively transmitted. Alternatively, there may be key receptor signals transmitted through sites other than the hypothalamus that we did not sample. Finally, our assay may not be sensitive enough to pick up STAT3 activation in a limited, but functionally important subpopulation of leptin-responsive hypothalamic neurons. Understanding the mechanism by which substantial resistance to leptin’s action on appetite and body weight coexists with persistent effects on neuroendocrine function should be an important goal of future research.
Leptin resistance is the hallmark of obesity induced by a high-fat diet in rodents, and occurs naturally in obese humans. If the factors limiting leptin action on appetite and body weight in human obesity are similar to those seen here in mice with DIO, then pharmaceutical development aimed at creating small-molecule leptin mimics with increased capacity to cross the blood-brain barrier might be effective in treating human obesity. On the other hand, a potential limitation of such an approach would be the factors that limit leptin signaling. It will be important in future studies to determine the precise molecular basis for this signaling defect.