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Pennington Biomedical Research Center (V.P., M.A.S., G.E.J., T.W.G.), Baton Rouge, Louisiana 70808; and Department of Medicine (P.M.W., I.C.F.), Medical University of South Carolina, Charleston, South Carolina 29425-2223
Address all correspondence and requests for reprints to: Thomas W. Gettys, Pennington Biomedical Research, 6400 Perkins Road, Baton Rouge, Louisiana 70808. E-mail: gettystw{at}pbrc.edu.
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Abstract |
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Introduction |
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Leptin resistance is a characteristic feature of most human obesity in that leptin is abundantly expressed but functions with reduced efficacy in obese patients. The biochemical mechanisms of leptin resistance are poorly understood, but mouse models of dietary obesity have provided excellent systems to explore how leptin functions and identify sites where leptin signaling becomes compromised. Leptin resistance typically develops progressively beginning with reduced leptin transport across the blood brain barrier, resulting in an apparent inability of circulating leptin to reach leptin-responsive nuclei in the hypothalamus (10, 11, 12, 13). Central leptin resistance develops in later stages of dietary obesity and involves compromised recognition or translation of the leptin signal through central effector systems (10, 14, 15, 16). Diet-induced changes in adipocyte signaling systems also produce peripheral leptin resistance by decreasing the ability of adipose tissue to respond to leptin-mediated increases in sympathetic nervous system (SNS) stimulation (9, 17, 18). Compromised function at any of these steps has the potential to limit leptin efficacy, but the relative functioning of each component has not been compared in obesity-prone and obesity-resistant mice during the period of rapid fat deposition that occurs after increases in caloric density. Using peripheral and central leptin administration in obesity-prone and obesity-resistant mice, we show that HF diets rapidly compromise the ability of obesity-prone C57BL/6J mice to detect and respond to leptin. The preservation of leptin responsiveness in obesity-resistant A/J mice is consistent with their documented ability to mount a more vigorous adaptive thermogenic response in the face of increased caloric density (9, 19, 20). It seems likely that strain differences in resistance to dietary obesity may be the product of strain-specific adaptations to increased caloric density that limit or preserve responsiveness to leptin.
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Materials and Methods |
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-[32P]-CTP was purchased from DuPont NEN Radiochemicals (Boston, MA). Immobilon-P polyvinylidene difluoride (PVDF) membranes were from Millipore Corp. (Bedford, MA). Total and phospho-specific signal transducers and activators of transcription (STAT)3 antibodies were purchased from Cell Signaling (Beverly, MA). A semipurified diet was prepared by Research Diets Inc. (New Brunswick, NJ) to contain 36% fat by weight (21), whereas the low-fat (LF) control diet contained 5% fat as described in detail previously (21, 22). The energy density of the LF and HF diets were 4.07 kcal/g and 5.56 kcal/g, respectively. The fat source for the diets was coconut and soybean oil (21). Male A/J and C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME).
Experimental animal protocol
A/J and C57BL/6J mice were obtained at weaning and representative mice of each strain were studied at 0 time and after consuming the HF diet described above for 4 or 8 wk. The mice were housed in solid bottom cages, room temperature was maintained at 22–23 C, and the lights were on a 12-h light, 12-h dark cycle. At each of the three time points, mice were given ip or intracerebroventricular (ICV) injections of vehicle or leptin for 3 d according to the protocols described below (23). Injections were given 2 h following the start of the light cycle, and all mice were killed 1 h after the last injection in the series. Food intake was recorded during the injection protocol, and tissues were harvested when the mice were killed. The hypothalamus from each mouse was carefully dissected, weighed, and processed as described by Scarpace et al. (24) for measurement of STAT3 phosphorylation by Western blot. The epididymal, retroperitoneal, and interscapular adipose tissue depots were dissected, weighed, and used for RNA isolation as previously described (6). The experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee.
ICV injections
Mice were anesthetized by inhalation of isoflurane and a guarded, blank 27-gauge 0.5-in. needle was used to create a guide injection site 0.7 mm posterior to bregma and 1.0 mm lateral to midline at a depth of 4.0 mm (25). In the experiments proper, a 10.0-µl Hamilton 1700 series gastight syringe (Hamilton, Reno, NV) was used to inject either artificial cerebrospinal fluid (aCSF), murine leptin, or rat neuropeptide Y (NPY) in a volume of 2–5 µl. Correct positioning of the guide injection site was confirmed before the start of each experiment by monitoring feeding behavior following injection of NPY [0.075 µg/g body weight (bw)]. Mice failing to respond to NPY were removed from the experiment, and experiments were begun on d 2 after NPY injections. aCSF, consisting of 70 mM NaCl, 6 mM KCl, 0.7 mM CaCl2, 0.85 mM MgCl2, 0.75 mM Na2HPO4, 0.10 mM NaH2PO4, and 0.1% untreated BSA, was injected in a volume of 5 µl. Thereafter, mice were monitored to ensure full recovery.
Experiment 1.
Twenty-four A/J and C57BL/6J mice were obtained at weaning, randomly assigned to one of four treatment groups, and acclimated for 2 d before study. Thereafter, mice in group 1 received ip injections of saline for 3 d, whereas mice in group 2 received ip injections of leptin (20 µg/g bw·d) during the same period. The mice in group 3 received ICV injections of aCSF for 3 d, whereas group 4 received ICV injections of mouse leptin (2 µg/mouse·d). Food intake was carefully measured during the injection protocol, and 1 h after the final injection, mice were killed and tissues were weighed and processed as described above.
Experiment 2.
Forty-eight A/J and C57BL/6J mice were obtained at 4 wk of age and weaned onto the HF diet described above. Mice were randomly assigned to one of four treatment groups as described in experiment 1, and representative mice from each group x strain combination were studied after 4 and 8 wk on the HF diet. Group 1 received ip injections of saline for 3 d, whereas mice in group 2 received ip injections of leptin (20 µg/g bw·d). The mice in group 3 received ICV injections of aCSF for 3 d, whereas group 4 received ICV injections of mouse leptin (2 µg/mouse·d). The HF diet was provided ad libitum, and food intake was carefully measured during the 3-d injection protocol. One hour after the final injection, the mice were killed and tissues was processed as described above.
Experiment 3.
Twenty-four A/J and C57BL/6J mice were obtained at 4 wk of age and weaned onto the LF diet described above. Mice were randomly assigned to one of four treatment groups as described in experiment 1 and representative mice from each group x strain combination were studied after 8 wk on the LF diet. Group 1 received ip injections of saline for 3 d, whereas mice in group 2 received ip injections of leptin (20 µg/g bw·d). The mice in group 3 received ICV injections of aCSF for 3 d, whereas group 4 received ICV injections of mouse leptin (2 µg/mouse·d). The LF diet was provided ad libitum, and food intake was carefully measured during the 3 d injection protocol. One hour after the final injection, the mice were killed and tissues was processed as described above.
Experiment 4.
In an additional experiment, representative mice from each strain received ip injections for 3 d with saline or CL 316,243 (1 µg/d·g bw) at weaning and after consuming the HF diet for 8 wk. The mice were killed 2 h after the last injection, and tissues were harvested and processed as before.
Preparation of RNA
After dissection, the interscapular, epididymal, and retroperitoneal fat pads were homogenized with Trizol LS reagent using an Ultraturax (Tekmar, Cincinnati, OH) according to manufacturer’s specifications. Total RNA was isolated, purified, and treated with deoxyribonuclease as before (26).
Ribonuclease protection assay
RNA probes complementary to uncoupling protein 1 (UCP1) and leptin mRNA were produced, labeled, and used as described previously to assay UCP1 and leptin mRNA (9). The mRNA species were quantitated using known amounts of sense strand standard as described previously (6, 9).
Real-time PCR
In some experiments, real-time PCR assays were used to measure UCP1 and leptin mRNA expression in samples where recovered amounts of RNA were limiting. To produce standards for each assay, primers for UCP1 and leptin were used to produce significant amounts of each cDNA fragment by PCR. The gene fragments were then purified, sequenced, quantitated by measuring Abs260/280, and used to construct standard curves for each gene. The slope of the standard curves for cyclophilin, UCP1, ß3-AR, and leptin did not differ from -3.3 in each case and dilutions of unknown cDNA samples produced changes in cycle thresholds that paralleled the standard curves. Pilot experiments established the amount of RNA needed for each gene and tissue type to place cycle thresholds for unknown samples in the middle portion of standard curves. In the assay proper, duplicate dilutions of each standard and unknown sample were amplified with the appropriate primer and probe sets and the mass of mRNA in unknown samples was estimated from standard curves relating cycle threshold to mass of each standard. Cyclophilin standard curves were used to determine the mass of this housekeeping gene in each sample and after correcting for cyclophilin mRNA differences, target gene mRNA levels were expressed as fmol/µg total RNA for analysis.
STAT3 activation
STAT3 activation was assessed by Western blot using an antibody specific for STAT3 phosphorylated on tyrosine 705 and a second antibody that detected total STAT3 (Cell Signaling, Beverly, MA). Duplicate hypothalamic extracts (15 µg) were resolved on 10% SDS-PAGE gels, transferred to PVDF membranes, and incubated with total and phospho-specific antibodies. Immunoreactivity was visualized by enhanced chemiluminescence and quantitated by laser densitometry. After adjusting for differences in total STAT3, the change in phosphorylated STAT3 induced by leptin was expressed as a function of phospho-STAT3 detected in vehicle-injected controls for each injection route.
Methods of analysis
The estimated concentrations of UCP1 and leptin mRNA were obtained by reverse calibration from standard curves as described in Materials and Methods. Group means for mRNA estimates, STAT3 activation, food consumption, and growth data were analyzed using a one-way ANOVA at each time point. Post hoc testing of group means within each time point was made with the Bonferroni correction using the pooled error term to calculate SE values. Protection against type I errors was set at 5%.
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Results |
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). The reduction of food consumption by ICV leptin in A/J mice was similar in magnitude to that produced by ip leptin, but the reduction in fat pad size was more pronounced with the ICV route of injection (Table 1). In weanling C57BL/6J mice, ICV leptin reduced food consumption by an amount additional to that seen with ip leptin and the fat pads were reduced by a corresponding additional increment (Table 1). After 4 and 8 wk on the HF diet, a different response pattern emerged in that ip leptin failed to reduce food intake or fat pad size in either strain (Table 1). Mice of both strains were responsive to centrally injected leptin after 4 and 8 wk on the HF diet, but the magnitude of reduction in food intake was significantly greater in A/J than C57BL/6J mice (Table 1). This difference translated into a 50% reduction in fat pad mass in A/J mice at both time points (Table 1). In contrast, the reduction in WAT mass in C57BL/6J mice failed to reach significance at either time point (Table 1).
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). Leptin injected ip and ICV produced comparable reductions in food intake in both strains (Table 2). In contrast, the ICV route was clearly more effective than the ip injection route at inducing UCP1 mRNA in both strains. Nevertheless, ip-injected leptin increased UCP1 mRNA to a comparable degree in BAT of both strains. In contrast, the effects of leptin on adipose tissue mass were less clear cut. Leptin injected ip reduced epididymal WAT in C57BL/6J mice, but the effect of ip leptin failed to reach significance in the A/J mice (Table 2). The reverse was seen with the ICV route of injection with WAT mass reduced in A/J but not in C57BL/6J (Table 2).
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and shows that ip leptin produced a comparable 2.4- and 2.9-fold increase in STAT3 phosphorylation over vehicle-treated controls in weanling A/J and C57BL/6J mice, respectively. ICV leptin produced a similar 2.8-fold increase in STAT3 phosphorylation in C57BL/6J mice, but the response to ICV leptin in weanling A/J was significantly larger (4.8-fold). A/J mice retained their responsiveness to ip leptin after 8 wk on the HF diet, as the increase in phosphorylated STAT3 was 2.2-fold. In contrast, ip leptin failed to increase STAT3 phosphorylation in hypothalamic extracts of C57BL/6J mice after 8 wk on the HF diet (Fig. 1). However, the ICV route of administration produced similar 1.9- and 1.8-fold increases in STAT3 phosphorylation in A/J and C57BL/6J mice (Fig. 1). Total STAT3 expression was evaluated using an antibody that recognized phosphorylated and nonphosphorylated forms of the transcription factor. Using this approach, total STAT3 did not differ in hypothalamic extracts among any of the groups (data not shown). Taken together, the results indicate that hypothalamic nuclei in both mouse strains retain the ability to initiate leptin-dependent signal transduction if leptin is delivered directly into the third cerebral ventricle. However, the results also indicate that after 8 wk on the HF diet, the ability of peripheral injections of leptin to activate responsive nuclei is differentially compromised in C57BL/6J compared with A/J mice.
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, A and B). This result complements the findings presented in Table 1
and Fig. 1 and shows that BAT is fully responsive in both strains to leptin-mediated increases in SNS stimulation. In contrast to the greater efficacy of ICV vs. ip leptin in reducing WAT (Table 1), ip and ICV leptin produced comparable increases in UCP1 mRNA in both strains (Fig. 2, A and B). This finding suggests that the hypothalamic nuclei that control this aspect of the leptin response are fully responsive in weanling mice.
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To better assess the magnitude of leptin effects on adipocyte gene expression at the 8-wk time point, UCP1 and leptin mRNA levels were measured in BAT and WAT samples using real-time PCR assays. Results presented in Fig. 3A show that ip leptin produced a 3-fold increase in BAT UCP1 mRNA in A/J mice but failed to increase UCP1 mRNA in C57BL/6J mice. The efficacy of ICV leptin was significantly higher than ip leptin in A/J mice, producing a 5-fold increase in UCP1 mRNA from 5.1–26.5 fmol/µg RNA (Fig. 3A). In contrast to the 4-wk time point, ICV leptin was able to produce a small but significant increase in UCP1 mRNA from 12.4–21.2 fmol/µg RNA in BAT from C57BL/6J mice (Fig. 3A). Of interest was the observation that basal UCP1 mRNA levels were higher in BAT from C57BL/6J compared with A/J mice (Fig. 3A). This result was also observed when UCP1 mRNA was assayed by ribonuclease protection assay (data not shown), but regardless of assay method, ICV leptin produced a greater increase in UCP1 in BAT from A/J compared with C57BL/6J mice.
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). Neither route of leptin administration increased UCP1 mRNA levels in C57BL/6J mice. Leptin injected ip produced a 2-fold increase in UCP1 mRNA in A/J mice (Fig. 3B), but the variability of UCP1 mRNA measurements precluded detection of significant differences. ICV leptin was far more effective and produced a 6- to 7-fold increase in UCP1 mRNA in retroperitoneal WAT of A/J mice to levels that were 80- to 90-fold higher than comparably treated C57BL/6J mice (Fig. 3B).
A second adipocyte gene known to be regulated by the SNS (7) was examined to see if its regulation would be similarly compromised. This was accomplished by looking at the ability of exogenous leptin to down-regulate leptin mRNA in retroperitoneal and epididymal WAT. Leptin mRNA levels were similar within retroperitoneal and epididymal WAT from control A/J and C57BL/6J mice, and ip leptin did not reduce leptin mRNA in either tissue of either strain (Fig. 4, A and B). ICV leptin produced a 50-fold reduction in leptin mRNA in retroperitoneal WAT of A/J mice but was without effect in C57BL/6J mice (Fig. 4A). In contrast, ICV leptin produced a comparable 4- to 5-fold reduction in leptin mRNA in epididymal WAT from A/J and C57BL/6J mice (Fig. 4B). Collectively, these results indicate strain and depot site-specific changes in leptin responsiveness and suggest that the compromised responses in retroperitoneal WAT from C57BL/6J mice originate in the tissue rather than in central recognition and response to the leptin signal. To distinguish between failure of leptin to increase SNS stimulation of adipose tissue and resistance of adipose tissue to sympathetic stimulation, gene expression was evaluated in mice of each strain after treatment with the selective ß3-AR agonist, CL316,243. In BAT, the ß3-AR agonist increased UCP1 mRNA in weanling mice (not shown) and in mice of both strains after 8 wk on the HF diet (Fig. 5). The magnitude of the increase in UCP1 mRNA was 2- to 3-fold in both strains, and the basal levels were also similar (Fig. 5). In contrast, basal UCP1 mRNA levels were significantly higher in retroperitoneal WAT of weanling A/J compared with C57BL/6J mice (Fig. 6). Despite this difference, CL316,243 produced a large 10- to 20-fold increase in UCP1 mRNA in weanling mice of both strains. After 8 wk on the HF diet, CL316,243 was equally effective in retroperitoneal WAT of A/J mice but failed to increase UCP1 mRNA in C57BL/6J mice (Fig. 6). Although not shown, the decline in responsiveness of C57BL/6J mice to CL316,243 was gradual in that the agonist still produced a partial response after 4 wk on the HF diet. Measurements of ß3-AR mRNA in retroperitoneal WAT from A/J (0.024 ± 0.015 fmol/µg RNA) and C57BL/6J mice (0.018 ± 0.006 fmol/µg RNA) after 8 wk on the HF diet indicate that decreased expression of the receptor is not the cause of the diminished responsiveness in C57BL/6J mice. These findings indicate that a combination of both central and peripheral mechanisms are involved in the development of leptin resistance in C57BL/6J mice. In contrast, a compromised ability of circulating leptin to reach leptin responsive nuclei in the hypothalamus appears to be the primary cause of leptin resistance in A/J mice.
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Discussion |
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-MSH release from leptin-responsive neurons within the arcuate nuclei, and these peptides increase sympathetic outflow by binding to melanocortin and cocaine amphetamine-related transcript receptors on preganglionic neurons of the SNS (28, 29, 30). The resulting increase in sympathetic stimulation of adipose tissue promotes fat oxidation through targeted effects on adipocyte gene expression (7, 27, 31). A second population of leptin-responsive neurons in the hypothalamus coexpress NPY and agouti-related peptide, and these orexigenic peptides are decreased by leptin (32, 33, 34). Thus, it is through reciprocal regulation of these two populations of hypothalamic nuclei that leptin produces its full effect on energy homeostasis. Its ability to fulfill this function is dependent upon its ability to reach and activate both sets of target neurons within the hypothalamus. Our primary goal is to test the hypothesis that the respective resistance and susceptibility of A/J and C57BL/6J mice to dietary obesity are conferred by their retention and loss of leptin responsiveness after weaning onto HF diets. In the present study, we have used obesity-resistant A/J and obesity-prone C57BL/6J mice to test this hypothesis by comparing the ability of leptin to affect responses linked to each set of target neurons during the development of dietary obesity. To distinguish between compromised access of leptin to responsive nuclei within the hypothalamus and compromised responsiveness of hypothalamic nuclei and/or downstream effector systems, we used a combination approach involving peripheral and central leptin injections in conjunction with the adipocyte-specific sympathomimetic ß3-AR agonist, CL316,243. Our results show clearly that mice of each strain were fully responsive to both routes of leptin administration and CL316,243 at weaning. This observation is important in that it establishes the absence of a strain difference in the ability of circulating leptin to cross the blood brain barrier, activate leptin-dependent responses, and affect changes in behavior and gene expression at the start of the study. By inference, these findings illustrate that the observed post weaning changes in leptin responsiveness are the product of strain-specific differences in response to the HF diet. Dietary effects on leptin signaling have not been reported in A/J mice, but previous studies have shown that C57BL/6J mice develop leptin resistance on HF but not LF diets (10, 11, 13, 35). In the present study, we found no evidence that A/J or C57BL/6J mice on LF diets develop leptin resistance during the time frame studied.
A different outcome is evident in C57BL/6J mice after consuming a HF diet for 4 or 8 wk, as peripherally injected leptin failed to affect any of the six leptin-dependent responses that were examined. In contrast, ICV leptin activated hypothalamic STAT3, reduced food consumption, increased UCP1 mRNA in BAT, and reduced leptin mRNA in epididymal WAT of C57BL/6J mice. These findings support the conclusion that target nuclei in the hypothalamus are responsive to leptin but fail to be activated by circulating leptin because of a defect in transport or access to the hypothalamus. Several studies have documented resistance to peripheral leptin during diet-induced obesity (10, 11, 13, 35), although attempts to identify the nature of the defect have so far proven inconclusive. For instance, defective transport of leptin across the blood brain barrier by short isoforms of the leptin receptor seems the most likely mechanism. And although recent studies support the involvement of this receptor in leptin transport, measurement of mRNA levels during dietary obesity failed to detect changes in its expression (36). It is also possible that elevation of serum leptin in dietary obesity saturates the transport system, making it unresponsive to subsequent changes in circulating leptin. This concept was tested by Maness et al. (37) who measured leptin transport across the blood brain barrier after injecting db/db and ob/ob mice with the peptide. Despite great differences in circulating leptin in these two models, leptin transport into the hypothalamus did not differ between them (37). This issue has also been addressed in a rat model of dietary obesity, where HF diet produced a 11-fold increase in the short isoform of the leptin receptor at the blood brain barrier (38). The effect on leptin transport was not assessed, but in other studies obesity was associated with decreased leptin transport across the blood brain barrier (39). Whereas the exact mechanism for decreased transport remains unknown, the present results are consistent with the idea that HF diets modify the ability of circulating leptin to reach target nuclei within the hypothalamus of C57BL/6J mice.
We also found evidence of resistance to peripheral leptin in A/J mice on the HF diet, but in this case the resistance was selective in that some responses were fully compromised, whereas others were only partially affected. For example, ip leptin failed to reduce food intake or decrease leptin mRNA in WAT but retained its ability to activate hypothalamic STAT3 and induce UCP1 in BAT and retroperitoneal WAT. Coupled with the observation that A/Js retained full responsiveness to ICV leptin, these findings suggest that leptin transport across the blood brain barrier may have been partially compromised by the HF diet. However, if this were the sole mechanism, we would predict that all responses to ip leptin would be similarly affected. The restoration of the anorexigenic response by ICV leptin and its increased efficacy to increase UCP1 is consistent with the idea that both signaling systems are fully capable of responding to leptin. The data further suggest that the amounts of leptin reaching the respective nuclei in ip-injected A/J mice were below the threshold for effects on food intake but at or above the threshold for effects on UCP1 induction. A similar finding was reported in young agouti mice where peripherally injected leptin was unable to reduce food intake but produced sympathoexcitatory actions comparable to those observed in control mice (40). The authors interpreted this finding as evidence of selective leptin resistance among subgroups of leptin-responsive nuclei (40). The comparable sympathoexcitatory effects in agouti vs. controls coupled with the lack of an anorexigenic effect argue that central leptin resistance rather than a deficit in transport is the primary mechanism in this model (40). This conclusion is consistent with a previous report showing decreased sensitivity to centrally injected leptin in agouti mice (12). Another possibility is that ip-injected leptin may act through leptin receptors in peripheral tissues, and either produce direct effects in these tissues or modify the ability of target tissues to respond to SNS stimulation. In the present studies, our findings are most consistent with a dual mechanism involving both decreased leptin transport into the hypothalamus and decreased sensitivity of specific leptin-responsive nuclei within the hypothalamus. El-Haschimi et al. (10) examined leptin-dependent STAT-3 activation and also found evidence of a dual component mechanism of leptin resistance in C57BL/6J after long term consumption of HF diets.
A particularly interesting difference in the response of A/J and C57BL/6J mice to ICV leptin was the robust induction of UCP1 in retroperitoneal WAT of A/Js and corresponding absence of this response in C57BL/6Js. The down-regulation of leptin mRNA in this depot shows a similar strain-specific dichotomy, whereas in epididymal WAT, ICV leptin produced a comparable down-regulation of leptin mRNA in A/J and C57BL/6J mice. This finding raises the interesting possibility that the HF diet may also have modified the ability of adipose tissue to respond to sympathetic stimulation in a strain and depot site-specific manner. To test the possibility of peripheral resistance to sympathetic stimulation, mice of each strain were treated with a ß3-AR agonist to produce a comparable level of ß3-adrenergic receptor activation. These findings showed that BAT was fully and equally responsive to the ß3-AR agonist at weaning, and after 8 wk on the HF diet in both strains. Retroperitoneal WAT from C57BL/6J was also responsive to the ß3-AR agonist at weaning, but after 8 wk on the HF diet this tissue showed no induction of UCP1 by the agonist. In contrast, the retroperitoneal WAT from A/J mice was fully responsive at both time points. A potential explanation for diet-induced transformation of the response could be changes in ß3-AR expression or function. Collins et al. (17) found that HF diets decreased ß3-AR expression in epididymal WAT and BAT of C57BL/6J mice after 16 wk on the diet (17). We also found decreased ß3-AR mRNA and function in epididydmal WAT after long-term consumption of the HF diet, but not at the 8 wk time point used in the present study (9). We also found no difference in ß3-AR mRNA in retroperitoneal WAT after 8 wk on the HF diet in the present study. Collectively, these studies show that consumption of the HF diet for 8-wk generated peripheral leptin resistance in C57BL/6J mice by altering the ability of retroperitoneal WAT to respond to sympathetic stimulation. A similar alteration is not seen in A/J mice, and when considered with their retention of responsiveness to peripheral leptin, is consistent with the more robust adaptive thermogenic response of A/J vs. C57BL/6J mice to HF diets.
An interesting feature of the difference in fat deposition between A/J and C57BL/6J mice is that it occurs at similar levels of food intake (8, 9). By default, this points to energy expenditure as the basis for their differences in fat deposition. Strain differences in adaptive thermogenesis have been documented in several labs (9, 17, 19), and we have argued that leptin plays a key role in modulating adaptive thermogenesis (6, 27). In the present study, both strains of mice were similarly unresponsive to the anorexigenic effects of peripheral leptin, and this finding is consistent with the lack of strain differences in food consumption (9, 41). In contrast, the strains did differ in their sympathetic responses to peripheral leptin in a manner that is consistent with the relative adaptive thermogenic responses of the two strains to the HF diet (9). The peripheral resistance of C57BL/6J mice to sympathetic stimulation is also likely to be a contributing factor in their higher propensity to obesity. This conclusion is supported by previous findings showing that ß3-AR agonists were unable to reduce fat deposition in C57BL/6J mice on HF diets (17). Therefore, it will be important in future studies to understand the cellular basis for diet-induced changes in adipocyte signaling systems that compromise responsiveness to sympathetic stimulation.
Collectively, our studies show that HF diets modify central and peripheral leptin response systems in a strain and depot site-specific manner. Strain differences in the severity of leptin resistance were consistent with the relative propensities of the mouse strains to become obese. Mice of each strain were equally responsive to leptin at weaning, but differences emerged during the post weaning period when significant differences in fat deposition occurred. The ability of A/J mice to resist obesity during this period is consistent with their retention of leptin responsiveness and is manifest as a more robust adaptive thermogenic response to increased caloric density.
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Acknowledgments |
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Footnotes |
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Abbreviations: aCSF, Artificial cerebrospinal fluid; A/J, obesity-resistant mice; BAT, brown adipose tissue; bw, body weight; C57BL/6J, obesity-prone mice; HF, high fat; ICV, intracerebroventricular; LF, low fat; NPY, neuropeptide Y; PVDF, polyvinylidene difluoride; SNS, sympathetic nervous system; STAT, signal transducers and activators of transcription; WAT, white adipose tissue.
Received August 12, 2002.
Accepted for publication December 4, 2002.
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in HIT-T15 cells. Diabetes 44:85–89[Abstract]
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A. Varma, J. He, B.-C. Shin, L. A. Weissfeld, and S. U. Devaskar Postnatal intracerebroventricular exposure to leptin causes an altered adult female phenotype Am J Physiol Endocrinol Metab, December 1, 2004; 287(6): E1132 - E1141. [Abstract] [Full Text] [PDF]
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 M. S. Winzell and B. Ahren The High-Fat Diet-Fed Mouse: A Model for Studying Mechanisms and Treatment of Impaired Glucose Tolerance and Type 2 Diabetes Diabetes, December 1, 2004; 53(suppl_3): S215 - S219. [Abstract] [Full Text] [PDF]
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J. W. Bullen Jr, M. Ziotopoulou, L. Ungsunan, J. Misra, I. Alevizos, E. Kokkotou, E. Maratos-Flier, G. Stephanopoulos, and C. S. Mantzoros Short-term resistance to diet-induced obesity in A/J mice is not associated with regulation of hypothalamic neuropeptides Am J Physiol Endocrinol Metab, October 1, 2004; 287(4): E662 - E670. [Abstract] [Full Text] [PDF]
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