BROWN adipose tissue (BAT) is the major site of nonshivering

0013-7227/99/$03.00/0 Vol. 140, No. 10 Endocrinology Printed in U.S.A. Copyright 1999 by The Endocrine Society Norepinephrine Is Required for Leptin Effects on Gene Expression in Brown and White Adipose Tissue* SCOTT P. COMMINS, DONALD J. MARSH, STEVEN A. THOMAS, PATRICIA M. WATSON, MARK A. PADGETT, RICHARD PALMITER, AND THOMAS W. GETTYS Departments of Medicine (P.M.W., M.A.P., T.W.G.) and Biochemistry and Molecular Biology (S.P.C., T.W.G.), Division of Gastroenterology and Hepatology, Medical University of South Carolina, Charleston, South Carolina 29425; and Howard Hughes Medical Institute, and Department of Biochemistry (D.J.M., S.A.T., R.P.), University of Washington, Seattle, Washington 98195 ABSTRACT Exogenous leptin enhances energy utilization in ob/ob mice by binding its hypothalamic receptor and selectively increasing peripheral fat oxidation. Leptin also increases uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT), but the neurotransmitter that mediates this effect has not been established. The present experiments sought to determine whether leptin regulates UCP1 expression in BAT and its own expression in white adipose tissue (WAT) through the long or short forms of leptin receptor and modulation of norepinephrine release. Mice lacking dopamine -hydroxylase (Dbh / ), the enzyme responsible for synthesizing norepinephrine and epinephrine from dopamine, were treated with leptin (20 g/g body weight/day) for 3 days before they were euthanized. UCP1 messenger RNA (mrna) and protein expression were 5-fold higher in BAT from control (Dbh / ) compared with Dbh / mice. Leptin produced a 4-fold increase in UCP1 mrna levels in Dbh / BROWN adipose tissue (BAT) is the major site of nonshivering thermogenesis in rodents (1, 2). Cold exposure increases the activity of sympathetic nerves innervating BAT, and the release of norepinephrine increases heat production by activating 3 -adrenergic receptors and increasing camp in brown adipocytes (1 3). The ability of BAT to produce heat and uncouple oxidative phosphorylation is conferred by the presence of uncoupling protein 1 (UCP1) on the inner mitochondrial membrane, where it serves to short circuit the proton gradient that normally drives ATP synthesis (4). The release of norepinephrine acutely activates UCP1 through a camp-dependent mechanism (5, 6) and simultaneously enhances thermogenic capacity by transcriptional activation of the Ucp1 gene (5, 7, 8). Mice lacking Ucp1 (9) or the ability to synthesize catecholamines (10) are cold intolerant because of their inability to induce thermogenesis. Received March 22, 1999. Address all correspondence and requests for reprints to: Thomas W. Gettys, 916-G Clinical Science Building, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29425. E-mail: gettystw@musc.edu. * This work was supported by a Research Grant from the American Diabetes Association (T.W.G.), United States Public Health Service Grants DK-53981 (to T.W.G.), GM-08716 (to S.P.C.), and HD-09172 (R.D.P.), and a Research Grant from the United States Department of Agriculture NRICGP/USDA no. 9800699 (to T.W.G.). Current address: Steven A. Thomas, Pharmacology Department, University of Pennsylvania, Philadelphia, Pennsylvania 19104. mice but had no effect on UCP1 expression in Dbh /. The 3 -adrenergic agonist, CL-316,243 increased UCP1 expression and established that BAT from both groups of mice was capable of responding to -adrenergic stimulation. Similarly, exogenous leptin reduced leptin mrna in WAT from Dbh / but not Dbh / mice. In separate experiments, leptin produced comparable reductions in food intake in both Dbh / and Dbh / mice, illustrating that norepinephrine is not required for leptin s effect on food intake. Lastly, db/db mice lacking the long form of the leptin receptor failed to increase UCP1 mrna in response to exogenous leptin but increased UCP1 mrna in response to CL-316,243. These studies establish that norepinephrine is required for leptin to regulate its own expression in WAT and UCP1 expression in BAT and indicate that these effects are likely mediated through the centrally expressed long form of the leptin receptor. (Endocrinology 140: 4772 4778, 1999) Adaptive thermogenesis has also been advanced as a way to increase energy utilization during periods of chronic caloric excess (reviewed in Ref. 6). Thus, the adaptive increase in thermogenic activity may provide a mechanism to defend body weight by matching rates of energy utilization with energy intake (11). Much of what is known about leptin has come from study of ob/ob mice, where repletion provides a sensitive readout of leptin-dependent responses (12 14). Leptin reduces body weight by decreasing food intake and stimulating energy utilization (12, 14), but the neural and peripheral pathways that mediate these responses have not been defined. Recent findings that exogenous leptin increases norepinephrine turnover (15, 16) and UCP1 expression (17 19) suggest that its ability to selectively increase fat oxidation (20, 21) occurs through sympathetic enhancement of thermogenic activity. However, the recent identification of signaling-competent forms of the leptin receptor in tissues outside the hypothalamus (22 24) raises the possibility that leptin may have additional direct effects in peripheral tissues (25) or provide peripheral sensory input to the brain (26, 27). Using mice lacking the enzyme responsible for converting dopamine to norepinephrine (10) and mice lacking the functional long form of the leptin receptor (28 30), we show that leptin regulates adipocyte gene expression through the long form of the leptin receptor and that norepinephrine is required for this effect. 4772

LEPTIN REGULATES FAT CELL GENES THROUGH NOREPINEPHRINE 4773 Materials Materials and Methods N-Tris(hydroxylmethyl)methyl-2-aminoethanesulfonic acid buffer (TES), EDTA, sodium cholate, Triton X-100, BSA, guanidinium thiocyanate, sucrose, and other common chemicals were from Sigma (St. Louis, MO). T1-RNase and Trizol LS Reagent were from Life Technologies, Inc. (Gaithersburg, MD). T7 RNA polymerase, SP6 RNA polymerase, Taq DNA polymerase, MMLV reverse transcriptase, and the pgem-3z cloning vector were from Promega Corp. (Madison, WI). The T7-Megashortscript kit was purchased from Ambion, Inc. (Austin, TX). 2-Mercaptoethanol was acquired from J. T. Baker, Inc. (Phillipsburg, NJ). Oligonucleotide primers were prepared by the DNA Core Facility at the Medical University of South Carolina. Na[ 125 I] and -[ 32 P]-CTP were purchased from NEN Life Science Products (Boston, MA). Immobilon-P PVDF membranes were from Millipore Corp. (Bedford, MA). CL-316,243 was a gift from Wyeth-Ayerst Laboratories, Inc. (Princeton, NJ). Recombinant methionyl mouse leptin and human leptin were kindly provided by Amgen, Inc. (Thousand Oaks, CA) and Zymogenetics (Seattle, WA), respectively. Experimental animal protocol Exp 1. Eight-month-old male mice with a targeted disruption of the dopamine -hydroxylase (Dbh / ) gene (10) and heterozygous controls (Dbh / ) were housed in pairs and acclimated at 28 C on a 12-h light, 12-h dark cycle for 10 days before the study. Two hours following the beginning of the light cycle on the mornings of day 10,11, and 12, the mice were given ip injections of either saline or recombinant mouse leptin (20 g/g bw/day). Three hours after the final injection on day 12, the mice were euthanized and interscapular BAT was removed and carefully dissected free of surrounding vessels and connective tissue. One fat pad from each animal was used to isolate total RNA, while the contralateral fat pad was used for isolation of mitochondria as described previously (19). Purina mouse chow and water were available ad libitum. Exp 2. Control (Dbh) and Dbh / knockouts were acclimated as described above and injected with saline, leptin (20 g/g bw/day), or the 3 -adrenergic receptor agonist, CL-316,243 (1 g/g bw/day) for 3 days according to the same protocol. Three hours after the final injection on day 12, the mice were euthanized and interscapular BAT was removed and processed as above. Epididymal WAT was also obtained and used to prepare total RNA. Exp 3. Lean ( /?) and diabetic (db/db) C57BLKS/J male mice were obtained from The Jackson Laboratory (Bar Harbor, ME) at 4 5 weeks of age and randomly assigned to one of three treatment groups. The mice were housed three to a cage and equilibrated at 23 C on a 12-h light, 12-h dark cycle for 4 days before beginning the experiment. Two hours after the start of the light cycle on day 5, and for 2 days thereafter, the mice received ip injections of saline, leptin (20 g/g bw/day), or CL-316,243 (1 g/g bw/day). On day 7, the mice were euthanized 3 h after the final injection and tissues were harvested as described above. Exp 4. Control (Dbh / ) and Dbh / knockouts were acclimated at 23 C and food intake was monitored for 5 days before the experiment to establish a baseline. Thereafter, mice of each genotype received ip injections of vehicle or human leptin at 8-h intervals for 2 days, followed by monitoring of food intake for an additional 5 days. In this protocol, mice received three injections of 2.5 g leptin/g bw per day for a total daily dose of 7.5 g/g bw/day. Body weight was monitored daily. Exp 5. Control (Dbh / ) and Dbh / knockouts were acclimated at 23 C and food intake was monitored for 4 days before the experiment to establish a baseline, and for the 2 days after leptin treatment began. At 0900 h on the morning of the fifth day, mice of each genotype received ip injections of vehicle or human leptin (2.5 g/g bw/injection) at 8-h intervals for 2 days, and at 0900 h on the third day. The total daily dose of leptin was 7.5 g/g bw. Three hours after the last injection, the mice were weighed and euthanized. The scapular, inguinal, epididymal, and retroperitoneal WAT depot sites were carefully removed, dissected free of connective tissue, and weighed. The epididymal WAT and interscapular BAT were used to prepare total RNA. Exp 6. Male C57BL/6 mice were obtained from The Jackson Laboratory at 7 weeks of age and randomly assigned to one of three treatment groups. The mice were allowed to acclimate for 1 week at 23 C, during which time food intake and body weights were monitored. Thereafter, mice in group 1 received ip injections of vehicle, and food was provided ad libitum. Mice in group 2 received mouse leptin (20 g/g bw/day) for 3 days while mice in group 3 received vehicle and were pair-fed with the mice in group 2 receiving leptin. Three hours after the last injection on day 3, the mice were weighed, euthanized, and adipose tissue was processed as described above. Ribonuclease protection assay (RPA) of uncoupling protein 1 and leptin RNA probes complementary to UCP1 messenger RNA (mrna) and leptin mrna were prepared, labeled, and used as described previously to quantitate the respective mrna species (19). Western blotting of UCP1 Mitochondria were isolated and extracted as described previously (19), and used for Western blotting of UCP1 using an affinity-purified antibody raised against a peptide corresponding to amino acids 145 159 in the mouse sequence (19). Methods of analysis The concentration of UCP1 and leptin mrna in each sample was determined by reverse calibration from standard curves as described previously (19), and group means were compared by one-way ANOVA. For Western blots of UCP1, densitometric values were expressed as a percentage of the control group. Group means for protein expression, food consumption, and body composition data were compared by ANOVA as above. The level of protection against Type 1 errors was set at 5%, and the P values for specific treatment comparisons of interest are presented in the Results. Results Effect of leptin on BAT UCP1 in dopamine -hydroxylase knockout mice To minimize initial genotypic differences in BAT UCP1, mice were acclimated at 28 C for 10 days. Results from Exp 1 illustrate that even under these conditions, UCP1 mrna levels were significantly higher (P 0.01) in vehicle-injected control mice (Dbh / ) compared with Dbh / knockouts (Fig. 1). The difference between Dbh / (0.87 0.06 fmol UCP1 mrna/ g RNA) and Dbh / mice (0.30 0.03 fmol UCP1 mrna/ g RNA) was approximately 3-fold, compared with a 10- to 20-fold difference when mice were housed at 22 C (10). Treatment of Dbh / mice with leptin significantly increased UCP1 mrna (P 0.01) by approximately 3-fold to 2.42 0.09 fmol UCP1 mrna/ g RNA (Fig. 1). In contrast, UCP1 mrna levels in Dbh / mice after leptin treatment (0.33 0.03 fmol UCP1 mrna/ g RNA) were essentially unchanged from levels noted in saline-injected Dbh / mice (Fig. 1). To test the capacity of BAT from Dbh / mice to respond to -adrenergic stimulation, Exp 2 was conducted using the 3 -adrenergic agonist, CL-316,243. As noted in the first experiment, UCP1 mrna was higher in Dbh / (0.53 0.07 fmol mrna/ g RNA) compared with Dbh / mice (0.26 0.06 fmol mrna/ g RNA). However, results presented in Fig. 2 demonstrate that CL-316,243 produced a comparable 5-fold increase in UCP1 mrna in both genotypes (P 0.01). These results establish that BAT in Dbh / mice can respond to -adrenergic stimulation by increasing UCP1 mrna.

4774 LEPTIN REGULATES FAT CELL GENES THROUGH NOREPINEPHRINE Endo 1999 Vol 140 No 10 FIG. 1. Ribonuclease protection assay of UCP1 mrna and 18S rrna in 1 g of total RNA from BAT of control (Dbh / ) and Dbh / knockout mice injected with saline or leptin (20 g/g bw/day) for 3 days as described in Materials and Methods. The relative abundance of UCP1 mrna was quantitated by comparing the densitometric intensities of protected fragments from each treatment group to known amounts of sense strand transcripts that were hybridized simultaneously. The UCP1 probe produces a protected fragment of 293 bp that corresponds to nucleotides 7 300 of the mouse UCP1 mrna, and a probe for 18S rrna is included to adjust for differences in RNA loaded between the lanes. Individual RNA samples from each animal were analyzed to calculate group means and representative samples are presented in the figure (Control saline, 0.87 0.06 fmol UCP1 mrna/ g RNA, n 8; Control leptin, 2.42 0.09 fmol UCP1 mrna/ g RNA, n 7; Dbh / saline, 0.30 0.03 fmol UCP1 mrna/ g RNA, n 8; Dbh / leptin, 0.33 0.02 fmol UCP1 mrna/ g RNA, n 7). To determine whether UCP1 protein expression paralleled the observed differences in UCP1 mrna associated with genotype and leptin treatment, mitochondrial extracts from contralateral fat pads were probed for UCP1 expression by Western blot. Figure 3 illustrates that UCP1 expression was 3- to 4-fold higher (P 0.01) in Dbh / compared with Dbh / mice. Figure 3 also shows that leptin did not increase UCP1 expression in BAT from Dbh / mice, but produced a significant 50 60% increase in UCP1 levels in Dbh / mice (P 0.05). Considered together, these data show that leptin increased UCP1 mrna and protein in BAT from Dbh / mice, but failed to do so in Dbh / mice. Effect of leptin on UCP1 in db/db mice Exp 3 was performed with db/db mice that express various short forms of the leptin receptor, but lack the functional long form of the receptor (28, 31). These experiments were conducted to determine if exogenous leptin could increase UCP1 mrna by direct effects on adipose tissue through the short form of the leptin receptor. To this end, we treated C57 lean and db/db mice with doses of leptin previously shown to induce UCP1 expression (17, 19). As predicted, both leptin and CL-316,243 produced significant (P 0.01) 2- to 4-fold increases in UCP1 mrna in BAT of lean mice compared with saline injected controls (data not shown). In contrast, leptin was without effect on UCP1 mrna in BAT from db/db mice (saline, 2.36 0.08 fmol UCP1 mrna/ g RNA; leptin, 2.23 0.07 fmol UCP1 mrna/ g RNA). However, the FIG. 2. Ribonuclease protection assay of UCP1 mrna in 1 g total RNA from BAT of control and Dbh / mice injected with vehicle or CL- 316,243 (1 g/g bw/day) as described in Materials and Methods. The UCP1 probe produces a protected fragment of 293 bp, and a probe for 18S rrna was included to adjust for differences in RNA loaded between the lanes. UCP1 mrna was quantitated by comparing the densitometric intensity of the protected fragment to the intensity of sense strand transcripts that were hybridized simultaneously. Individual RNA samples from each animal were analyzed to calculate group means and representative samples are presented in the figure (Control saline, 0.53 0.07 fmol UCP1 mrna/ g RNA, n 8; Control CL-316,243, 2.81 0.14 fmol UCP1 mrna/ g RNA, n 3; Dbh / saline, 0.26 0.06 fmol UCP1 mrna/ g RNA, n 8; Dbh / CL-316,243, 1.17 0.09 fmol UCP1 mrna/ g RNA, n 3). FIG. 3. Western blot of UCP1 in BAT from control mice (Dbh / ) injected with saline (lanes 1 2) or leptin (lanes 3 4) and Dbh / knockout mice injected with saline (lanes 5 6) or leptin (lanes 7 8) as described in Materials and Methods. Mitochondrial extracts (5 g/lane) were prepared from the brown fat pads that were contralateral to the fat pads used to measure UCP1 mrna in Fig. 1, and subjected to Western blotting using affinity-purified UCP1 antibody raised against a peptide corresponding to AA 145 159 of mouse UCP1. The detected proteins were visualized with 125 I-labeled goat antirabbit IgG and the autoradiograms were scanned by laser densitometry. Densitometric intensities were expressed as a percentage of control values and individual mitochondrial extracts from each animal were analyzed to calculate group means. The autoradiogram is representative of eight replicates for the saline-injected mice of each genotype and seven replicates for the leptin-injected mice. significant induction (P 0.01) of UCP1 mrna in CL-316,243-treated db/db mice (5.10 0.25 fmol UCP1 mrna/ g RNA) demonstrates that their BAT is capable of responding to -adrenergic stimulation. These results also show that the long form of the leptin receptor is required for leptin to increase UCP1 expression in BAT. Effect of leptin on food intake and tissue weights In Exp 4, food intake in control (Dbh / ) and Dbh / mice was comparable between groups during the preinjection protocol, and leptin produced a similar and significant decrease in food consumption in both genotypes on days 6 and 7 (Fig.

LEPTIN REGULATES FAT CELL GENES THROUGH NOREPINEPHRINE 4775 4). Figure 4 also shows that food consumption returned to preinjection levels on days 8 12. It should be noted that despite the leptin-induced reduction in food intake, the reduction of body weight was evident but not significant (preand postleptin body wts; vehicle Dbh /, 30.28 1.44 g; leptin Dbh /, 29.04 1.56 g; vehicle Dbh /, 23.90 0.40 g; leptin Dbh /, 23.03 0.41 g; Exp 4). Comparable results were obtained in a second replicate of this experiment (Exp 5, Table 1). Leptin reduced food consumption from 4.4 0.2 g/d to 3.1 0.4 g/d in Dbh / mice, and produced a comparable decrease from 4.2 0.1 g/day to 2.8 0.2 g/day in the Dbh / group (Table 1). The initial body weights were approximately 5 g lower in Dbh / than Dbh / mice, but as before, body weight was unaffected by leptin treatment in either group (Table 1). It should be noted that despite this difference in body weight, food consumption was comparable between Dbh / and Dbh / mice (Fig. 4 and Table I). This means that Dbh / mice were consuming more food per unit body weight than control mice. Thus, if Dbh / and Dbh / mice are studied at comparable body weight, the Dbh / group would be considered hyperphagic, as noted in the original report (10). Although leptin had no discernible effect on body weight, it was important to determine whether leptin produced FIG. 4. Food intake over time in Dbh / and Dbh / mice injected with vehicle on days 1 5, human leptin on days 6 and 7 (s), and vehicle on days 8 12 of the experimental protocol. Mice of each genotype were injected on days 6 and 7 with 2.5 g leptin/g bw at 09:00, 17:00, and 01:00 h for a total daily leptin dose of 7.5 g/g bw. Average daily food consumption ( SE) is presented for 10 Dbh / ( ) and 9 Dbh / (E) mice. changes in the amount or distribution of fat in the carcass. Careful dissection of the various WAT depots indicated that our short injection protocol with leptin produced no significant changes in any of the WAT depots examined in either genotype (Table 1). Effect of exogenous leptin on leptin mrna in WAT To test the hypothesis that leptin regulates its own expression in WAT through a feedback mechanism involving norepinephrine, we measured leptin mrna in epididymal WAT from leptin-treated mice. In Dbh / mice from Exp 5, exogenous leptin produced a highly significant reduction (P 0.01) of leptin mrna in epididymal WAT from 0.085 0.004 fmol leptin mrna/ g RNA to 0.018 0.001 fmol leptin mrna/ g RNA in leptin-injected mice (Fig. 5). Figure 5 also shows that treatment of Dbh / mice with CL-316,243 reduced leptin mrna in epididymal WAT to near the detection limits of the assay (P 0.01). In contrast, exogenous leptin had no effect on leptin mrna in epididymal WAT (vehicle, 0.099 0.004 fmol/ g RNA; leptin, 0.090 0.003 fmol/ g RNA) from Dbh / mice (Fig. 5). However, the robust reduction (P 0.01) of leptin mrna by CL-316,243 confirmed that epididymal WAT from these mice was fully responsive to -adrenergic stimulation (Fig. 5). Effects of food intake and leptin on leptin mrna in WAT To separate the effects of leptin on food intake from the effects of leptin on leptin mrna that are independent of food intake, vehicle-injected mice were either fed ad libitum or pair-fed to leptin-injected mice (Exp 6). Leptin produced a 20 30% reduction in food intake compared with the ad lib group over the 2-day treatment protocol, but the decrease in body weight was small and not significant (data not shown). The body weight and fat pad masses from the pair-fed group were also unchanged in comparison to the ad lib and leptininjected groups (not shown). In contrast, leptin mrna in the pair-fed group (0.036 0.005 fmol/ g RNA) was lower than levels (0.063 0.007 fmol/ g RNA) in the ad libitum group (P 0.05, Fig. 6). However, leptin produced an additional 3-fold decrease in leptin mrna to 0.011 0.003 fmol/ g RNA compared with the pair-fed group (P 0.01, Fig. 6). These results show that modest food restriction produced a TABLE 1. Food consumption, body weight, and fat pad weight in control (Dbh / ) and Dbh / mice treated with leptin a Treatment group Control (Dbh / ) Knockouts (Dbh / ) Saline (n 5) Leptin (n 5) Saline (n 4) Leptin (n 5) Initial body wt (g) 29.9 1.7 30.2 1.5 24.7 1.0 24.7 0.7 Final body wt (g) 31.1 1.7 29.1 1.8 24.8 0.8 24.4 0.6 Food consumption (g/d) 4.4 0.2 3.1 0.4 b 4.2 0.1 2.8 0.2 b Scapular WAT (g) 0.61 0.10 0.57 0.12 0.83 0.11 0.76 0.11 Inguinal WAT (g) 0.46 0.06 0.52 0.14 0.70 0.08 0.69 0.11 Epididymal WAT (g) 0.77 0.21 0.81 0.23 0.58 0.08 0.52 0.07 Retroperitoneal WAT (g) 0.32 0.08 0.35 0.09 0.27 0.03 0.26 0.03 Total adipose tissue (g) 2.29 0.43 2.36 0.58 2.62 0.30 2.42 0.31 a Control (Dbh / ) and Dbh / knockout mice received ip injections with vehicle or human leptin (7.5 g/g body wt/day) on each of two successive days as described in Materials and Methods. The mice were euthanized 3 h after the last injection on the morning of the third day. Mice were weighed at the beginning of the experiment and at the time they were euthanized. The indicated tissues were removed at the time they were euthanized, dissected free of connective tissue, and weighed. The data were subjected to ANOVA. b Food consumption in Dbh / and Dbh / mice injected with leptin was significantly less (P 0.01) than vehicle-injected mice of each genotype.

4776 LEPTIN REGULATES FAT CELL GENES THROUGH NOREPINEPHRINE Endo 1999 Vol 140 No 10 FIG. 5. Ribonuclease protection assay of leptin mrna in 5 g total RNA extracted from EWAT of control (Dbh / ) and Dbh / mice injected with either saline, human leptin (7.5 g/g bw/day), or CL- 316,243 (1 g/g bw/day) as described in Materials and Methods. The leptin probe produces a 355 bp protected fragment corresponding to nucleotides 1 355 of the mouse leptin mrna, and a probe for 18S rrna was included to adjust for differences in RNA loaded between the lanes. Leptin mrna was quantitated by comparing the densitometric intensity of the protected fragment to the intensity of sense strand transcripts that were hybridized simultaneously. Individual RNA samples from each animal were analyzed to calculate group means and representative samples are included in the figure (control saline, 0.09 0 0.004 fmol leptin mrna/ g RNA, n 5; control leptin, 0.018 0.003 fmol leptin mrna/ g RNA, n 5; control CL-316,243, 0.002 0.002 fmol leptin mrna/ g RNA, n 3; Dbh / saline, 0.099 0.004 fmol leptin mrna/ g RNA, n 4; Dbh / leptin, 0.090 0.004 fmol leptin mrna/ g RNA, n 5; Dbh / CL-316,243, 0.002 0.001 fmol leptin mrna/ g RNA, n 3). decrease in leptin mrna that was independent of any changes in fat pad size. Leptin produced a further reduction in leptin mrna in WAT that is independent of both changes in food intake and the size of the fat pad. Discussion In the present study, we have used mice lacking the ability to synthesize catecholamines from dopamine to address the mechanism used by leptin to regulate gene expression in both brown and white adipose tissue. The initial description of Dbh / mice reported that they were unable to increase UCP1 mrna in response to cold (10). Cold exposure activates thermogenesis by increasing norepinephrine release from sympathetic nerves, which induces UCP1 expression through a 3 -adrenergic receptor-mediated and campdependent mechanism (4 8). Recent studies indicate that leptin also regulates UCP1 expression in BAT (17 19, 32, 33). This response has been studied using surgical (34) or chemical (35) ablation of the nerves innervating BAT, and both treatments blocked leptin s ability to increase UCP1. Leptin signaling mechanisms were also addressed using multifiber recording (16) and norepinephrine turnover (15) as endpoints. These findings complement a recent study that mapped the hypothalamic pathways mediating leptin s effects on the sympathetic nervous system and found that leptin activates neurons in the retrochiasmatic area and lateral arcuate nucleus (36). This study further established that FIG. 6. Ribonuclease protection assay of leptin mrna in 5 g total RNA extracted from epididymal WAT of C57BL/6J mice given ip injections with vehicle and fed ad libitum (lanes 1 2), injected with vehicle and pair-fed to mice receiving leptin (lanes 3 4), or injected with leptin (lanes 5 6) as described for Exp 6 in Materials and Methods. Leptin mrna was quantitated by comparing the densitometric intensity of the protected fragment to the intensity of sense strand transcripts that were hybridized simultaneously. Individual RNA samples from each animal were analyzed to calculate group means and representative samples are included in the figure (vehicle ad libitum, 0.063 0 0.007 fmol leptin mrna/ g RNA, n 6; vehicle pair-fed, 0.036 0.005 fmol leptin mrna/ g RNA, n 6; leptin, 0.011 0.003 fmol leptin mrna/ g RNA, n 6). the neurons in these regions innervate sympathetic preganglionic neurons in the thoracic spinal cord (36). Together with the present demonstration that norepinephrine is required for leptin to increase UCP1 expression, these results support an emerging consensus that the sympathetic nervous system is a common conduit for regulating both temperature- and leptin-dependent UCP1 expression. It should also be noted that treatment with DOPS, a synthetic amino acid that can be converted to norepinephrine through a mechanism independent of dopamine -hydroxylase, restores norepinephrine levels and UCP1 expression in BAT from Dbh / mice (37). Leptin produced comparable reductions of food intake in both genotypes in the current study. These findings establish that Dbh / mice are responsive to leptin, and that their failure to regulate UCP1 or leptin mrna in response to leptin is not due to leptin resistance. This conclusion is also supported by the original report that indicated that serum leptin levels were comparable between Dbh / and Dbh / mice (10). Previous studies have established that 3 -adrenergic receptor-mediated lipolysis acutely suppresses food intake in mice (38, 39), raising the possibility that leptin may suppress food intake indirectly by mobilizing FFA. Based on previous studies (21), it seems likely that leptin s ability to increase serum FFA would have been compromised in Dbh / mice. Therefore, the finding that leptin-mediated inhibition of food intake was intact suggests that leptin s behavioral effects are not mediated by peripheral mobilization of fat. In db/db mice, a point mutation in the leptin receptor gene generates a new splice donor site that suppresses expression of the long form of the leptin receptor (28, 29, 31). The long isoform (Ob-Rb) is expressed preferentially in the hypothalamus and contains the putative cytoplasmic signaling do-

LEPTIN REGULATES FAT CELL GENES THROUGH NOREPINEPHRINE 4777 main (31), while the short forms (Ob-Ra) are widely expressed in central and peripheral tissues (22 24). We used db/db mice in the current study to determine whether leptin could influence UCP1 expression in adipose tissue through the short forms of the leptin receptor. As previously reported (40 42), the absence of Ob-Rb led to significant up-regulation of leptin mrna and circulating leptin in db/db mice. This observation is relevant for two reasons. First, UCP1 should be down-regulated, as we have seen in ob/ob mice (19), if Ob-Rb is the primary mediator of leptin s effects on UCP1. On the contrary, if leptin is capable of acting through the short form of the leptin receptor, expression of UCP1 should be significantly increased in db/db mice. The outcome was intermediate in the sense that UCP1 mrna was the same or slightly lower in BAT from db/db mice compared with their lean littermates. Coupled with a robust response to CL-316,243 and the absence of any effect of injected leptin, the results indicate that the absence of leptin effects on adipocyte gene expression in db/db mice are not due to an inability of the adipocyte to respond to sympathetic stimulation. Thus, in addition to norepinephrine and epinephrine, our studies show that the long form of the leptin receptor is required for leptin to increase UCP1 expression in BAT. The recent detection of Ob-Rb outside the central nervous system (43), coupled with evidence for direct effects of leptin in peripheral sites (22 24) broadened our understanding of how leptin may be working. As such, the present studies do not distinguish between a peripheral vs. a central site of action. However, the additional requirement for norepinephrine argues against direct effects of leptin on UCP1 expression in adipose tissue, even though Ob-Rb has been readily detected in both isolated adipocytes and stromovascular cells (unpublished data). It seems far more likely that leptin induces UCP1 expression in BAT through its hypothalamic receptor and increased sympathetic output. Studies with genetic models of obesity demonstrate that the absence of leptin (ob/ob) or its receptor (db/db) results in up-regulation of leptin mrna in WAT (40 42), indicating that both components are required to produce an efferant signal from the brain which feeds back on leptin production. The up-regulation of leptin mrna is disproportionate to the size of the fat pads, suggesting that the efferant signal may be inhibitory. These observations raise the interesting possibility that sympathetic stimulation, acting through adrenergic receptors, inhibits leptin expression through a camp-dependent pathway. This concept is supported by studies showing that 3 -adrenergic receptor agonists or cold exposure decrease leptin expression in vivo (39, 44, 45) and in vitro (46). Rayner et al. (47) presented complementary findings by showing that administration of -methyl- tyrosine to block norepinephrine synthesis produced hyperleptinemia in mice. Another recent study reported that exogenous leptin reduced leptin mrna in WAT (34). Although the significance of sympathetic innervation of WAT has been questioned for many years, recent studies with Siberian hamsters (48, 49) and fasted rats (50) provide direct evidence of sympathetic stimulation of WAT. Results from the present study support this concept by demonstrating that Dbh / mice unable to synthesize catecholamines do not downregulate leptin mrna in response to exogenous leptin. Viewed in tandem with the robust decrease in leptin mrna in CL-316,243-treated Dbh / mice and leptin-treated heterozygous controls (Dbh / ), the present results make a compelling case that leptin regulates its own expression through sympathetic stimulation of -adrenergic receptors. It is also possible that leptin increases catecholaminergic stimulation of WAT through an indirect mechanism involving the adrenal medulla. Although adrenal medullary cells express primarily the short form of the leptin receptor, these receptors were colocalized to epinephrine-secreting cells in the adrenal medulla (51). By binding to these peripheral receptors, it is possible that leptin could increase epinephrine release through this mechanism. However, in view of the high circulating leptin levels in db/db mice, which express the short form of the leptin receptor, the significance of this mechanism is questionable. 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