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APPETITE, OBESITY, DIGESTION, AND METABOLISM
Department of Foods and Nutrition, University of Georgia, Athens, Georgia
Submitted 30 May 2006 ; accepted in final form 10 July 2006
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-methyl-para-tryrosine. The centrally injected leptin-treated animals demonstrated more dramatic reductions in food intake, body weight, and fat pad size and an increase in NETO compared with the peripherally infused animals. Neither route of leptin administration caused a uniform increase in NETO across all fat pads tested, and in both treatment conditions leptin decreased the size of certain fat pads independent of an increase in NETO. Similar discrepancies in white fat NETO were found for rats pair fed to leptin-treated animals. These results demonstrate that leptin acting either centrally or peripherally selectively increases sympathetic outflow to white fat depots and that a leptin-induced change in fat pad weight does not require an increase in NETO. white adipose tissue; brown adipose tissue; sympathetic nervous system
The amount of energy that is available for storage in fat depots is determined both by the energy balance status of the animal and by the partitioning of nutrients between different tissue types. The loss of body fat in leptin-treated animals could be associated with changes in a number of different pathways, including those that reduce the number of preadipocytes (40), those that promote lipid mobilization out of adipocytes (14), and those that inhibit accumulation of triglyceride in fat cells (23). In vivo and in vitro studies indicate that leptin has the potential to influence each of these pathways (13, 17, 40, 41).
All of these changes in adipose metabolism can be activated in response to peripheral infusions of leptin; however, it is not clear whether the in vivo response is because of a direct effect of leptin on adipocytes or whether leptin crosses the blood-brain barrier and acts centrally to initiate the changes in metabolism. Adipocytes express leptin receptors (3), making it reasonable to assume that an increase in circulating leptin could directly reduce the size of fat depots; however, adipose tissue also is innervated by the sympathetic nervous system (SNS; see Ref. 44), providing the opportunity for central regulation of adipose tissue mass. Because sympathetic nerves in white adipose tissue regulate both lipolysis and the number of cells present and because some of the nuclei that have been identified as sites of initiation of sympathetic outflow to white fat (2) also express leptin receptors (11), this provides an obvious pathway by which leptin might influence body fat content. Haynes et al. (22) have clearly demonstrated that intracerebroventricular leptin can activate renal and lumbar sympathetic outflow, but it is not clear that leptin activates the sympathetic supply to white adipose tissue. Physiological stimuli promote SNS activity in a tissue-specific manner; therefore, activation of SNS outflow to one organ does not necessarily represent outflow to all organs (29). Measurements of norepinephrine turnover (NETO) in specific tissues has been used as a measure of site-specific sympathetic activity in conditions such as fasting (28) and seasonal changes in energy balance in hibernating animals (27). Collins et al. (7) found only a small nonsignificant increase in NETO to retroperitoneal (RP) white adipose tissue of mice given an intraperitoneal injection of leptin, despite a significant increase in intrascapular brown adipose tissue (IBAT) NETO.
Previously, we attempted to determine the importance of sympathetic innervation of white fat in mediating leptin-induced changes in body fat mass by chemically sympathectomizing one fat pad in leptin-treated mice or rats (34). The outcome of this study was complicated by the finding that sympathectomy of one fat pad influenced both the norepinephrine (NE) content and leptin responsiveness of other neurally intact fat pads in the same animal. Therefore, the studies described here measured NETO in different fat depots of rats that received either central injections or peripheral infusions of leptin to test whether there was a direct association between a leptin-induced increase in NETO and a reduction in fat pad mass. Because of the high probability that peripheral and central leptin activate different subpopulations of leptin receptors and because it is impossible to calculate how much of the peripheral leptin would reach hypothalamic sites activated by third ventricle infusions of leptin, we did not attempt to match the treatments for degree of response in the animals but compared the effects of peripheral vs. central leptin administration on a qualitative rather than a quantitative level. Measurements in multiple fat depots allowed us to determine whether the relative differences in leptin responsiveness of individual fat depots was associated with differences in NETO in these depots and whether peripheral leptin produced significant changes in adipose NETO, which would imply that some, or all, of the metabolic response to peripheral leptin is mediated by centrally located receptors.
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METHODS |
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Experiment 1: Effect of varying peripheral doses of leptin on food intake, body weight, fat pad weight, and serum leptin.
The objective of this study was to identify a dose of leptin that produced a significant change in body composition after 13 days of peripheral infusion. Twenty-five male Sprague Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) with body weights of 
250 grams were individually housed in hanging wire cages in a humidity- and temperature-controlled room on a 12:12-h light-dark cycle with lights on at 0700. Animals had free access to tap water and standard rat chow (LabDiet 5012; PMI Nutrition International, Brentwood, MO). After 1 wk of adaptation to the environment, baseline daily food intakes, corrected for spillage, and body weights were recorded for 6 days before rats were divided into three weight-matched groups. Under isofluorane anesthesia, each rat was then fitted with an intraperitoneal Alzet miniosmotic pump (model 2002; Durect, Curpertino, CA) that delivered either 40 µg leptin (recombinant rat leptin; R&D Systems, Minneapolis, MN), 75 µg leptin, or an equal volume of 0.01 M PBS/day for 13 days. Daily body weights and food intakes were measured, and, on day 13, trunk blood was collected for measurement of serum leptin (Leptin RIA; Linco Research, St. Charles, MO). Epididymal (EPI), RP, mesenteric (MES), and IBAT were excised and weighed.
The results of this experiment demonstrated that 13-day infusions of 40 or 75 µg of leptin were both capable of reducing fat pad size to a similar degree even though 40-µg levels of leptin did not significantly increase serum leptin concentration above control levels. Therefore, the lower dose of leptin was used in experiment 2.
Experiment 2: Effect of peripherally infused leptin on NETO in specific fat depots.
Fifty-five male Sprague-Dawley rats (Harlan Sprague-Dawley) with body weights of 300 grams were housed in similar conditions as described in experiment 1. After 3 days of adaptation to the environment, baseline daily food intakes, corrected for spillage, and body weights were recorded for 7 days before rats were divided into three weight-matched groups: leptin (n = 19), pair fed (n = 18), and control (n = 18). Rats were fitted with intraperitoneal miniosmotic pumps (model 1007D) on the 7th day that delivered either 40 µg of leptin or an equal volume of 0.01 M PBS/day. Daily food intakes and body weights were measured for 4 days. Pair-fed rats were fed the average amount of food that the leptin rats had eaten on the previous day. Food was given to the pair-fed rats in two meals: one at the start of the light cycle and one at the start of the dark cycle.
On day 4 of infusion, food was removed from the cages at 0800. Starting at 1000, one-half of the rats in each treatment group was decapitated (time 0), whereas the remaining rats were given the first of two intraperitoneal injections of -methyl-para-tyrosine (-MPT; Sigma, St. Louis, MO), a tyrosine hydroxylase inhibitor that blocks NE synthesis. -MPT was first dissolved in glacial acetic acid (0.5 µl/mg -MPT) and then diluted to a final concentration of 300 mg/ml in sterile PBS. The solution was kept in the dark and on ice. At time 0, the rats were injected with 300 mg -MPT/kg body wt, and 2 h later they were given a second injection of 150 mg -MPT/kg body wt. Exactly 4 h after the first injection of -MPT, the rats were decapitated (time 4). Only two time points were used for the measurement of turnover, since previous publications have shown linearity of the logarithmic decline in NE content over periods of 6 or 8 h (28).
Trunk blood was collected for measurement of serum leptin. IBAT, MES, and combined left and right RP, inguinal (ING), and EPI fat pads were weighed. Approximately 50 mg of IBAT, 100 mg of MES, and 200 mg of each of the other pads were snap-frozen for measurement of tissue NE by reverse-phase HPLC, as described below. For bilateral depots, approximately equal quantities of fat were taken from the same anatomic location of each pad and combined for analysis to control for differences in cellularity and morphology within a depot. Remaining tissue was snap-frozen for RNA extraction.
Experiment 3: Effect of centrally infused leptin on NETO in specific fat depots.
Fifty-seven male Sprague-Dawley rats (Harlan Sprague Dawley) with body weights of 185 grams were housed as described for experiment 1. After 1 wk of adaptation, animals were fitted with third ventricle cannulas. They were anesthetized by intraperitoneal injections of ketamine (90 mg/kg) and xylazine (10 mg/kg). Guide cannulas (22 gauge, 15 mm long) were placed using the stereotaxic coordinates applied to a flat skull: anteroposterior –2.8, lateral 0.0, ventral –8.1 from bregma (38). Injection cannulas (28 gauge) were designed to project 1 mm beyond the tip of the guide cannula. The rats were given an injection of analgesic (2 mg/kg Ketoprofen) immediately after surgery and on the day after surgery. Proper cannula placement was confirmed 10 days later by monitoring water intake during the 5 min after infusion of ANG II (10 ng delivered over 2 min). Later (3 days), baseline body weights and food intakes, corrected for spillage, were recorded for 5 days, and then animals were divided into three weight-matched groups: leptin (n = 19), pair fed (n = 19), and control (n = 19). Rats in the leptin group received twice daily third ventricle infusions of 1.5 µg of leptin, in a volume of 2 µl infused over 2 min. Infusions were given at 0900 and 1700 for 2 days, and then a final infusion was given on the 3rd day 2 h before time 0. The control and pair-fed animals were infused with an equivalent volume of 0.01 M PBS. On the 3rd day, animals were injected with -MPT, and tissue samples were collected exactly as described for experiment 2. Tissue NE concentration was measured as described below.
NE measurements. NE content of white fat depots and IBAT was measured by reverse-phase HPLC with electrochemical detection. EPI, MES, or RP fat was sonicated on ice three times for 30 s in 800 µl 0.2 M perchloric acid containing 3 µg/ml ascorbic acid and 25 ng/ml 3,4-dihydroxybenzylamine hydromide (DHBA) as an internal standard. ING and IBAT were processed in an identical manner except that they were sonicated in 1 ml of DHBA. The sonicated fat mixture was centrifuged for 15 min at 9,000 revolutions/min at 4°C. After centrifugation, the infranate was filtered through a 0.2-µm Nylon Sterile 25-mm syringe filter. NE was assayed using an ESA HPLC system (Bedford, MA) that consisted of a model 582 Solvent Delivery Module, a model 542 auto sampler maintained at 6°C, and a model 5600A CoulArray detector at 350 mV. The column was a Phenomenex (150 x 4.6 mm) SYNERGI 4 µm Max RP-80A, and the mobile phase consisted of 0.1 M sodium phosphate monobasic, 0.1 mM disodium EDTA, 0.3 mM 1-octanesulfonic acid, and 4% acetonitrile, pH of 3.1. Chromatograms were analyzed with CoulArray for Windows, version 1.04, and NE content was calculated from a standard curve. Data are expressed as nanograms of NE per fat depot.
Calculation of NETO. Fractional turnover rates of NE were measured based on the rate of decline in tissue NE content after inhibition of synthesis. This method has been widely used in measuring NE fractional degradation rate and turnover time and is an application of the principles of steady-state kinetics to the change with time of NE concentration (5). NETO was calculated using the following formula:
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where the fractional turnover rate (k) = {log(mean [NE])0 – log(mean [NE]4)}/(0.434 x 4); [NE]0 is NE concentration at time 0, and [NE]4 is NE concentration at time 4.
Real-time PCR. MES and ING tissue from time 0 rats in each experiment were used to measure hormone-sensitive lipase (HSL) and leptin mRNA because these were the white fat depots that were the least and the most responsive to leptin, respectively, in terms of increased NETO. IBAT was used to measure uncoupling protein 1 (UCP1) mRNA. Total RNA was extracted using Tri-Zol reagent according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA). RNA concentration and quality were determined by the spectrophotometric 260:280 ratio and by agarose gel electrophoresis.
After extraction, 1 µg of RNA was transcribed for cDNA as described previously (20). Primers used to amplify leptin, UCP1, HSL mRNA, and 18S rRNA are described in Table 1. The SYBR green reaction was prepared according to the manufacturer’s protocol (Bio-Rad, Hercules, CA), and PCR was performed using the Bio-Rad iCycler system. Amplification conditions were as follows: 95°C for 3 min; 40 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s. Immediately after Q-PCR amplification, a melt curve analysis was performed to ensure the specificity of the amplification. 
Ct (dCt) values or differences in threshold cycles for the mRNA of interest and 18S rRNA were determined for each sample, and relative mRNA expression was expressed as 1/dCt.
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RESULTS |
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The changes in white fat NETO were depot specific and different from those induced by peripheral leptin infusions. In contrast to experiment 2, ING NETO was not different between treatment groups, but leptin significantly increased NETO in EPI and MES fat (Fig. 3B). Pair feeding increased NETO in EPI, MES, and IBAT but dramatically inhibited NETO in RP fat (Figs. 3B and 4B). NETO was higher in MES fat than in other white fat depots (Fig. 3B). Differences in NETO between fat depots was because of the difference in both tissue NE content and k. In contrast, when there was a treatment effect on NETO for a specific depot, it was because of a change in k rather than in tissue NE concentration at time 0 (Table 4).
In contrast to experiment 2, we found significant effects of leptin and of pair feeding on white fat mRNA expression. Pair feeding stimulated HSL mRNA expression in ING fat (Fig. 5B), and both leptin and pair feeding inhibited leptin mRNA expression in ING fat (Fig. 5D). HSL and leptin mRNA expression was greater in the ING than the MES fat depot (Fig. 5B). There were no significant differences in IBAT UCP1 mRNA expression (Fig. 6B).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The measures of NETO in experiments 2 and 3 cannot be compared quantitatively because the pattern of receptor activation is likely to be different between peripheral and central leptin administration, NE measurements for each of the studies were made at different times, and the duration of leptin treatment was different for experiments 2 and 3. NETO varied between white fat depots in both experiments, but the pattern of response was different. This could not simply be attributed to a generalized response to the more energetically stressful condition produced by central leptin infusions and the resulting more extreme food restriction of the pair-fed controls in experiment 3 because differences between the two experiments were not consistent among white fat depots. NETO was relatively low in ING fat from centrally infused rats but high in ING from peripherally infused rats, whereas MES and RP fat NETO were relatively low in peripherally infused animals but high in centrally infused rats. In addition, leptin and pair feeding had opposing effects on NETO in RP fat from the centrally infused animals. Because the size of all white fat depots except MES decreased in both the centrally leptin-infused and pair-fed animals in experiment 3, these results indicate that leptin does not depend on the SNS to reduce the size of all fat depots, consistent with the results of a study in which rats with one sympathetically denervated fat pad were infused with leptin (34). The results from experiment 2 also support this conclusion because peripherally infused leptin caused a significant decrease in the size of the MES tissue, whereas ING fat was the only depot to show even a tendency for an increase in NETO. The results from experiment 1 demonstrated that a 13-day infusion of 40 µg of leptin significantly reduced fat pad weight; therefore, we can conclude that the absence of a significant decrease in size of the majority of fat pads in rats in experiment 2 was because of the short duration of treatment, which was selected to ensure that measurements were made during the dynamic phase of leptin-induced SNS activation.
The variability in NETO across different fat depots confirms previous findings that physiological stimuli promote sympathetic activity in a tissue-specific manner (43) and suggests either a variation in the degree of innervation or a difference in the concentration and ratio of adrenergic receptors in the different tissues (24). Others have reported variations in the degree of innervation of different white fat pads (45) and that catecholamine concentrations (33, 34), oxidative capacity (9), and cellularity (10) are different in different fat depots. These region-specific differences imply that the response to endocrine and neural factors varies between depots and that the variable responses of the different fat pads were not unique to the experiments reported here.
How leptin caused a decrease in pad size without increasing NETO is not clearly understood. We have reported that the size of the sympathetically denervated EPI pad of mice and RP pad of rats was reduced in response to peripheral infusion of physiological doses of leptin (34), and others reported that transplanted EPI fat, with no apparent reinnervation, decreased in size in rats expressing adenovirus leptin (42). Additionally, in vitro studies have shown that direct administration of leptin stimulates lipolysis in isolated fat pads (15, 37, 41, 42) and that the sensitivity of fat pads to the lipolytic effects of leptin appears to be depot specific (15). In contrast, adipocyte lipogenesis has been shown to be inhibited in mice infused (17) or injected (6) with leptin, but leptin does not directly inhibit adipocyte lipogenesis in vitro (17). The direct actions of leptin on adipose tissue require a functional long-form leptin receptor (Ob-Rb), since several in vitro studies have shown that, in contrast to fat from lean rats, white fat from obese Zucker rats (fa/fa), which have a mutation in the extracellular domain of the leptin receptor, fails to respond to doses of leptin ranging from physiological to supraphysiological (15, 37, 41).
We measured white fat HSL mRNA expression in the studies reported here because the SNS is thought to be a primary regulator of lipolysis (25). Neither peripheral nor central infusions of leptin influenced MES or ING HSL mRNA expression even though centrally infused leptin caused a substantial increase in MES NETO and produced a significant decrease in the weight of the ING pad. These observations do not account for posttranslational changes in HSL activity or for regulation of lipolysis by lipases other than HSL (12), both of which could have a significant impact on adipose tissue lipolysis. Leptin mRNA expression was measured because both circulating leptin (8) and activation of 
-adrenoreceptors (39) inhibit adipose tissue leptin mRNA expression. The results reported here are consistent with previous reports that subcutaneous fat has greater levels of leptin expression than visceral fat (31, 36), but we did not find a specific effect of either peripheral or central leptin on white fat leptin mRNA expression. In experiment 3, ING expression of leptin mRNA was inhibited in both the leptin and pair-fed groups, probably because of the reduction in fat depot size, since there was no treatment effect on NETO in this pad and the two groups of rats had very different circulating levels of leptin.
NETO in IBAT was higher than in any of the white fat pads but was similar across the two experiments. Leptin did not increase IBAT NETO in either peripherally or centrally infused rats. These results do not exclude the possibility that leptin increased thermogenesis in this tissue independent of an increase in the transcription of the protein. An unexpected finding in both experiments was the significant increase in IBAT NETO of only the pair-fed animals despite them being food restricted during the experimental period and not receiving any food for >15 h before death. Neither peripherally nor centrally infused pair-fed animals showed an increase in UCP1 mRNA expression to correspond with the increased NETO. It has been reported that the thermoregulatory processes of IBAT are depressed in response to 10 days of food restriction (35) but also that there is an increase in expenditure of lean subjects during an 84-h fast (46). Because we did not measure energy expenditure of the rats in this study, it is unclear whether the increase in NETO was associated with an increase in thermoregulation or was associated with an unrelated aspect of IBAT metabolism.
A surprising finding in the centrally infused animals was the elevation of serum leptin above control levels. This most likely resulted from the intracerebroventricular leptin infusion leaking into the periphery because there was no increase in leptin expression in the ING or MES tissues of leptin-infused animals. Others have reported that serum leptin is increased after a central leptin infusion (26, 32). The half-life of leptin is estimated to be 36.3 min (21). With the rats having an average weight of 272 grams, administration of 1.5 µg leptin 2 h before death, and the average serum leptin concentration for control rats at 1.5 ng/ml, it can be calculated that serum leptin would rise to 8.4 ng/ml, and our actual serum leptin was measured at 7.6 ng/ml.
In summary, the results of the experiments reported here indicate that leptin can reduce body fat independent of stimulation of SNS activity in white fat depots. Experiment 1 demonstrates that chronic peripheral infusions of physiological doses of leptin reduce body fat content, but experiment 2 shows that these doses of leptin do not increase NETO in any white fat depot. Infusion of leptin in the third ventricle, an area adjacent to nuclei that express leptin receptors and also initiate sympathetic outflow to white fat, reduced the size of most white fat depots but produced selective increases in NETO. Although leptin increased NETO in some fat depots, we did not determine whether this directly increased lipolysis or inhibited lipogenesis in these tissues. Further studies are needed to identify the metabolic pathways that lead to a reduction in fat mass in normal-weight animals treated with leptin, how the individual white fat depots respond to leptin, and which aspects of leptin action go awry in obesity or other conditions of leptin resistance.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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