Chemical sympathectomy alters regulation of body weight during prolonged ICV leptin infusion

Robert L. Dobbins1, Lidia S. Szczepaniak1, Weiguo Zhang1, and J. Denis McGarry1,2

Departments of 1 Internal Medicine and 2 Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9135


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To assess the importance of the sympathetic nervous system in regulating body weight during prolonged leptin infusion, we evaluated food intake, body weight, and physical activity in conscious, unrestrained rats. Initial studies illustrated that prolonged intracerebroventricular (ICV) infusion of leptin enhanced substrate oxidation so that adipose tissue lipid stores were completely ablated, and muscle triglyceride and liver glycogen stores were depleted. After neonatal chemical sympathectomy, changes in weight and food intake were compared in groups of sympathectomized (SYM) and control (CON) adult animals during ICV infusion of leptin. CON animals lost 60 ± 9 g over 10 days vs. 25 ± 3 g in the SYM animals when food intake was matched between the two groups. Greater weight loss despite similar energy intake points to an important role of the sympathetic nervous system in stimulating energy expenditure during ICV leptin infusion by increasing the resting metabolic rate, since no differences in physical activity were observed between CON and SYM groups. In conclusion, activation of the SNS by leptin increases energy expenditure by augmenting the resting metabolic rate.

lipid oxidation; sympathetic nervous system; intracerebroventricular


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ADMINISTRATION OF LEPTIN in normal rodents activates the ventromedial hypothalamus, resulting in both anorexia and increased sympathetic stimulation of peripheral tissues (7, 19, 42). Seminal studies indicate that leptin infusion enhances daily energy expenditure in food-restricted animals by preventing circadian decreases in resting energy expenditure that are normally observed during the first few hours of the light cycle (14, 22, 39, 51, 52). It is postulated that this effect is mediated via disinhibition of sympathetically mediated thermogenesis (14). On the other hand, fatty (fa/fa) rats with a mutation in the gene encoding the long form of the leptin receptor that disrupts intracellular signaling events (11, 24, 25, 44, 55, 56) develop profound obesity that is linked to impaired sympathetic regulation of peripheral metabolism (33, 34, 37).

Despite the straightforward results obtained in the animal models just cited, questions remain regarding the role that sympathetic activation plays in regulating body weight. Decreased sympathetic activity is associated with weight gain in rodent models of obesity, but muscle sympathetic nerve activity measured by direct recordings obtained from the peroneal nerve is increased in obese human subjects (49, 50). Furthermore, peripheral sympathetic nerve terminals have been destroyed in rats by triggering an immune-mediated response with repeated injections of guanethidine or 6-hydroxydopamine (27-29). Surprisingly, chemically sympathectomized rats do not gain more weight than untreated littermates, even when challenged with a high-fat diet (27, 29, 35, 45). Given the complex nature of the multiple processes controlling energy balance, it is likely that additional mechanisms are triggered that compensate for the loss of peripheral sympathetic nerves. If a second factor is introduced, such as anorexia induced by leptin, the metabolic consequences of chemical sympathectomy may be unmasked. The following experiments test the hypothesis that chemical sympathectomy blunts metabolic responses and limits weight loss during prolonged intracerebroventricular (ICV) leptin infusion.

ICV infusion of leptin places the hormone directly in the central nervous system (CNS), where it can stimulate the ventromedial hypothalamus without achieving circulating concentrations that could directly affect peripheral target tissues. Although ICV infusion is an important technique for distinguishing CNS effects mediated through autonomic signals from peripheral actions of leptin, it is important to carefully characterize the response to this relatively nonphysiological mode of hormone administration for comparison with other models of hyperleptinemia. Therefore, a separate important goal of these studies is to measure the time course for metabolic and hormonal changes during prolonged ICV leptin infusion in normal rats.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal care. Male Sprague-Dawley rats weighing 250-300 g were obtained from Harlan Sprague Dawley, Indianapolis, IN. They were housed in approved animal facilities at a temperature of 22°C and were maintained on a 12:12-h light-dark cycle (lights on from 10 AM to 10 PM) with ad libitum access to water and powdered standard (4% fat) rat chow prepared by Harlan Teklad, Madison, WI. Studies were performed after the rats had been acclimated to the new facilities for >= 3 days, and all protocols were approved by the University of Texas-Southwestern Medical Center at Dallas Institutional Animal Care and Research Advisory Committee.

Surgical procedures. The rats were anesthetized with methoxyflurane and an intraperitoneal injection of pentobarbital sodium at 75 mg/kg body wt before they were placed on a Stoelting stereotaxic device. A midline incision of the scalp exposed the periosteum, which was scraped away to reveal the skull. A hole was drilled in the skull at a point centered 2.2 mm posterior to bregma to visualize the superior sagittal sinus, and two smaller holes were then drilled posterolaterally for the fixation of 1/8-in. 0-80 machine screws (Small Parts, Miami Lakes, FL). The dorsal third ventricle was cannulated at stereotaxic coordinates CV = bregma - 2.2 mm and AP = bregma - 4.5 mm using a 28-gauge, 6-mm-long osmotic pump connector cannula (Plastics One, Wallingford, CT). The apparatus was cemented into place with dental cement, and the ICV cannula was connected to an osmotic minipump (model 2002, Durect, Cupertino, CA) by a PE-60 catheter. The pump was placed in a subcutaneous pouch made in the interscapular region, and the incision was closed. Surgery was performed using aseptic technique, and the overall success rate of the procedure was 85-90%. In initial investigations, correct placement of the catheter was confirmed by autopsy to determine the ventricular distribution of methylene blue dye injected via the infusion catheter.

An arterial catheter was required to measure plasma parameters in selected protocols. While the rats were still anesthetized immediately after insertion of the ICV catheter, a sampling catheter was placed into the right common carotid artery by using a previously established protocol (12, 54).

ICV leptin administration and fat-free mass. We obtained highly purified recombinant leptin from Novo Nordisk Pharmaceutical, prepared as previously described (57). The stock was diluted to the desired concentration in phosphate-buffered saline (pH = 7.4) containing 0.2% bovine serum albumin. Preliminary experiments revealed that continuous ICV leptin infusion at rates ranging from 5 to 500 ng/h (0.12-12 mg/day) significantly reduced food intake and body weight in normal rats. A dose of 50 ng/h was chosen for all subsequent experimental protocols, because it was the smallest dose to yield consistent results in all animals studied. Twelve rats were acclimated to metabolic cages (Nalge Nunc International, Rochester, NY) for 3 days before any surgery was performed. They received either ICV leptin (n = 4; L) or the dilution vehicle (n = 8) for 10 days. One-half of the animals receiving vehicle were fed ad libitum (V), and the other one-half were pair fed to the leptin group (PF). Body weight, food intake, water ingestion, and urinary nitrogen excretion were monitored daily. Food spillage was accounted for by weighing any food that fell through the floor of the cage into the collecting apparatus. On days 3, 6, and 10, the animals were transported to the NMR facility and briefly anesthetized with methoxyflurane. 1H spectra of the entire animal were collected with a 4.7-T OMEGA system by use of established pulse sequences (13, 17, 23, 40, 53), and the resulting water and fat resonances were quantified after line-fitting with NUTS software (ACORNNMR, Fremont, CA). Fat mass and fat-free mass were computed from the ratio of fat and water signal areas and known ratios of proton densities of fatty acid chains and water, with the assumption that water comprises 72% of fat-free mass (48). Fat-free mass = body mass divide  (1 + fat/water ratio × 0.72), and fat mass = body mass - fat-free mass. The NMR technique was validated by previous investigators, with the correlation coefficient between total body lipid and water content measured by whole body proton magnetic resonance spectroscopy (HMRS), and classical carcass analysis being 0.97 (40).

ICV leptin administration and substrate oxidation. Separate experiments characterized key components of glucose and lipid metabolism in a single set of animals. Three groups of rats were studied: 8 animals receiving leptin, 10 pair-fed control rats, and 8 ad libitum-fed control rats receiving the dilution vehicle only. All animals received ICV and arterial catheters, as we have described, and were infused with either leptin or the dilution vehicle. The animals remained in metabolic cages, and food intake and weight were recorded daily for 5 days. Urine was collected daily for determination of nitrogen excretion, and blood was sampled daily between 10 AM and 12 noon for measurement of plasma glucose, insulin, free fatty acid (FFA), and triglyceride (TG) concentrations. Between 12 noon and 5 PM, the animals were placed in random order in an airtight glass container for 1 h for indirect calorimetry. Room air warmed to 28°C was pumped through the container at 400 ml/min, and changes in O2 and CO2 content were measured in the effluent while the animals were calm and rested. On the 6th postoperative day, the animals were anesthetized for HMRS measurements and killed to obtain the liver and gastrocnemius and soleus muscles.

Chemical sympathectomy. Normal male and female Sprague-Dawley rats were bred in our animal facility. Male pups were identified on day 5 after birth and received subcutaneous injections of guanethidine (50 mg · kg-1 · day-1) prepared daily in phosphate-buffered saline (pH = 7-7.4) at a concentration of either 5 or 10 mg/ml. A total of 15 injections were given 5 days a week, as originally described by Johnson and colleagues (27, 29). This treatment protocol results in a permanent sympathectomy that has been verified by numerous investigators (27, 29, 35, 45-47), and we validated the functional integrity of postganglionic sympathetic nerve responses in representative adult animals from our treated group by measuring the increment in blood pressure after a 250 µg/kg intravenous injection of tyramine. Arterial and venous catheters were inserted, and the rats had 5-7 days to recover before experiments were performed in conscious, unrestrained animals. The arterial line was connected to a continuous blood pressure transducer for 30 min before the first tyramine bolus was administered, and cardiovascular responses were monitored. We performed 2-3 trials in each animal separated by 10-15 min. Untreated littermates served as controls.

To test whether metabolic responses to chronic ICV leptin administration were dependent on the activation of peripheral sympathetic neurons, guanethidine-treated and control rats were separated into individual plastic shoebox cages when they reached 200-250 g to avoid any excess stress associated with the metabolic cages. After 3-7 days to measure basal food intake, ICV catheters were placed for infusion of leptin (catalog no. L-4146, Sigma, St Louis, MO) or the dilution vehicle. To limit confounding effects of stress from additional procedures, we monitored only food intake and body weight for 10 days in four initial groups of rats: control + vehicle (n = 7), control + leptin (n = 12), sympathectomy + vehicle (n = 7), and sympathectomy + leptin (n = 11). A fifth group, control + leptin + pair-fed animals (n = 8), was added to account for differences in food intake between the two leptin-treated groups.

Physical activity during leptin administration was evaluated using the Opto-Varimex-3 Auto-Track System from Columbus Instruments, Columbus, OH, which has been previously described (16). Briefly, separate animals (n = 5-6/group) were placed into individual cages at 5 PM on the fourth postoperative day with free access to food and water, and experiments were initiated at 10 PM when the lights were turned off. Data was collected and analyzed to assess total active time and total distance traveled during the first 8 h of the dark cycle.

Analytical procedures and materials. Plasma glucose concentrations were measured by the glucose oxidase method on a Beckman Glucose Analyzer II machine purchased from Beckman Industries, Fullerton, CA. Plasma FFA concentrations were determined by a colorimetric assay incorporating fatty acyl-CoA synthetase, acyl-CoA oxidase, and a peroxidase-linked reagent (Boehringer Mannheim, Indianapolis, IN). Plasma TG concentrations were measured using a baseline correction for glycerol (Sigma Diagnostics). Urinary nitrogen content was calculated as the sum of urine ammonia, uric acid, and creatinine content measured using kits obtained from Sigma Diagnostics. Insulin and leptin levels in plasma were determined by radioimmunoassays with kits specific for rat insulin and leptin purchased from Linco Research, St. Charles, MO. The interassay coefficients of variation were <9 and 6% for insulin and leptin, respectively.

Statistical analyses. Experimental results were analyzed using multiple analysis of variance, with a probability for type I error set at P < 0.05%. Procedures for repeated measures were incorporated when appropriate. All calculations were made with SigmaStat software for Windows (SPSS, Chicago, IL).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Time course of leptin's effect in dissipating adipose tissue. The initial objective of these investigations was to characterize closely the time course for the metabolic actions of ICV leptin in normal rats maintained in metabolic cages. Figure 1 illustrates the changes in food intake, body mass, and percent body fat that were observed as leptin-treated (L), vehicle-treated (V), and pair-fed (PF) rats were followed for 10 days. Treatment with leptin inhibited food intake, but the anorexic effect waned from days 6 to 10 compared with the response noted earlier in the course of treatment. The control rats receiving the dilution vehicle had a stable body weight compared with a loss of 50 ± 5 g in PF and 73 ± 5 g in L animals. Total body fat mass as measured by HMRS fell to essentially undetectable levels at days 6 and 10 in animals receiving leptin, whereas it was easily detected in the other two groups. As body mass continued to fall from day 6 to day 10 in the L group without any additional fall in body fat, estimated fat-free mass decreased significantly by 21 g in L vs. PF animals. The urinary nitrogen excretion provided additional evidence for diminished fat-free mass in L animals. It was not significantly different in the groups from days 1-5, but it increased with leptin infusion from days 7 to 9 (277 ± 11 vs. 215 ± 11 mg/72 h in L and PF rats, respectively).


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Fig. 1.   Effect of leptin on food intake (A), body mass (B), and whole body fat content (C) for normal Sprague-Dawley rats maintained in plastic metabolic cages. Surgery was performed on day 0 to place a catheter in the dorsal 3rd ventricle for the infusion of either leptin (n = 4) or the dilution vehicle (n = 8). One-half of the animals receiving the vehicle infusion were pair fed to individual rats in the leptin group. Percent body fat was determined by nuclear magnetic spectra obtained from anesthetized animals placed within the magnetic coil. The day 0 value for body fat represents an average for normal rats before any surgery or treatment. Measurements were made on the postoperative days listed. Despite matched food intake, it is evident that leptin-treated animals lost more body mass and body fat over the 10-day treatment period than the pair-fed controls. * P < 0.05 vs. control and pair-fed groups.

The measured changes in fat mass and dry fat-free mass were contrasted with the caloric intake to provide a comparison of the caloric balance throughout the treatment period in the three treatment groups. Total food intake in the V group was 88.5 ± 6.1 g over the initial 5 days and 209.6 ± 3.0 g over 10 days after surgery. Because the diet contained 3.1 kcal/g of metabolizable energy, this converted to 274 ± 18 and 650 ± 9 kcal of food energy consumed. The caloric intake of the PF and L animals was diminished by 149 kcal over 5 days and 318 kcal over 10 days of treatment. Using conversion factors of 4 kcal/g change in dry fat-free mass and 9 kcal/g change in fat mass, it was estimated that the L animals experienced a net loss of 145 kcal in excess of the value observed in the control group during the initial 5 days of treatment. Because the decline in caloric intake matched the net energy deficit, energy expenditure seemed to be similar in the L and V groups. The PF rats experienced a net energy deficit of only 80 kcal during a time when caloric intake was reduced by 149 kcal vs. the V group. Such a difference was consistent with the oft-described limitation of energy expenditure during food restriction. Over the 10 days of treatment, the net negative energy balance as determined from changes in body composition was 253 and 166 kcal in the L and PF groups, respectively.

Leptin enhances substrate oxidation and depletes intramyocellular lipids. These experiments directly measured key components of glucose and lipid metabolism in a single set of animals as adipose tissue fat stores were depleted over the initial 5 days of leptin treatment. Again, three groups of animals were studied. Rats receiving ICV leptin at 50 ng/h (L) were compared with littermates receiving the dilution vehicle that were either eating ad libitum (V) or were pair fed (PF) to the L group. Figure 2 shows the unique effect of chronic ICV leptin administration on the respiratory quotient (RQ) and resting metabolic rate (RMR) in normal rats. RQ in L, V, and PF rats was initially quite similar. The RQ in the V rats steadily climbed to expected levels as the animals recovered from surgical placement of ICV and arterial catheters, but it remained equally suppressed in the PF and L groups. As predicted by the aforementioned body composition and caloric balance calculations, O2 consumption fell in the PF rats to levels below values observed in the L or V rats having ad libitum access to food.


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Fig. 2.   Intracerebroventricular (ICV) leptin infusion significantly impacts metabolic rate (A) and fuel partitioning (B) as measured by indirect calorimetry in normal Sprague-Dawley rats maintained in plastic metabolic cages. Surgery was performed on day 0 to place a catheter in the dorsal 3rd ventricle for the infusion of either leptin (filled bars) or the dilution vehicle (open bars). Separate animals receiving the vehicle infusion were pair fed to individual rats in the leptin group (hatched bars). Respiratory gas exchange was measured in resting animals between 12 noon and 5 PM. Measurements were obtained on postoperative days 1-5 as shown (n = 5-8/group). * P < 0.05 vs. control group and +P < 0.05 vs. pair-fed group.

Daily measurements of plasma glucose, insulin, FFA, and TG concentrations (Fig. 3) highlighted the depletion of readily available energy substrates during continuous leptin infusion. After maintaining normal plasma glucose concentrations for 4 days, the L group became hypoglycemic even though insulin concentrations fell to nearly undetectable levels. In addition, plasma TG and FFA levels were significantly reduced. Profound changes in tissue energy stores were also evident (Fig. 4). Liver glycogen content was substantially diminished, falling from 44.9 ± 3.1 to 21.6 ± 0.8 to 7.8 ± 1.3 mg/g tissue in V, PF, and L groups, respectively. Proton spectroscopy revealed that these animals depleted their adipose tissue mass to nearly undetectable levels, whereas the lipid content in the muscle tissue decreased fivefold and twofold in soleus and gastrocnemius muscle, respectively.


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Fig. 3.   Effect of leptin on plasma glucose (A), insulin (B), free fatty acid (FFA, C), and triglyceride (TG, D) concentrations in normal Sprague-Dawley rats maintained in plastic metabolic cages. Surgery was performed on day 0 to place a catheter in the dorsal 3rd ventricle for infusion of either leptin (filled bars) or dilution vehicle (open bars). Separate animals receiving vehicle infusion were pair fed to individual rats in leptin group (hatched bars). Blood was drawn while the animals were in the postabsorptive state between 10 AM and 12 noon. Measurements were made on the postoperative days listed (n = 5-8 per group). * P < 0.05 vs. control group, +P < 0.05 vs. pair-fed group, and ** P < 0.05 vs. both control and pair-fed groups.



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Fig. 4.   Depletion of liver glycogen stores and muscle TG content in normal Sprague-Dawley rats after 5 days of chronic ICV leptin infusion. Surgery was performed on day 0 to place a catheter in the dorsal 3rd ventricle for the infusion of either leptin (L) or dilution vehicle (V). Separate animals receiving the vehicle infusion were pair fed (PF) to individual rats in the L group. Measurements were made with tissues excised from 4-8 animals in each experimental group. Liver glycogen was analyzed using a modification of the method of Chan and Exton (8). Skeletal muscle TG content was assessed by proton magnetic resonance spectroscopy. ** P < 0.05 vs. both control and pair-fed groups.

ICV leptin administration in chemically sympathectomized rats. We also examined the extent to which leptin alters food intake and body weight after the interruption of sympathetic efferent activity in rats treated with guanethidine as neonates. As previous investigators have noted, the weights of the pups receiving guanethidine were reduced by 10-15% during the injection period, but the rate of growth returned to normal after the injections concluded. Also, other distinct physical features were observed, including a reversible ptosis that peaked at 4-6 wk of age, and some modest diarrhea in the treated animals (27, 29, 35, 45). We validated the completeness of sympathectomy in guanethidine-treated animals by measuring the peak blood pressure responses to bolus injections of tyramine. Treatment with guanethidine during the neonatal period suppressed the response to central stimulation of vasoconstrictor mechanisms by 65 ± 5% (range 42-88%).

Figure 5 shows the changes in body weight and food intake that occurred in sympathectomized (SYM) or control (CON) rats that received continuous ICV infusions of leptin or the dilution vehicle under circumstances in which additional experimental procedures were avoided to minimize anxiety experienced by the animals. The two groups of rats behaved similarly when the dilution vehicle was administered. The weight gain over 10 days was 45 ± 5 and 47 ± 6 g in the CON and SYM groups, respectively, while the average daily food intake was 26.0 ± 1.2 vs. 26.8 ± 0.7 g. The interruption of sympathetic efferent activity had no discernible impact on weight loss during ICV leptin infusion, but there was an unexpected difference in food intake. Figures 5 and 6 show that the SYM animals lost 25 ± 4 g over 10 days vs. 29 ± 3 g in the CON group, but they also ate significantly less food (149 ± 5 vs. 171 ± 5 g). Metabolic efficiency, calculated as the total change in weight divided by the decrease of food intake from baseline, was significantly different between the two groups (-0.21 ± 0.03 and -0.32 ± 0.04 in SYM and CON groups, respectively). These results indicated that weight loss during prolonged ICV leptin infusion approximated estimated fat mass. More importantly, they pointed to an important role of the sympathetic nervous system (SNS) in stimulating energy expenditure by increasing either the RMR or physical activity. We evaluated physical activity during the dark cycle using an open-field apparatus to record total active time and distance traveled (Fig. 7). No significant differences were observed between CON or SYM groups receiving ICV leptin.


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Fig. 5.   Impact of neonatal chemical sympathectomy on body weight (A) and food intake (B) in response to prolonged ICV infusion of leptin or the dilution vehicle. Sympathectomized rats received subcutaneous guanethidine injections 5 days/wk for 3 wk starting at 5 days of age. When the rats reached 8-9 wk of age, surgery was performed on day 0 to place a catheter in the dorsal 3rd ventricle for the infusion of either leptin or the dilution vehicle. Animals were individually housed in plastic shoebox cages, and food intake and body weight were measured daily for a total of 10 days. Control animals lost nearly the same amount of weight as the sympathectomized animal, even though they ate significantly more.



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Fig. 6.   Impact of neonatal chemical sympathectomy on total changes in food intake, body weight, and metabolic efficiency in response to prolonged ICV infusion of leptin. Change in body weight represents total weight loss during 10 days of leptin infusion; change in food intake represents the average daily decrease from baseline food intake over the 10-day treatment period. Metabolic efficiency was calculated as the total change in weight divided by the total decrease of food intake from baseline during the 10-day experiment. * P < 0.05 vs. control group.



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Fig. 7.   Neonatal chemical sympathectomy did not alter physical activity measured with an open-field apparatus in rats receiving ICV leptin infusion. Animals (n = 5-6/group) were placed into individual cages at 5 PM on the 4th postoperative day, with free access to food and water, and experiments were initiated at 10 PM when the lights were turned off. Data were collected and analyzed to assess total active time and total distance traveled during the 1st 8 h of the dark cycle.

An additional control study verified the physiological significance of the diminished food intake in the chemically sympathectomized rats receiving ICV leptin. Littermates (n = 8) that had not been treated with guanethidine were given ICV leptin for 14 days. We restricted food intake by pair feeding each rat to an individual animal in the SYM + leptin group for 10 days before allowing the animals to eat ad libitum for an additional 4 days. Figure 8 shows that the CON + leptin + PF rats lost considerably more weight, 60 ± 9 g, than their paired counterparts in the SYM + leptin group. When they were allowed free access to food on postoperative day 11, food intake increased, and their weight jumped from 208 ± 8 to 229 ± 3 g.


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Fig. 8.   Impact of neonatal chemical sympathectomy on body weight in response to prolonged ICV infusion of leptin when food intake is matched between groups. Surgery was performed on day 0 to place a catheter in the dorsal 3rd ventricle for the infusion of leptin. Animals were individually housed in plastic shoebox cages, and food intake and body weight were measured daily. For 10 days, the control + leptin + pair-fed animals had access to an amount of food equivalent to that eaten by a paired sympathectomy + leptin animal. Arrow at day 11 indicates point at which pair-fed animals were given ad libitum access to food. Control rats lost significantly more weight than sympathectomized littermates when food intake was matched between the 2 groups.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The current experiments were designed to characterize the time course for the metabolic actions of ICV leptin in normal rats and to assess the importance of the SNS in regulating body weight during prolonged leptin infusion. The key finding of the initial characterization study was that leptin-treated animals failed to downregulate resting energy utilization during the early hours of the light cycle to compensate for diminished food intake. The end result was excessive depletion of tissue lipid stores and hepatic glycogen in leptin-treated animals. The experiments in rats after chemical ablation of peripheral sympathetic nerves revealed that an intact SNS is critical for regulating body weight and peripheral metabolism during prolonged ICV leptin infusion. When sympathectomized rats were treated with ICV leptin for 10 days, their weight loss was similar to that of nonsympathectomized, leptin-treated littermates, even though they ate significantly less food. Restricting rats with intact sympathetic responses to the same food intake as the sympathectomized animals resulted in additional weight loss.

The current report focuses on a scenario in which leptin activates neurons in hypothalamic nuclei and is believed to stimulate peripheral substrate utilization via an increase in sympathetic activity. ICV leptin infusion was chosen because this method of hormone administration stimulates the ventromedial hypothalamus without achieving circulating concentrations that could directly affect peripheral target tissues. In our hands, ICV leptin infusion in normal rats prevented the decrease in resting energy expenditure that is normally observed in food-restricted animals during the first few hours of the light cycle. Similar alterations in the regulation of energy expenditure have been observed by other investigators in mice after subcutaneous (14), intraperitoneal (51, 52), and ICV (39) administration of leptin. Our experiments extended previous reports by demonstrating that leptin-induced changes in carbohydrate and lipid metabolism were associated with depletion of tissue energy stores and a progressive decline in plasma insulin, glucose, FFA, and TG concentrations. It is widely accepted that chronic hyperleptinemia diminishes body weight and adipose tissue content as well as plasma concentrations of insulin, glucose, and lipid substrates below levels observed in pair-fed animals (1, 6, 18, 32). Although smaller effects of leptin are seen when low physiological levels of plasma leptin are achieved (2), large metabolic perturbations appear when supraphysiological doses of peripheral leptin are given or when the hormone is delivered directly to the CNS via an ICV catheter (1, 18, 32, 43). The metabolic responses in our experiments, particularly the loss of fat-free mass in the initial characterization studies, were generally more dramatic than those observed by most other investigators. Part of the explanation lies in the exquisite sensitivity to centrally administered leptin vs. peripheral infusion (18), but ICV leptin infusion also increases interleukin-1beta generation in the hypothalamus, triggering a secondary neuroimmune response that may augment the effect of central leptin administration beyond that which can be achieved with peripheral hormone injections (36).

The physiological stress of multiple experimental procedures is a more basic complicating factor. Maintaining animals in metabolic cages, drawing daily blood samples from indwelling catheters, administering anesthesia, and transporting animals between buildings were all necessary components of the experiments, but the rigors associated with these procedures accentuated weight loss. Notably, when chemical sympathectomy experiments were performed under conditions that minimized stress, leptin-induced weight loss was approximately equal to the estimated total body fat mass. Therefore, it is important to emphasize that, although the magnitude of the responses in this ICV leptin infusion model may not have matched all previous results, the qualitative differences between leptin-treated and pair-fed rats undergoing identical procedures were consistent with other models of leptin administration.

After the model of ICV leptin infusion was adequately established, we progressed to elucidating the importance of the SNS in regulating body weight during chronic ICV leptin infusion. Because chemical sympathectomy with guanethidine should not result in any structural changes in the CNS or the adrenal gland (27-29), it was anticipated that the central anorexic effect of leptin would be similar in treated and untreated animals. Surprisingly, the decline in food intake for the sympathectomized group was 2.2 g/day (22%) greater than that of the control group. Additional evidence supporting the significance of this small difference in food intake was supplied by an experiment in which the food intake of control and sympathectomized rats receiving ICV leptin was matched. These data indicated that chemical sympathectomy modulated the anorexic effect of ICV leptin. Additional experiments are required to delineate whether this action occurs directly at the level of the hypothalamus or is indirectly mediated via other factors such as plasma insulin, glucose, or FFA concentrations.

Despite the influence of sympathectomy on food intake in this study, we found that intact peripheral sympathetic nerve responses were necessary for the normal metabolic response to leptin. The data provided two ways to estimate what portion of weight loss during ICV leptin infusion was mediated by sympathetic stimulation of peripheral metabolism. First, metabolic efficiency, calculated as the total change in weight over 10 days divided by the total decrease of food intake from baseline, was increased by 34% in the sympathectomized rats. Also, when control rats receiving ICV leptin were food restricted to match the intake of guanethidine-treated littermates, their weight dropped a net 108 g over 10 days compared with vehicle-treated control animals. The net difference in the guanethidine-treated groups was 70 g over 10 days, so chemical sympathectomy, independent of changes in food intake, again limited weight loss by about one-third. These values likely provide a minimal estimate of the importance of the SNS activity during leptin infusion due to nonuniformity of tissue responsivity and because functional studies indicated that the guanethidine protocol did not completely ablate sympathetic responses (46). Other investigators observed that tyrosine hydroxylase activity in the superior cervical ganglion was virtually absent in adult rats treated with guanethidine as neonates (27, 29, 45), whereas norepinephrine content was reduced in brown adipose tissue and soleus, gastrocnemius, and heart muscle by 65-95% (35). It is possible that some of the disparity between norepinephrine release and vasomotor responses resulted from a compensatory increase of the adrenergic receptor number in target tissues.

Two general views prevail regarding the physiological mechanisms through which leptin regulates body weight. The most prevalent viewpoint states that the effect of leptin is mediated by activation of neurons within the hypothalamus. Information is then transmitted to peripheral tissues via changes in food intake and nutrient fluxes or by modulating neuroendocrine and autonomic nervous system signals (30). Sympathetic nerve terminals directly innervate brown and white adipose tissue and skeletal muscle (3-5, 26), and intravenous infusion of leptin has been shown to increase sympathetic nerve traffic to brown adipose tissue, kidney, adrenal medulla, and the hindlimb of rats (10, 21). Although measures of increased sympathetic activity are consistent with an important role of the SNS in mediating the effects of leptin on energy utilization in peripheral tissues, they are not sufficient to establish that sympathetic nerve signals are necessary for normal leptin action. Previous experiments performed in normal rats after chemical sympathectomy with either guanethidine or 6-hydroxydopamine raise a note of caution. The treated rats did not gain more weight or gain weight at a faster rate than untreated littermates, even when high-fat diets were provided. The conclusion of the historical experiments was that the presence of a fully competent SNS was not required for weight maintenance under the majority of conditions (35).

An alternative viewpoint holds that leptin acts directly on beta -cells, liver, muscle,and fat. Leptin receptors (OB-Rb) have been detected in brown and white adipocytes by RT-PCR,and leptin treatment of cultured adipocytes activates the JAK/STAT pathway (15). Incubated soleus muscle also responds to high doses of leptin by increasing the oxidation of fatty acids by 42% (41). Moreover, studies with cultured C2C12 myotubules indicate that leptin can activate JAK-2,which induces tyrosine phosphorylation of IRS-2 leading to activation of phosphatidylinositol 3-kinase (PI 3-kinase) in this skeletal muscle model system. Finally, human hepatocellular carcinoma cell lines also express OB-Ra, and these cells have increased IRS-1-associated PI 3-kinase after exposure to physiological concentrations of leptin (9).

The current results and other recent studies favor the view that leptin stimulation of sympathetic activity is a key element promoting substrate utilization in tissues. Leptin has been implicated in the stimulation of the alpha 2 catalytic subunit of the 5'AMP-activated protein kinase (alpha 2 AMPK) in skeletal muscle (38). Intravenous leptin injection (1 mg/kg body wt) stimulated alpha 2 AMPK activity in mouse soleus muscle and increased phosphorylation of acetyl-CoA carboxylase, suppressing its ability to form malonyl-CoA, the endogenous inhibitor of carnitine palmitoyltransferase I. Prolonged activation of alpha 2 AMPK 6 h after intravenous leptin administration was ablated in soleus muscle that had been denervated by cutting the soleus, femoral, and obturator nerves (38). Other acute denervation experiments delineated specific effects of nerve activity on leptin action in peripheral tissues. ICV leptin administration stimulated glucose uptake in the extensor digitorum longus and soleus muscles, but this response was decreased after the femoral nerve was surgically interrupted (31). Similarly, local denervation of interscapular brown adipose tissue impaired the ability of intrahypothalamic injections of leptin to potentiate insulin-stimulated glucose uptake in that tissue (20).

The current study is unique, because peripheral sympathetic nerve terminals were specifically destroyed without the negative impact on motor function that would be expected from surgical nerve disruption. These techniques permitted close characterization of the importance of the SNS in regulating weight loss during prolonged leptin infusion. Specific destruction of peripheral sympathetic nerves impaired leptin acceleration of energy expenditure and limited weight loss when food intake was matched between control and sympathectomized rats. When animals had free access to food, compensatory mechanisms modulated food intake during prolonged ICV leptin infusion and minimized weight loss. Therefore, it is evident that the SNS played a critical role in increasing energy expenditure and promoting weight loss in response to leptin, but even this effect was difficult to unmask unless additional factors curbed changes in food intake. In the same way, weight loss interventions that only target energy expenditure will yield disappointing results unless compensatory mechanisms that regulate energy intake are subverted as well.


    ACKNOWLEDGEMENTS

We appreciate the excellent technical assistance provided by Nelda Parker, Ben Alexander, and Courtney Halbrooks.


    FOOTNOTES

The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (DK-57558), the Donald W. Reynolds Cardiovascular Clinical Research Center, and a Career Development Award from the American Diabetes Association supported this work. Additional support was received from NIDDK Grant DK-18573 and the Forrest C. Lattner Foundation.

Address for reprint requests and other correspondence: R. L. Dobbins, Univ. of Texas Southwestern Medical Center, Dallas, TX 75390-9135 (E-mail: robert.dobbins{at}utsouthwestern.edu).

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.

First published December 27, 2002;10.1152/ajpendo.00128.2002

Received 22 March 2002; accepted in final form 13 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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