Accelerated substrate cycling: a new energy-wasting role for leptin in vivo

Shannon P. Reidy and Jean-Michel Weber

Biology Department, University of Ottawa, Ottawa, Ontario, Canada K1N 6N5


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Simultaneous lipolysis and reesterification form the triacylglycerol/fatty acid (TAG/FA) cycle, a substrate cycle commonly used for thermogenesis. Its rate was measured in vivo by indirect calorimetry and continuous infusion of [2-3H]glycerol and [1-14C]palmitate, after injection of leptin or vehicle saline in rabbits. Leptin stimulated in vivo lipolysis from 9.66 ± 0.62 to 14.78 ± 0.93 µmol · kg-1 · min-1, the rate of appearance of FA from 20.69 ± 2.14 to 29.03 ± 3.03 µmol · kg-1 · min-1, and TAG/FA cycling from 24.82 ± 1.73 to 37.09 ± 2.49 µmol FA · kg-1 · min-1. This large increase in total cycling was caused by an 85% rise in primary cycling (reesterification without transit in the circulation) and accounted for 14% of the difference in metabolic rate between the controls and the leptin-treated animals. This study shows that leptin causes a strong activation of TAG/FA cycling, lipolysis, and FA oxidation, shifting fuel preference from carbohydrates to lipids. Therefore, the acceleration of substrate cycling is a new mechanism triggered by leptin to increase metabolic rate, besides the known induction of uncoupling proteins.

futile cycling; energy expenditure; lipid reserve homeostasis; body weight regulation; obesity


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

LEPTIN IS THOUGHT TO MAINTAIN normal body mass by modulating rates of energy intake and expenditure (9, 18), and it plays a fundamental role in the regulation of fat reserves [adipose triacylglycerol (TAG)] (21). This lipostatic hormone adjusts the size of lipid stores through multiple mechanisms acting on metabolic rate (7, 14, 15, 23, 37), fuel selection (17), and food consumption (4, 15, 28). The hydrolysis of lipid stores, or lipolysis, releases fatty acids (FA) that can be either oxidized or reesterified (TAG resynthesis). Simultaneous lipolysis and reesterification form the TAG/FA cycle (40, 41), a substrate cycle known to dissipate energy for thermogenesis or weight reduction (26, 38). Concurrent flux through these opposing reactions catalyzed by different enzymes uses energy without net conversion of substrate to product.

Most of the information available on leptin-induced changes in lipid metabolism has been obtained in vitro (29), and it is unclear how TAG/FA cycling might be influenced by this hormone in vivo. For example, leptin could reduce fat mass by decreasing reesterification to channel more FA toward oxidation, a strategy supported by some findings on isolated cells (25, 31, 33) but not by others (39). Alternatively, it could stimulate reesterification to promote TAG/FA cycling as another means to increase energy expenditure besides the well-recognized pathway involving uncoupling proteins (32, 43). Leptin is thought to increase energy expenditure primarily by driving the hypothalamic-pituitary-thyroid axis to produce more triiodothyronine (T3) (20), a key regulator of standard metabolic rate (16). One of the ways T3 can stimulate energy expenditure is by inducing uncoupling proteins (UCPs) to increase the proton leakiness of mitochondria and reduce the efficiency of ATP production from metabolic fuels (30). A lot of attention has been focused on the role of UCPs, because leptin increases the expression of their mRNA, and a clear link between UCP-1 and the thermogenic capacity of brown adipose tissue has been established (31, 32, 43). Because hormones often elicit the same physiological response through redundant pathways, we have investigated the possible stimulation of substrate cycling, a UCP-independent mechanism to increase energy expenditure. The TAG/FA cycle is one such potential target that plays a known thermogenic role (26, 38, 40), is sensitive to hormones other than leptin (24), and is located at a strategic biochemical junction for the regulation of lipid stores. Experiments on isolated adipocytes show that the ratio of glycerol to FA released by these cells increases after leptin treatment (39) and that TAG/FA cycling is negatively correlated with obesity (3, 42). This indirect evidence suggests that TAG/FA cycling can influence basal energy expenditure and, therefore, that it may play a role in determining individual tendency to become obese. In this study, our goal was to quantify the integrated effects of hyperleptinemia on TAG/FA cycling, lipolysis, and oxidative fuel preference in vivo in an attempt to find a new functional link between leptin, substrate cycling, and energy expenditure.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and surgery. Adult male New Zealand White rabbits were used. They were fed rabbit chow (52% carbohydrates, 3% fat, 16% protein, 14% crude fiber, and 15% water). Rates of food and water intake were recorded for each individual pre- and postsurgery. The animals were given two subcutaneous injections of buprenorphine (0.02 mg/kg each) on the day before and two injections on the day after surgery to eliminate surgical pain. Just before catheterization, the animals received an intramuscular injection of ketamine (15 mg/kg), midazolam (0.5 mg/kg), and robinel (0.005 mg/kg), and they were intubated under isoflurane anesthesia with oxygen. Polyethylene catheters (PE-50) were placed in the right jugular vein and right carotid artery. They were inserted 7 cm toward the heart, filled with heparinized saline (40 U/ml), and penicillin G (125,000 U/ml). They were sutured to the vessels, tunneled under the skin, and exteriorized between the scapulas. The catheters were flushed daily to keep them patent. The saline containing heparin and penicillin filling the catheters was never injected; it was withdrawn and discarded before flushing. The metabolic measurements were made 5-7 days after surgery, only when food and water consumption had returned to presurgery levels for >= 3 days. The animals were randomly assigned to a treatment group (n = 6, body mass 2.59 ± 0.11 kg) or a control group (n = 6, 2.79 ± 0.10 kg) that received an intravenous injection of leptin or vehicle saline, respectively.

Indirect calorimetry. Each rabbit was placed individually in a closed Plexiglas respirometer (54 × 38 × 67 cm). Measurements of whole animal oxygen consumption (VO2) and carbon dioxide production (VCO2) were carried out using a calibrated Columbus Instruments system, as described previously (10). The urine produced was collected to quantify rates of nitrogen excretion. After 30 min in the respirometer, a 5-ml solution of physiological saline containing recombinant rat leptin was injected in the treatment group (1 mg/kg from R&D Systems, Minneapolis, MN), whereas the control group received the same volume of vehicle saline alone. After injection, the animals were left quiet in the respirometer for 12 h with access to water but without food.

Tracer infusions and sample analyses. After 12 h, a continuous infusion of [2-3H]glycerol (8.06 ± 0.34 µCi/ml) and [1-14C]palmitate (8.96 ± 0.33 µCi/ml) (Amersham, Baie d'Urfé, QC, Canada) was started through the venous line with a calibrated syringe pump (Harvard Apparatus) set at 5 ml/h. The infusate was prepared individually for each animal by mixing [1-14C]palmitate with a saline solution of rabbit albumin, adding [2-3H]-glycerol, and diluting to a total volume of 5 ml with saline. The activity of each infusate was quantified by counting on a Tri-Carb 2500 scintillation counter (Canberra Packard, Mississauga, ON, Canada). Indirect calorimetry measurements were continued throughout the tracer infusion. Arterial blood samples (1.5 ml each) were drawn 40, 50, and 60 min after the beginning of infusion, when steady state had been reached [i.e., when no changes in metabolite concentration, radioactivity, or rate of appearance (Ra) were observed over time]. Therefore, the metabolic fuel kinetics reported in this paper were measured between 12 and 13 h after saline (control group) or leptin injection (treatment group). Plasma was separated immediately after sampling and was stored at -20°C in glass tubes for <= 7 days before analysis. The concentrations and activities of plasma palmitate, total nonesterified fatty acid (NEFA), and glycerol were quantified as detailed previously (2, 22). Plasma leptin concentration was measured with a commercial immunoassay kit (Quantikine M; R&D Systems). Total urinary nitrogen was measured by the Kjeldahl method using Tecator analyzers (Kjeltec System 1002 and 1007).

Calculations. Rates of total lipid and total carbohydrate oxidation were calculated from VO2, VCO2, and nitrogen excretion (11). FA oxidation was obtained by multiplying the molar rate of TAG oxidation by 3. Only four rabbits produced urine during the infusions, and their values were averaged with the 24-h urinary nitrogen excretion rate of a separate group of animals held under the same conditions. Therefore, a mean excretion rate of 24 µmol N · kg-1 · min-1 (n = 11) was used in all calculations. The Ras of glycerol and palmitate were calculated with the steady-state equation of Steele (36). Ra glycerol is commonly used as an indirect measurement of lipolysis, because adipose tissue lacks significant glycerokinase activity and cannot metabolize free glycerol (1, 19, 38, 41). Ra NEFA was determined by dividing Ra palmitate by the fractional contribution of palmitate to total plasma NEFA. Total reesterification (or total TAG/FA cycling) is the sum of primary reesterification (defined as reesterification without FA exit from intracellular sites of lipolysis) and secondary reesterification (defined as reesterification after FA transit in the circulation). These rates of TAG/FA cycling were calculated as follows, using previously published equations (1, 38, 41)
total TAG<IT>/</IT>FA cycling<IT>=</IT>(3<IT>×</IT>R<SUB>a</SUB> glycerol)<IT>−</IT>FA oxidation

primary TAG<IT>/</IT>FA cycling<IT>=</IT>(3<IT>×</IT>R<SUB>a</SUB> glycerol)<IT>−</IT>R<SUB>a</SUB> NEFA

secondary TAG<IT>/</IT>FA cycling<IT>=</IT>total cycling<IT>−</IT>primary cycling
These calculations assume that FA released by lipolysis are either reesterified or exported in the circulation. The direct oxidation of newly released NEFA within the adipocytes could therefore lead to the underestimation of Ra NEFA and the overestimation of primary cycling (43). However, this has only a minor impact, because intra-adipocyte NEFA oxidation is low (41) and the rate of total TAG/FA cycling is not affected by this process (i.e., its calculation is independent of Ra NEFA).

Statistics. Differences in rates of VO2 were assessed with repeated-measures analysis of variance (ANOVA) and the Bonferroni post hoc test. Student's unpaired t-test was used to identify other statistical differences. All percentages were transformed to the arcsine of their square root before analysis. Significant differences are indicated when P < 0.05, and all results are presented as means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

VO2 and plasma leptin. The control animals showed a progressive decrease in VO2 over the 12 h after saline injection (P < 0.05). However, this decline in VO2 normally seen in fasting individuals was not observed in the rabbits treated with leptin, which maintained preinjection metabolic rates throughout the experiments (P > 0.05, Fig. 1). At the end of the respirometry measurements, plasma leptin concentration was 0.56 ± 0.05 ng/ml in the controls and 4.84 ± 1.16 ng/ml in the leptin group (P < 0.05). For comparison, plasma leptin concentration was also measured for a separate group of catheterized New Zealand White rabbits (n = 5) in the postabsorptive state (0.76 ± 0.06 ng/ml) and after a 6-day fast (0.39 ± 0.10 ng/ml).


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Fig. 1.   Oxygen consumption (VO2) of adult New Zealand White rabbits before (time 0) and for 12 h after intravenous injection of vehicle saline () or recombinant leptin (open circle ). Values are means ± SE; n = 6. *Significant differences from the preinjection value within each group.

Glycerol kinetics. The two experimental groups showed no difference in plasma glycerol concentration (P > 0.05, Table 1), but leptin treatment caused a large increase in lipolytic rate (P < 0.05, Table 1).

                              
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Table 1.   Glycerol and NEFA concentrations and their Ra in rabbits 12 h after treatment with saline or leptin

NEFA kinetics. Hyperleptinemia caused an increase in plasma NEFA concentration (P < 0.05, Table 1), but had no effect on plasma NEFA composition (P > 0.05, Table 2). Palmitate (16:0) and oleate (18:1) were the most abundant FA found in plasma and represented 50% of total NEFA in both groups. Leptin treatment stimulated Ra NEFA significantly (P < 0.05, Table 1).

                              
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Table 2.   Plasma FA composition (%total FA) in rabbits 12 h after treatment with saline or leptin

TAG/FA substrate cycling and FA oxidation. Leptin treatment caused an increase in the rate of TAG/FA cycling compared with the controls (P < 0.01, Table 3). The difference in cycling rate between the two groups was due to a strong leptin-induced stimulation of primary cycling (P < 0.01, Table 3), because secondary cycling was not significantly different between the two groups (P = 0.184, Table 3). The partitioning between the two pathways available for FA disposal, namely oxidation and reesterification (= TAG/FA cycling), is presented in Fig. 2. Absolute rates of FA oxidation and FA reesterification were both increased after leptin treatment (Fig. 2A). Interestingly, however, when these rates were expressed as percentages of total FA released by lipolysis, no difference was found between the controls and the leptin-treated group (P > 0.05, Fig. 2B). This is because hyperleptinemia caused equivalent relative changes in lipolytic rate (Table 1), FA oxidation (Fig. 2A), and TAG/FA cycling (Table 3).

                              
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Table 3.   Rates of primary, secondary, and total TAG/FA cycling in rabbits 12 h after treatment with saline (control) or leptin



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Fig. 2.   Metabolic fate of fatty acids (FA) after their release from triacylglycerol (TAG) stores. Absolute (A) and relative (B) rates of FA oxidation and reesterification in control (vehicle saline injection) and leptin-treated animals. Values are means ± SE; n = 6. Significant differences between the control and leptin-treated groups, * P < 0.05; ** P < 0.01.

Oxidative fuel selection. No difference in carbohydrate or lipid metabolism was present between the two groups before injection. Twelve to thirteen hours after injection, the hyperleptinemic animals showed a shift in the contribution of the different oxidative fuels available. Absolute rates of carbohydrate oxidation (µmol glucose · kg-1 · min-1) and lipid oxidation (µmol FA · kg-1 · min-1) are presented in Fig. 3A. Lipid oxidation was stimulated by leptin treatment (7.15 ± 0.83 vs. 4.63 ± 0.42 µmol FA oxidized · kg-1 · min-1, P < 0.05), but the observed decrease in mean carbohydrate oxidation was not significant (P > 0.05). Because metabolic rate was higher in the leptin-treated rabbits than in the controls (Fig. 1), rates of lipid and glucose oxidation were also calculated as percentages of total VO2. Figure 3B shows the relative contributions of carbohydrate and lipid oxidation to VO2. After leptin treatment, the relative importance of lipids was elevated (P < 0.05), but the observed decrease in mean carbohydrate oxidation did not reach statistical significance (P > 0.05).


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Fig. 3.   Oxidative fuel selection in adult rabbits 12 h after injection of vehicle saline (filled bars) or recombinant leptin (open bars). A: absolute rates of carbohydrate oxidation (µmol glucose · kg-1 · min-1) and lipid oxidation (µmol FA · kg-1 · min-1). B: relative contributions of carbohydrate and lipid oxidation to total metabolic rate (%VO2). Values are means ± SE; n = 6. *Significant increase in lipid oxidation (absolute and relative) between the controls and the leptin-treated group (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study uncovers a novel physiological link between leptin, substrate cycling, and total energy expenditure that may play a role in the natural prevention of obesity. TAG/FA cycling contributes significantly to the stimulation of metabolic rate caused by hyperleptinemia and represents an alternative mechanism activated by this hormone in parallel with the known induction of UCPs (18, 32, 43). We show that, in vivo, leptin shifts oxidative fuel preference toward lipids through the integrated activation of lipolysis, FA release, and FA oxidation.

Stimulation of susbtrate cycling. The VO2 of the control animals decreased progressively, whereas no such metabolic depression was observed after leptin administration (Fig. 1). Hyperleptinemia prevented the decline in energy expenditure normally caused by several hours of fasting, a stimulating effect of leptin on VO2 previously observed during various hypometabolic states like sleep, torpor, and food deprivation (7, 14, 15, 17). Flux through the TAG/FA cycle and its associated energy cost were 50% higher in the leptin-treated animals than in the controls (Table 3). This effect was achieved almost entirely by activating primary cycling (+84%) because mean secondary cycling was not elevated significantly. In this context, it is interesting to note that both cycling pathways can be stimulated in humans, because burn trauma mainly increases primary cycling (40), whereas cold stress only affects secondary cycling (38). Here, we have calculated the contribution made by the stimulation of TAG/FA cycling to the difference in metabolic rate between the controls and the leptin-treated animals, knowing that 8 mol of ATP (or 602 kJ) are required to reesterify each mole of TAG (see details of calculations in Refs. 8 and 41). The differences in rates of TAG/FA cycling and VO2 between the two groups were 4.33 µmol TAG · kg-1 · min-1 (or 2.6 J · kg-1 · min-1; Table 3) and 40 µmol O2 · kg-1 · min-1 (or 18 J · kg-1 · min-1, assuming an energy equivalent of 450 kJ/mol O2 consumed; Fig. 1). Therefore, by itself, the change in TAG/FA cycling observed in our experiments accounted for 2.6 divide  18, or 14% of the difference in metabolic rate. If several other substrate cycles not measured in this study were also stimulated by leptin, it is conceivable that the cumulative effect of all the futile cycles potentially involved in this response would be quantitatively as important as UCP induction for the overall increase in energy expenditure elicited by leptin. However, additional experiments are needed to measure the direct effect of leptin on other substrate cycles (e.g., the glucose and fructose cycles) to determine their overall quantitative impact on changes in metabolic rate. Also, the plasma leptin concentrations measured in the treated animals show that the dose selected brought leptin levels above the physiological range for nonobese individuals. Therefore, the measurement of dose-response curves will be needed to further the understanding of leptin effects on susbtrate cycles.

The role of substrate cycling as a physiological furnace increasing energy expenditure has been previously demonstrated in diverse situations (26), including healthy humans after exercise or cold stress (1, 38, 41), burn patients (40), malignant-hyperthermic pigs (6), and flying insects (5). Therefore, it appears logical that such a ubiquitous thermogenic mechanism be sensitive to leptin, one of the main hormones thought to be responsible for long-term homeostasis of body weight in mammals. Indirect evidence from obesity research lends further support for the "energy-wasting" role of the TAG/FA cycle demonstrated here in rabbits. Adipocytes isolated from obese humans generate less heat (27, 35) and show lower rates of TAG/FA cycling (3) than adipocytes from lean individuals. Furthermore, in vivo measurements show that this cycle is 64% more active in lean than in obese humans when expressed per kilogram of fat mass (42). The hormonal regulation of substrate cycles may therefore play a role in determining the metabolic efficiency of each individual and, as a result, set personal propensity towards obesity. If humans also show the responses observed in this study, the known leptin resistance of obese people (18) would impair their ability to mobilize substrate cycles.

In vivo effects of leptin on lipid metabolism. FA themselves stimulate cellular respiration, and leptin may therefore also increase metabolic rate indirectly through its lipolytic effects (Table 1). In vivo leptin administration stimulated lipolysis (+53%) and Ra NEFA (+40%). Although similar responses were observed in vitro (39), this is the first direct demonstration of leptin-induced mobilization of TAG reserves at the whole organism level. The stimulation of lipolytic rate by leptin was reported in adipocytes isolated from lean rats or mice and ob/ob mice, but not from db/db mice or fa/fa rats, both of which lack functional leptin receptors (12, 13, 34). Those studies showed that at least some of the lipolytic effects of leptin on adipocytes do not depend on hypothalamic, neural, or adrenergic control, although these systems may still modify leptin effects in vivo.

The absolute rate of fat oxidation was 55% higher in the leptin-treated rabbits than in the controls (Fig. 3A). However, because the metabolic rates of the two groups were different, relative rates of fuel oxidation may be more relevant (i.e., %VO2 accounted for by lipid or carbohydrate oxidation, see Fig. 3B). Leptin administration increased the relative contribution of lipids to total metabolism from 40 to 53% of VO2, but the observed decrease in carbohydrate oxidation was not significant (34-24% of VO2). A similar leptin-induced switch in fuel preference was reported previously but in an animal model lacking endogenous leptin production (17). Such changes in lipolytic rate and FA supply provide the additional substrate necessary to support both the increase in energy expenditure and the switch in oxidative fuel preference. Circulating leptin can adjust the idling rate of the metabolic machinery through its parallel effects on substrate cycling and uncoupling proteins. The relative importance of each one of these mechanisms in modulating basal energy use remains to be established.

In conclusion, through in vivo measurements in rabbits, we show that leptin causes a strong activation of TAG/FA cycling, lipolysis, and FA oxidation, shifting fuel preference from carbohydrates to lipids. The stimulation of substrate cycling is, therefore, a new mechanism triggered by leptin to decrease metabolic efficiency.


    ACKNOWLEDGEMENTS

We thank two anonymous reviewers for their generous help in improving this paper.


    FOOTNOTES

This work was supported by grants from the University of Ottawa and National Sciences and Engineering Research Council (Canada) to J.-M. Weber.

Address for reprint requests and other correspondence: J.-M. Weber, Biology, Univ. of Ottawa, 30 Marie Curie, Ottawa, ON, Canada K1N 6N5 (E-mail: jmweber{at}science.uottawa.ca).

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.

10.1152/ajpendo.000037.2001

Received 29 January 2001; accepted in final form 10 September 2001.


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ABSTRACT
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METHODS
RESULTS
DISCUSSION
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