Biology Department, University of Ottawa, Ottawa, Ontario, Canada K1N 6N5
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ABSTRACT |
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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 · kg1 · 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
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INTRODUCTION |
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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.
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METHODS |
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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 (O2) and carbon dioxide
production (
CO2) 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 O2,
CO2, 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)
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Statistics.
Differences in rates of O2 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.
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RESULTS |
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O2 and plasma leptin.
The control animals showed a progressive decrease in
O2 over the 12 h after saline
injection (P < 0.05). However, this decline in
O2 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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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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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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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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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 · kg1 · 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
O2.
Figure 3B shows the relative contributions of carbohydrate and lipid oxidation to
O2. 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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DISCUSSION |
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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 O2 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
O2
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
O2 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
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.
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., % ![]() |
ACKNOWLEDGEMENTS |
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We thank two anonymous reviewers for their generous help in improving this paper.
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FOOTNOTES |
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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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