1 Department of Social and Preventive Medicine, Laval University, Ste-Foy, Quebec, Canada; and 2 Department of Diabetes and Endocrinology, Princess Alexandra Hospital, Brisbane, Australia
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ABSTRACT |
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In this study, we
examined whether weight loss-induced changes in plasma organochlorine
compounds (OC) were associated with those in skeletal muscle markers of
glycolytic and oxidative metabolism. Vastus lateralis skeletal muscle
enzyme activities and plasma OC (Aroclor 1260, polychlorinated
biphenyl 153, p,p'-DDE, -hexachlorocyclohexane, and
hexachlorobenzene) were measured before and after a weight loss program
in 17 men and 20 women. Both sexes showed a similar reduction in body
weight (~11 kg) in response to treatment, although men lost
significantly more fat mass than women (P < 0.05).
Enzymatic markers of glycolysis, phosphofructokinase (PFK) activity,
and oxidative metabolism,
-hydroxyacyl-CoA dehydrogenase (HADH), citrate synthase (CS), and cytochrome c oxidase (COX)
activities, remained unchanged after weight loss. A significant
increase in plasma OC levels was observed in response to weight loss,
an effect that was more pronounced in men. No relationship was observed between changes in OC and those in PFK activity in either sex [
0.31 < r < 0.12, not significant (NS)].
However, the greater the increase in plasma OC levels, the greater the
reduction in oxidative enzyme (HADH, CS, COX) activities was in
response to weight loss in men (
0.75 < r <
0.50, P < 0.05) but not in women (
0.33 < r < 0.33, NS). These results suggest that the weight loss-induced increase in plasma pollutant levels is likely to be
associated with reduced skeletal muscle oxidative metabolism in men but
not in women.
organochlorine compounds; polychlorinated biphenyl congeners; pesticides; mitochondria; caloric restriction
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INTRODUCTION |
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OBESITY IS COMMONLY
ASSOCIATED with an increase in skeletal muscle triglyceride
deposition (9, 14), a feature associated with insulin
resistance (12, 18, 24). Recently, Kelley et al.
(11) outlined the mechanism that could account for the
increase in skeletal muscle lipid deposition occurring in obesity by
reporting reduced skeletal muscle capacity for fat oxidation rather
than increased fatty acid uptake in obese individuals. In this previous study, it was also reported that the fasting pattern of fatty acid
oxidation by muscle did not improve after weight loss. This observation
is supported by recent data showing alterations in mitochondrial
skeletal muscle markers of fatty acid metabolism after dietary
restriction (25). Such markers include -hydroxyacyl-CoA dehydrogenase (HADH), citrate synthase (CS), and cytochrome
c oxidase (COX) activities. This finding appears consistent
with previous data having reported persistent alteration of fatty acid metabolism after weight loss (1, 27), an observation of
clinical importance because a decreased reliance on lipid oxidation has already been shown to be a risk factor for weight gain
(29).
We recently reported that an adequate diet aiming at weight loss induced a significant increase in plasma organochlorine (OC) levels (3). OC are man-made chemicals, which include agricultural and industrial compounds as well as by-products of industrial processes involving chlorine chemistry and combustion of fuels. Because of their persistence and their lipophilicity, these compounds preferentially bioaccumulate in higher trophic levels of the food chain (15, 22). Consequently, OC are found in virtually every person on the planet and may have adverse effects on human health (8, 22). In this regard, OC have previously been shown to induce inhibiton in enzyme activities of the mitochondrial electron transport chain (19). More recently, Narasimhan et al. (17) also reported that polychlorinated biphenyls (PCBs) have multiple inhibitory sites on the mouse liver mitochondrial electron transport system. So far, little is known regarding the effect of OC on the mitochondrial bioenergetic capacity of human skeletal muscle. On the basis of the previous observations, it is likely that weight loss-induced increase in plasma pollutant levels could be associated with changes in determinants of the capacity for fatty acid utilization in human skeletal muscle. The objective of the current study was to explore this hypothesis.
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MATERIAL AND METHODS |
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Subjects.
Seventeen men and twenty women, all Caucasians, were recruited through
the media and gave their written informed consent to participate in
this study, which was approved by the Laval University Medical Ethics
Committee. All individuals underwent a medical evaluation by a
physician, which included a medical history. Subjects with
cardiovascular disease, diabetes mellitus, and endocrine disorders, or
those on medication that could have influenced triglyceride metabolism
(such as -blockers and antihypertensive drugs), were excluded from
the study. All participants were sedentary (i.e., fewer than two
exercise sessions of 30 min/wk), nonsmokers, and moderate alcohol
consumers (i.e., <140 g/wk). None was working in an environment where
the risk of exposure to OC was high. None had recently been on a diet
or involved in a weight-reducing program, and their body weight had
been stable during the last 6 mo before the study.
Anthropometric evaluation. Body weight was taken with a standard beam scale. Abdomen circumference was taken according to Lohman et al. (13). Body density was determined by the underwater weighing technique, from which percent body fat was derived with the Siri formula (26). Pulmonary residual volume was measured using the helium dilution method (16). Fat mass and fat-free mass were derived from the percentage of body fat and total body weight.
Skeletal muscle biopsies.
Muscle samples were obtained before and 4-6 wk after the weight
loss intervention. Biopsies were taken from the middle region of the
vastus lateralis muscle (15 cm above the patella) and ~2 cm away from
the fascia by use of the percutaneous needle biopsy technique
previously described by Evans et al. (5). Muscle samples
were frozen in liquid nitrogen and kept at 80°C until they were
assayed for enzyme activities.
Skeletal muscle enzyme activities. Small pieces of the muscle sample (~10 mg) were homogenized in a glass-glass Duall homogenizer with 39 vol of ice-cold extracting medium (0.1 M Na-K-phosphate, 2 mM EDTA, pH 7.2). Homogenate was transferred into 1.5-ml polypropylene tubes to be magnetically stirred on ice for 15 min and finally sonicated six times for 5 s at 20 W on ice with pauses of 85 s between pulses. The resulting homogenate was used for determination of activity (Vmax) of phosphofructokinase (PFK; EC 2.7.1.11), HADH (EC 1.1.1.35), CS (EC 4.1.3.7), and COX (EC 1.9.3.1), as previously described (7).
Chemical analysis.
On the basis of our previous observations (3) and to
simplify the statistical analyses, only the most abundant OCs found in
plasma were considered. Briefly, one PCB congener (International Union
of Pure and Applied Chemistry no. 153), one commercial PCB mixture
formerly used in electrical transformers (Aroclor 1260), and three
chlorinated pesticides
[2,2'-bis(4-chlorophenyl-1,1-dichloroethene), (p,p'-DDE), -hexachlorocyclohexane (
-HCH),
and hexachlorobenzene (HCB)] were determined in plasma samples
at the Quebec Toxicology Center. Blood samples were taken before and
after weight loss and were centrifuged to extract plasma (2 ml), which
was cleaned up by chromatography on an acidic silica gel column and a
deacrivated (0.5%) Florisil column. Plasma samples were eluted from
the columns using methylene chloride-hexane (25:75, vol/vol) and
analyzed on an HP-5890 gas chromatograph equipped with dual capillary
columns (Ultra-1 and Ultra-2) and dual 63Ni electron
detectors. Peaks were identified by their relative retention times
obtained on the two columns using a computer program developed by the
Quebec Toxicology Center. Depending on the compounds, detection limits
varied from 0.02 to 0.3 µg/l. Plasma lipid concentration was also
determined by enzymatic methods on a Technicon automatic analyzer
(RA-500) with the following test packs: Randox for total cholesterol
and triglycerides, BMC for free cholesterol, and Wako for
phospholipids. Plasma total lipids were calculated with the equation
recommended by Patterson et al. (20)
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Statistical analyses. Pretreatment sex differences were tested for significance with the Student's t-test. Multivariate analysis of variance (MANOVA) for repeated measures was performed on all variables to assess the effects of treatment and gender over time. As no treatment and time interaction was noted for all variables investigated, data from placebo- and drug-treated individuals were pooled. Identification of a significant sex × time interaction led to further analysis of a simple main effect for sex, and post hoc analysis was tested with a paired t-test. Univariate associations between variables were quantified using Pearson's product-moment correlation coefficients. Finally, partial correlation was performed to assess the relationship between two variables with the effect of a third variable eliminated. The change in variables was determined as the difference between post- minus preweight loss values. Results of the present study were similar when changes were expressed in percentage. Statistical significance was defined as P < 0.05. All analyses were performed using JMP software from SAS Institute (Cary, NC) on Macintosh computers.
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RESULTS |
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The physical characteristics of obese men and women before and
after weight loss are presented in Table
1. Pretreatment body weight and fat-free
mass levels were higher in men, whereas percent body fat and fat mass
were higher in women (P < 0.01). Men and women showed
a similar reduction of body weight and fat-free mass in response to
treatment. However, significant sex × time interactions were
found for abdomen circumference, percent body fat, and fat mass,
revealing that men lost significantly more of these parameters than
women.
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Skeletal muscle enzyme activities of men and women before and after
weight loss are shown in Table 2. PFK
levels were greater in men than in women before and after weight loss
(P < 0.001). In both sexes, mean PFK, HADH, CS, as
well as COX activities, remained unchanged in response to treatment.
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Plasma OC levels were comparable in men and women before weight loss,
as shown in Fig. 1. Except for -HCH,
which increased in a similar way in response to weight loss in both
sexes, sex × time interactions were found for all OC investigated
in response to weight loss. Plasma levels of p,p'-DDE,
Aroclor 1260, and PCB 153 were significantly increased in response to
caloric restriction in both sexes (P values ranging from
0.001 to 0.05), this effect being more pronounced in men. A significant
increase in plasma levels of HCB was also observed in men
(P < 0.05), whereas plasma concentrations of this
pollutant remained unchanged after weight reduction in women.
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Before weight loss, no significant correlation was observed between
plasma OC levels and skeletal muscle enzyme activities in either sex
(0.41 < r < 0.35; NS) (not shown). As shown in Table 3, no significant relationship was
observed between the change in the marker enzyme of glycolysis (PFK)
and changes in plasma OC levels (
0.31 < r < 0.10, NS). We found that the greater the increase in plasma OC levels,
the greater the decrease in marker enzymes of skeletal muscle oxidative
capacity (HADH, CS, COX) was in men (
0.75 < r <
0.50, P < 0.05) but not in women [
0.33 < r < 0. 33, not significant (NS)] (Table 3). To verify whether the previous significant relationships in men were independent of changes in body weight, partial correlations were performed. Significant negative partial relationships were observed between variations in HCB, Aroclor 1260, and PCB 153 levels and changes in
HADH, CS, and COX activities (
0.68 < partial r <
0.48, P < 0.05), as shown in Table
4. Changes in HCB, Aroclor 1260, and PCB
153 levels and changes in HADH, CS, and COX activities also remained
significantly correlated after correction for fat mass (
0.70 < partial r <
0.50, P < 0.05) (not
shown).
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DISCUSSION |
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This study was undertaken to examine whether weight loss-induced increases in plasma pollutant levels were correlated to changes in determinants of the capacity for fatty acid utilization in human skeletal muscle. To our knowledge, this is the first study to report clear associations between variations in environmental pollutants and those in skeletal muscle markers of oxidative capacity in humans. Indeed, we showed that increased plasma OC levels were associated with decreased skeletal muscle oxidative enzyme activities (HADH, CS, and COX) in men even after control for body weight loss. No significant relationship was observed between changes in plasma OC and muscle oxidative capacity in response to weight loss in women.
A decreased reliance on lipid oxidation has previously been highlighted as a metabolic predictor of weight gain (29), and this has also been related to a decrease in skeletal muscle lipoprotein lipase activity (6), an enzyme promoting the uptake of free fatty acid in muscle after its catabolic action on circulating triglyceride-rich lipoproteins. Recent studies have supported the proposal that decreased oxidative enzyme capacity of skeletal muscle might be responsible for reduced fasting rates of fatty acid oxidation in obesity (11, 25). In these previous studies, it was also reported that a dietary restriction aiming at weight loss without change in baseline levels of physical fitness did not correct the predisposition toward fat esterification of skeletal muscle in obesity. These authors suggested that this could be due to the parallel reduction observed in CS and COX activities, two markers of the mitochondrial oxidative capacity. Although only associative in nature rather than truly mechanistic, the findings of the current study suggest that a rise in OC levels could be a plausible mechanism whereby the capacity for fatty oxidation in muscle remained unchanged after weight loss, at least in men. This observation is concordant with previous data that have shown an impaired mitochondrial bioenergetic capacity in animals exposed to PCB (17, 19). In the current study, the specific impact on the oxidative capacity of skeletal muscle also appeared to be reinforced by the absence of a significant relationship observed between changes in OC levels and those in PFK activity, an enzyme of the glycolytic system.
It is not immediately clear why changes in pollutants and those in oxidative enzyme activities in response to weight loss were not significantly correlated in women. This absence of relationship may be due to the smaller increase in plasma OC levels in response to weight loss observed in women as opposed to men, as previously suggested (10). This could imply that a certain weight loss-induced increase in threshold of plasma OC levels is requisite for altering skeletal muscle oxidative capacity. Because of their lipophilicity, one could also hypothesize that a larger remaining fat mass in women as opposed to men after the weight loss program may render OC sequestration more favorable, thus dampening their mobilization. Further studies are required to elucidate this issue.
We have recently observed that weight loss-induced rise in OC levels was associated with decreased serum triiodothyronine (T3) and resting metabolic rate (21). Thyroid hormones are well recognized to act as major regulators of oxidative energy metabolism at the level of the mitochondria. Short et al. (23) recently showed that T3 increased oxidative skeletal muscle enzyme activities (CS and COX) in rodents. On our retrospective analyses, we did not observe any relationship between changes in serum thyroid hormone levels and changes in markers of muscle oxidative capacity. Bearing in mind that the effect of thyroid hormone on mitochondrial oxidative capacity is regulated through the thyroid hormone receptor, it is also biologically plausible that OC may alter oxidative muscle enzyme activities because of their known interaction with the human thyroid receptor (2). Although speculative, it is likely that these mechanisms are triggered in response to weight loss to promote weight regain, which in turn would prevent further release of OC into the circulation as a result of the redistribution of OC to adipose tissue. The absence of change in fasting pattern of fat oxidation by muscle in response to weight loss recently reported (11) could also be a mechanistic strategy taken by the myocyte to preserve its TG content for local buffering of OC. This assumption would be concordant with a recent report on a subsample of the present study, from which we observed that intramyocellular lipid concentration was not significantly reduced after weight loss (14).
In summary, the current study indicates that an increase in plasma OC levels derived from a weight loss is likely to be associated with a reduction in determinants of the capacity for fatty acid utilization in skeletal muscle. This finding is supported by the fact that an increase in OC levels is correlated with a decrease in HADH, CS, and COX activities in response to weight loss, at least in men. Further studies are, however, needed to verify whether these observations are causally related. Together, these intervention data suggest that environmental pollutants may be involved in the fatty acid metabolism perturbations commonly reported in human skeletal muscle in response to weight loss.
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ACKNOWLEDGEMENTS |
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This work was supported by the Fonds FCAR-Québec and Servier Canada. P. Imbeault is a recipient of a Natural Sciences and Engineering Research Council of Canada fellowship.
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. Imbeault, Dept. of Diabetes and Endocrinology, Princess Alexandra Hospital, Ground floor, Wing C, Ipswich Rd., Woolloongabba, Brisbane, 4102, Australia (E-mail: pascal.imbeault{at}kin.msp.ulaval.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.00394.2001
Received 31 August 2001; accepted in final form 13 November 2001.
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