Development of leptin resistance in rat soleus muscle in response to high-fat diets

Greg R. Steinberg and David J. Dyck

Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Direct evidence for leptin resistance in peripheral tissues such as skeletal muscle does not exist. Therefore, we investigated the effects of different high-fat diets on lipid metabolism in isolated rat soleus muscle and specifically explored whether leptin's stimulatory effects on muscle lipid metabolism would be reduced after exposure to high-fat diets. Control (Cont, 12% kcal fat) and high-fat [60% kcal safflower oil (n-6) (HF-Saff); 48% kcal safflower oil plus 12% fish oil (n-3)] diets were fed to rats for 4 wk. After the dietary treatments, muscle lipid turnover and oxidation in the presence and absence of leptin was measured using pulse-chase procedures in incubated resting soleus muscle. In the absence of leptin, phospholipid, diacylglycerol, and triacylglycerol (TG) turnover were unaffected by the high-fat diets, but exogenous palmitate oxidation was significantly increased in the HF-Saff group. In Cont rats, leptin increased exogenous palmitate oxidation (21.4 ± 5.7 vs. 11.9 ± 1.61 nmol/g, P = 0.019) and TG breakdown (39.8 ± 5.6 vs. 27.0 ± 5.2 nmol/g, P = 0.043) and decreased TG esterification (132.5 ± 14.6 vs. 177.7 ± 29.6 nmol/g, P = 0.043). However, in both high-fat groups, the stimulatory effect of leptin on muscle lipid oxidation and hydrolysis was eliminated. Partial substitution of fish oil resulted only in the restoration of leptin's inhibition of TG esterification. Thus we hypothesize that, during the development of obesity, skeletal muscle becomes resistant to the effects of leptin, resulting in the accumulation of intramuscular TG. This may be an important initiating step in the development of insulin resistance common in obesity.

pulse-chase; fatty acid oxidation; triacylglycerol; hydrolysis; esterification


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

LEPTIN, THE PRODUCT OF THE ob GENE, is a peptide hormone that is produced by adipose tissue and has been shown to regulate food intake and energy expenditure (47). In ob/ob mice, the absence of leptin results in a phenotype characterized by obesity and insulin resistance (32). Daily injection with exogenous recombinant leptin results in a rapid reduction in food intake, body adiposity, and restoration of insulin sensitivity (32). It is important to note that the leptin-induced decreases in serum glucose, insulin, and lipids at low dosages occur before changes in body mass (32), suggesting that leptin may have a direct effect on peripheral tissues such as skeletal muscle. Numerous peripheral tissues express leptin receptors (8, 43).

Leptin has been demonstrated to reduce triacylglycerol (TG) content in various peripheral tissues such as liver, muscle, and pancreatic cells (39) and to partition free fatty acids (FFA) toward oxidation and away from storage in oxidative skeletal muscle (28, 29). Although the acute effects of leptin on glucose uptake and metabolism in muscle have been equivocal (6, 20, 36, 48), there is evidence of convergence between the leptin and insulin signaling pathways in this tissue (3, 29). Therefore, the direct effects of leptin on skeletal muscle metabolism appear to be well established. Skeletal muscle, by virtue of its mass, is the major tissue responsible for insulin-stimulated glucose uptake and also accounts for a large proportion of whole body energy metabolism and may be an important tissue in mediating leptin's effects on energy homeostasis.

In humans and rodents, serum concentrations of leptin are primarily dependent on adipose cell size (25). High levels of circulating leptin, even when normalized for body fat, characterize most cases of human obesity (25). This suggests the development of central and/or peripheral resistance to leptin in obesity. High-fat diets in rodents result in a diminished metabolic response to peripheral leptin injections (44) and impaired leptin transport across the blood-brain barrier (2). In addition, several studies have demonstrated that high-fat diets lead to an increase in circulating leptin (1, 14, 24) that can occur in as few as 2 days (24). However, changes in plasma insulin subsequent to the consumption of high-fat diets may alter leptin production from adipocytes (9, 23). Thus increases in plasma leptin may not be an accurate reflection of the development of leptin resistance. It is important to note that direct evidence for the development of leptin resistance during obesity in skeletal muscle does not exist.

Therefore, in this study, we have used the pulse-chase technique in an isolated muscle preparation to examine the effect of high-fat diets on lipid metabolism and, specifically, whether diets high in n-6 and n-3 fatty acids alter the sensitivity of skeletal muscle to leptin. For the purposes of this study, we will define the development of leptin resistance as either an inability or reduced ability of leptin to alter lipid metabolism in skeletal muscle (i.e., stimulate FFA oxidation and decrease TG esterification). Diets high in saturated and n-6 polyunsaturated fatty acids (PUFA) induce insulin resistance in skeletal muscle (15, 49), whereas partial substitution with up to 12% of the total kilocalories with n-3 fatty acids restores insulin sensitivity (41). The effect of high-fat diets of varying fatty acid composition on leptin sensitivity in muscle has not been investigated. Therefore, we chose to use a dietary model that has been employed successfully to investigate the development of insulin resistance in muscle.

The isolated muscle preparation used in this study permits precise control of hormone and substrate concentrations, allowing us to study the effects of leptin in the absence of changes in the hormonal milieu as a result of the high-fat diets. We hypothesize that 1) diets high in PUFA will cause a compensatory increase in muscle lipid oxidation, 2) diets high in n-6 PUFA will reduce the sensitivity of oxidative muscle to the stimulatory effects of leptin on lipid metabolism, and 3) the partial substitution of n-3 PUFA in the high-fat diet will restore the sensitivity of muscle to leptin.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and Diets

Female Sprague-Dawley rats (Charles River Laboratories, Quebec, Canada) were used in all experiments. Upon arrival, animals were weighed (165 ± 4 g) and assigned to individual cages in a controlled environment with a reverse 12:12-h light-dark cycle. Animals were fed Purina rat chow ad libitum for a 7-day acclimation period. Animals were then assigned to one of the following three isocaloric and isonitrogenous diets for 4 wk: high-safflower oil (HF-Saff; 60% kcal fat), high-safflower oil with fish oil (HF-Fish; n-6 48% kcal fat/n-3 12% kcal fat), and a high-carbohydrate control diet (Cont: 12% kcal fat, 16% kcal protein, and 72% kcal carbohydrate). The composition of these diets is summarized in Table 1. All diets met the American Institute of Nutrition guidelines for vitamin and mineral content. Diets were prepared as previously described (37), vacuum sealed, and stored at -20°C.

                              
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Table 1.   Composition of experimental diets

Pulse-Chase Studies

Preequilibration. Rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (6 mg/100 g body wt), and the soleus (Sol) muscle was carefully dissected into longitudinal strips from tendon to tendon using a 27-gauge needle. Two Sol strips were used from each Sol muscle and were placed in a 20-ml glass scintillation vial containing 3 ml of warmed (30°C), pregassed (95% O2-5% CO2, pH = 7.4) modified Krebs Henseleit buffer containing 4% fatty acid-free BSA (ICN Biomedicals), 2 mM pyruvate, and 0.5 mM palmitate. This was the base buffer used in all experiments. Vials were gently agitated in a shaking water bath (PolyScience; Fisher Scientific) throughout the experiment.

Pulse and wash. The pulse and chase procedures used have been described previously (12). After a preincubation of 20 min, all muscles were transferred to the pulse buffer. The pulse buffer consisted of the base buffer plus 2 µCi of [9,10-3H]palmitate in ethanol (Amersham, Oakville, ON, Canada). Muscles were pulsed with [9,10-3H]palmitate for 40 min to prelabel all endogenous lipid pools [TG; diacylglycerol (DG); phospholipid (PL)]. After the pulse, muscles were washed for 30 min with incubation medium containing no radiolabeled palmitate. At the end of the pulse and wash, one Sol strip from each pair was removed, blotted, weighed, and extracted for endogenous lipids.

Experimental phase (chase). The remaining muscle strips were transferred to the experimental (chase) phase of the incubation and exposed to either 0 or 10 µg/ml of murine leptin (Amgen, Thousand Oaks, CA). This concentration was selected as it has previously been demonstrated to elicit a maximal response on skeletal muscle lipid metabolism (28). In a separate set of experiments, it was confirmed that leptin did not degrade over the course of the incubations (0 min, 10.1 ± 0.9 µg/ml; 90 min, 9.3 ± 0.6 µg/ml). The chase buffer contained 0.5 µCi/ml of [1-14C]palmitate (Amersham Life Science). During the 90-min chase phase, exogenous palmitate oxidation and esterification were monitored by the production of 14CO2 and incorporation of [1-14C]palmitate into endogenous lipids. Intramuscular lipid hydrolysis and oxidation were monitored simultaneously by measuring the decrease in lipid [3H]palmitate content and production of 3H2O, respectively.

Extraction of muscle lipids. Muscles were placed in 13-ml plastic centrifuge tubes containing 5.0 ml of ice-cold 1:1 chloroform-methanol (vol/vol) and homogenized using a Polytron (Brinkman Instruments, Mississauga, ON, Canada). After homogenization, connective tissue was removed, weighed, and subtracted from the total wet weight. Samples were then centrifuged at 2,000 g (4°C) for 10 min. The supernatant was removed with a glass Pasteur pipette and transferred to a clean centrifuge tube. Distilled water (2.0 ml) was added, and samples were shaken for 10 min and centrifuged as before to separate the aqueous and lipophilic phases. One milliliter of the aqueous phase was quantified by liquid scintillation counting to determine the amount of 14C-labeled oxidative intermediates resulting from isotopic exchange. This represented a twofold correction factor for exogenous [14C]palmitate oxidation, as previously described (12).

The chloroform phase, containing the total lipids extracted from muscle, was gently evaporated under a stream of N2 and was redissolved in 100 µl of 2:1 chloroform-methanol containing ~5 mg of lipids (TG, DG, and PL; Sigma Chemical, St. Louis, MO) to identify the lipid bands on the silica gel plates. Fifty microliters of each sample were spotted on an oven-dried silica gel plate (Fisher Scientific Canada, Mississauga, ON). Silica gel plates were placed in a sealed tank containing solvent (60:40:3 heptane-isopropyl ether-acetic acid) for 40 min. Plates were then permitted to dry, sprayed with dichlorofluorescein dye (0.02% wt/vol in ethanol), and visualized under long-wave ultraviolet light. The individual lipid bands were marked on the plate with a scalpel and scraped into vials for liquid scintillation counting.

Measurement of endogenous and exogenous oxidation. 3H2O produced from the endogenous oxidation of [9,10- 3H]palmitate was separated from the labeled substrate by transferring 1.0 ml of the chase incubation medium to a plastic centrifuge tube containing 5.0 ml of 2:1 chloroform-methanol (vol/vol). Samples were shaken for 10 min before adding 2.0 ml of 2 M KCl-HCl, were shaken again for 10 min, and were then centrifuged at 2,000 g (4°C) for 15 min. A 0.5-ml aliquot was removed from the aqueous phase and quantified by liquid scintillation counting.

Gaseous 14CO2 produced from the exogenous oxidation of [1-14C]palmitate during the incubation was measured by transferring 1.0 ml of the chase incubation medium to a 20-ml glass scintillation vial containing 1.0 ml of 1 M H2SO4 and a 0.5-ml microcentrifuge tube containing 1 M benzethonium hydroxide. Liberated 14CO2 was trapped in the benzethonium hydroxide over 60 min, and the microcentrifuge tube containing trapped 14CO2 was placed in a scintillation vial and counted. In a separate experiment, complete recovery of [14C]bicarbonate (98.3 ± 3%) was confirmed. Labeled CO2 released from the incubation buffer during the chase was captured in a center well containing benzethonium hydroxide, which was suspended from a rubber septum that sealed the vial. The 14CO2 captured in the well during the incubation was added to our measurement of exogenous oxidation.

Serum Leptin and Insulin

Blood samples were collected from animals at three time points (0, 2, and 4 wk). All samples were collected between 0900 and 1200 to eliminate variability due to diurnal rhythm. Blood was collected from the tail vein after immersion in warm water for both the 0- and 2-wk time points. The final blood sample at 4 wk was collected via cardiac puncture after excision of the Sol. Serum leptin and insulin levels were assayed in duplicate using RIA kits specific for rat leptin and insulin (Linco, St. Charles, MO).

Carcass Analysis

Frozen animal carcasses were cut into four or five small pieces and freeze-dried for 5 days. Samples were finely ground and vacuum-dried in an oven overnight. Percent body fat was assessed using the technique of Bligh and Dyer (4). Briefly, ~2 g of ground sample were added to a homogenizing tube containing 14 ml of distilled H2O, 40 ml of methanol, and 20 ml of chloroform and homogenized for 5 min. The homogenate was filtered, and the tube was rinsed with 20 ml of chloroform and 20 ml 1:1 of chloroform-methanol (vol/vol). The filtrate was added to a 100-ml graduated cylinder containing 25 ml of distilled H2O. The filter flask was rinsed with 5 ml of 1:1 chloroform-methanol to remove any remaining lipids, and the solution was allowed to settle overnight. The top aqueous layer was aspirated, leaving the chloroform layer, which was poured into a weighed 50-ml beaker. The chloroform layer was evaporated off in the fume hood, leaving the lipid residue, which was weighed. Percent body fat for each animal was measured in triplicate.

Calculations and Statistics

The quantity of palmitate (nmol) esterified and oxidized was calculated from the specific activity of the incubation medium (i.e., radiolabeled palmitate in dpm/total palmitate in nmol). Intramuscular hydrolysis was calculated as the loss of preloaded [3H]palmitate from each lipid pool between paired Sol strips.

Results were analyzed using ANOVA procedures, and a Tukey's post hoc test was used to test significant differences revealed by the ANOVA. Significance was accepted at P<= 0.05. All data are reported as means ±SE.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal Characteristics

Body composition. Body mass of rats fed Cont, HF-Fish, and HF-Saff diets are shown in Fig. 1A. Body mass did not differ among the groups at any time during the feeding protocol. Despite similar body masses, the mean percent body fat after 4 wk was significantly higher in animals fed the HF-Saff diet compared with animals on the other two diets (Fig. 1B).


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Fig. 1.   Body mass of animals over 4-wk feeding protocol (A) and percent body fat (B) after 4 wk of diets. aSignificantly different from control (Cont) or 48% kcal safflower oil + 12% fish oil (HF-Fish; Fish) diet. Saff, 60% kcal safflower oil (HF-Saff) diet.

Serum leptin and insulin. Serum leptin increased in all dietary groups over the 4-wk feeding period in relation to body mass gain (r2 = 0.35, P < 0.001). Leptin levels were significantly elevated in the HF-Saff group at both 2 and 4 wk compared with the Cont (2 wk, P < 0.001; 4 wk, P < 0.007) and HF-Fish (2 wk, P = 0.036; 4 wk, P = 0.037) dietary groups (Fig. 2A). Serum leptin levels after 4 wk (normalized per gram of body fat) were significantly greater in the HF-Saff compared with Cont group (P = 0.043; Fig. 2B). Serum insulin increased significantly (P < 0.001) in both high-fat diet groups after 4 wk (HF-Saff, 2.5 ± 0.2 ng/ml; HF-Fish, 2.0 ± 0.15 ng/ml), whereas insulin levels in the Cont group remained unchanged (0.65 ± 0.12 ng/ml).


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Fig. 2.   Serum leptin concentrations over 4-wk feeding protocol (A) and serum leptin concentrations normalized per g body fat after 4-wk feeding protocol (B). aSignificantly different from Cont and HF-Fish diet. bSignificantly different from high carbohydrate diet.

Basal Adaptations to High-Fat Diet

Under basal conditions (i.e., absence of leptin), exogenous palmitate oxidation increased in the HF-Saff group by 45% (P = 0.034) compared with the Cont group (Fig. 3A). High-fat diets had no effect on basal palmitate esterification into any of the endogenous lipids, or hydrolysis. Total incorporation of palmitate into Sol (lipid esterification plus oxidation) was unchanged after high-fat diets.


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Fig. 3.   Exogenous palmitate oxidation (A) and palmitate esterification (B) to triacylglycerol (TG) during 90 min in the presence or absence of leptin. aSignificantly different from basal condition without leptin. bSignificantly different from Cont diet.

Metabolic Responses to Leptin

Exogenous palmitate oxidation and esterification. Exogenous palmitate oxidation in the Cont group was increased by 80% (P = 0.0188) in the presence of leptin (Fig. 3A). This stimulatory effect was eliminated in the HF-Saff and HF-Fish groups (Fig. 3A). Palmitate esterification into TG was reduced in the presence of leptin by 25% (P = 0.043) in the Cont and 26% (P = 0.043) in the HF-Fish group (Fig. 3B). Leptin did not decrease TG esterification in the HF-Saff group. Esterification of [14C]palmitate into DG (Table 2) and PL (data not shown) was unaffected by leptin.

                              
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Table 2.   Esterification of exogenous [14C]palmitate into diacylglycerol during 90-min chase in resting soleus muscle in the presence and absence of leptin

Intramuscular lipid hydrolysis and oxidation. Hydrolysis of the DG (Table 3) and TG (Fig 4) pools was increased by 48% (P = 0.04) and 33% (P = 0.043), respectively, in the presence of leptin in the Cont group. Leptin had no effect on DG or TG hydrolysis in either the HF-Saff or HF-Fish groups. Leptin also had no affect on PL hydrolysis in any of the groups (data not shown). Endogenous lipid oxidation, monitored by the production of 3H2O, was insignificantly elevated in the presence of leptin in all groups (Table 4).

                              
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Table 3.   Hydrolysis of endogenous diacylglycerol pool (loss of [3H]palmitate) during 90-min chase in resting soleus muscle in the presence and absence of leptin



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Fig. 4.   Triacylglycerol hydrolysis during 90 min in the presence (filled bars) or absence (open bars) of leptin. aSignificantly different from basal condition without leptin.


                              
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Table 4.   Endogenous palmitate oxidation as monitored by the production of 3H2O during 90-min chase in resting soleus muscle in the presence and absence of leptin


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we used the pulse-chase technique in isolated Sol strips to examine the effect of 4 wk of high-fat diets on muscle lipid turnover and oxidation and, more specifically, whether the consumption of high-fat diets resulted in the loss of leptin's effects on muscle lipid metabolism. The use of an isolated muscle preparation allows for the examination of the direct consequences of a high-fat diet on the responsiveness of muscle to leptin in the absence of other metabolic perturbations (i.e., altered circulating insulin, FFA, etc.). To our knowledge, this is the first study to demonstrate direct evidence of the development of leptin resistance in peripheral tissues such as skeletal muscle. Several novel observations were made in the present study: 1) diets high in safflower oil (n-6 PUFA) induce a compensatory increase in basal lipid oxidation in oxidative muscle; 2) this compensation is not observed when 12% of the safflower component is replaced with fish oil (n-3 PUFA); 3) in Cont animals, leptin significantly stimulates the hydrolysis of muscle DG and TG and repartitions FFA toward oxidation and away from esterification; 4) the stimulatory effects of leptin on muscle lipid oxidation and hydrolysis were eliminated after consumption of both high-fat diets; 5) the inhibitory effect of leptin on lipid esterification was eliminated in the High-Saff group but was restored when fish oil was included in the high-fat diet; and 6) the increase in serum leptin during the 4-wk dietary treatment was greater than in Cont only in the High-Saff group, suggesting that increases in circulating leptin are not an accurate indication of leptin resistance in skeletal muscle.

Effect of High-Fat Diets on Muscle Lipid Metabolism

An increase in basal whole body lipid oxidation in response to high-fat diets has been demonstrated in rodents (7) and humans (38). However, there is relatively little evidence documenting the effect of fatty acid composition on muscle lipid oxidation. In this study, we provide evidence that 4 wk of feeding a HF-Saff, but not a HF-Fish, diet leads to a compensatory increase in exogenous palmitate oxidation. This discrepancy is unexpected given existing evidence that diets high in n-3 PUFA stimulate whole body lipid oxidation in humans (11) and stimulate muscle carnitine palmitoyltransferase (CPT) I activity in rats (34). However, we are unaware of any studies that have directly examined the effects of PUFA composition on muscle lipid metabolism. The increase in lipid oxidation in the HF-Saff animals also seems paradoxical given the increase in body fat in this group. This may in part be due to the fact that our observations of increased lipid oxidation were made in oxidative muscle and may not be relevant to the whole organism. Alternatively, it is possible that a greater degree of insulin resistance was induced in skeletal muscle in the HF-Saff group, which may have provided more glucose for disposal in adipocytes. A recent study has clearly demonstrated that high-fat diets induce insulin resistance in skeletal muscle but not adipose tissue (46).

Although there is a paucity of information regarding the effects of dietary fat on the regulation of muscle lipid metabolism, the beneficial effects of fish oil on basal and insulin-stimulated glucose utilization are well documented. Therefore, under our experimental conditions, it is possible that glucose utilization was most severely impaired in the High-Saff group, which may have resulted in a greater reliance on lipid oxidation. This is supported by a recent study in humans which found that safflower oil impaired pyruvate dehydrogenase activity, but fish oil did not (19).

In addition, it may be possible that the HF-Saff diet increased muscle lipid oxidation through an increase in the expression of uncoupling protein 3 (UCP-3), which has been shown to be increased by FFA and leptin (16, 45). In the present study, serum leptin was elevated to the greatest extent in the High-Saff group, raising the possibility that UCP-3 expression was increased, thus enhancing lipid oxidation. Alternatively, it is possible that different PUFA have variable direct effects on the expression of UCP-3. However, studies examining the effect of high-fat diets on UCP-3 expression in rodent skeletal muscle have been equivocal (10, 26). Because the expression of UCPs was not measured in the present study, our hypothesis cannot be confirmed. We are also unaware of any studies that have examined the effects of PUFA composition on UCP expression in skeletal muscle. Our study suggests that the composition of the high-fat diet may have an important effect on UCP expression and warrants further investigation.

Effect of Leptin on Lipid Metabolism in Skeletal Muscle

In this study, we demonstrate that leptin increases exogenous lipid oxidation by 80% while reducing lipid esterification by 25% in Cont animals. Thus the ratio of FFA esterification to oxidation was reduced by 140% (P < 0.001), indicating a repartitioning away from esterification and toward oxidation (Fig. 5). These findings are similar to data recently reported by Muoio and colleagues (28, 29) in mouse Sol muscle. In an extension to the findings of Muoio and colleagues, we have also provided the first direct evidence that leptin stimulates the hydrolysis of the intramuscular lipids DG and TG. Furthermore, our results demonstrate that leptin's effects on lipid metabolism are not specific to the fatty acid oleate, as used previously (28, 29).


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Fig. 5.   Fatty acid partitioning ratio of incorporated palmitate. aSignificantly different from basal condition without leptin. bSignificantly different from Cont diet.

To date, the mechanism by which leptin repartitions FFA toward oxidation in skeletal muscle is unknown. In pancreatic cells incubated for 2 days in leptin, a 20% decrease in the expression of acetyl-CoA carboxylase (ACC) was observed; the decrease coincided with an increase in lipid oxidation (47). This is presumably due to a decrease in malonyl-CoA, which is a potent inhibitor of CPT I (27); however, direct changes in malonyl-CoA were not measured. Although the role of malonyl-CoA in regulating muscle lipid oxidation remains controversial, particularly in human muscle (30), recent evidence using isolated rodent muscle has indicated that acutely decreasing the activity of ACC directly leads to an elevation in lipid oxidation (18). Leptin was also observed to significantly increase the expression of acyl-CoA oxidase and CPT I in this study (47). There are no data regarding the potential effects of leptin on rate-limiting enzymes regulating TG hydrolysis (e.g., hormone-sensitive lipase); however, a decrease in glycerol 3-phosphate expression in pancreatic islet cells has been observed (47). In the present study, total incorporation of FFA (esterification and oxidation) was unaffected by leptin in Cont muscle (no leptin, 220.4 ± 32.0; leptin, 184.5 ± 16.9 nmol/g), suggesting that the transport of FFA across the sarcolemma is not affected. Nevertheless, the effects of leptin on the putative muscle FFA transporters [fatty acid translocase (FAT/CD36), fatty acid transport protein (FATP), plasma membrane-bound fatty acid binding protein (FABPpm)] have not been investigated. Interestingly, the effect of leptin on TG turnover (synthesis-degradation) was greater than the increase in total (endogenous and exogenous) palmitate oxidation (Fig. 6), indirectly suggesting that leptin may have a direct effect on the synthesis and hydrolysis of intramuscular TG.


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Fig. 6.   Triacylglycerol turnover (synthesis - hydrolysis; A) and total palmitate oxidation (endogenous + exogenous oxidation; B) during 90 min in the presence or absence of leptin. aSignificantly different from basal condition without leptin.

Development of Leptin Resistance

Effect of high-fat diets on serum leptin. It has been suggested that the elevation in circulating leptin levels in obesity and subsequent to the consumption of high-fat diets may reflect the development of resistance to leptin's metabolic effects. Although leptin resistance at the level of the blood-brain barrier has been demonstrated in response to high-fat diets (2, 44), it may not be accurate to presume that elevations in plasma leptin are necessarily indicative of peripheral resistance. High-fat diets may result in elevated serum leptin due to a spontaneous increase in energy consumption (22) or secondary to increases in insulin (7), which have been demonstrated to stimulate leptin production in adipocytes from rodents and humans (15, 22). Furthermore, the effects of high-fat diets on circulating leptin levels are variable (1, 17, 42), which may be due to the type of fat used in the diet. Supplementation with docosahexanoic acid in rats has been demonstrated to reduce adipocyte leptin mRNA (35). This may explain the findings of the present study that a HF-Saff diet leads to a significant elevation in serum leptin at both 2 and 4 wk compared with the Cont and HF-Fish groups.

Although food consumption and body mass gain were similar in the three groups, animals fed a HF-Saff diet accumulated ~100 and 40% more carcass fat than pair-fed animals receiving the Cont and HF-Fish diets, respectively. These results are also in agreement with previous findings of reduced fat depots in rats fed diets supplemented with fish oil (31). As discussed below, rats fed a HF-Saff diet with partial substitution of fish oil in the present study maintained most aspects of leptin resistance demonstrated in the HF-Saff group. Thus the significant differences in serum leptin in the HF-Saff and HF-Fish groups indicate that elevations in circulating leptin are not an accurate indicator of leptin resistance.

Direct evidence for leptin resistance in skeletal muscle. One of the major objectives of the present study was to determine whether high-fat diets result in an impaired response of skeletal muscle to leptin. This phenomenon has not been investigated in any study to date. In the present study, we have defined leptin resistance in skeletal muscle as an impaired ability of leptin to stimulate FFA oxidation and TG hydrolysis and to blunt TG esterification.

Leptin has been demonstrated to have profound effects on lipid metabolism in skeletal muscle (28, 29, 39). However, the present study indicates that these effects can be almost completely eliminated after 4 wk of diets high in n-6 and n-3 PUFA. It must be acknowledged that the basal increase in FFA oxidation in the HF-Saff group complicates the interpretation of the lack of further stimulation in the presence of leptin. However, we have previously demonstrated that resting Sol muscle can oxidize palmitate at a rate of ~100 nmol · g-1 · h-1 when the concentration of palmitate is increased to 1.0 mM (12). The rate of ~20 nmol/g in 90 min observed in the HF-Saff group is well below the capacity of Sol to oxidize FFA at rest. Therefore, we interpret the inability of leptin to further stimulate palmitate oxidation in Sol from the HF-Saff group as a resistance to the effects of leptin. It is important to note that substitution of fish oil results in the restoration of leptin's inhibitory effect on TG synthesis. Thus the inclusion of dietary n-3 PUFA may help to counter some of the deleterious effects of n-6 PUFA on leptin sensitivity in muscle. Based on the results of the present study, the possibility that a higher substitution of fish oil would have elicited a greater restoration of the sensitivity of muscle to leptin cannot be eliminated.

Numerous studies have demonstrated that high-fat feeding results in a reduction in insulin sensitivity in skeletal muscle (15, 21, 49). It is important to note that these results are consistent even in studies such as our own in which animals are pair-fed to minimize differences in body mass (41, 46). Insulin resistance as a result of high-fat feeding has been correlated with an increase in intramuscular TG (13, 33). Partial substitution of n-6 PUFA with n-3 PUFA has been found to reduce intramuscular TG accumulation and restore insulin sensitivity (40, 41). The restoration of insulin sensitivity in ob/ob mice after leptin administration also appears to be related to a reduction in intramuscular TG (5). Changes in membrane fluidity as a result of a relative reduction in the percentage of highly unsaturated n-3 PUFA in PLs has also been associated with impaired activation of the insulin receptor (15, 21, 49). It is reasonable to hypothesize that the leptin receptor would be affected similarly, although direct evidence for altered leptin signaling in response to changes in membrane lipid composition are lacking.

In conclusion, the present study has demonstrated that diets high in n-6 PUFA stimulate basal lipid oxidation in skeletal muscle, but not when fish oil is substituted for 12% of the total kilocalories. Serum leptin was elevated significantly by the diet high in safflower oil, indirectly suggesting the development of leptin resistance in this group. However, pulse-chase studies using incubated Sol strips revealed that the antilipogenic effects of leptin were virtually eliminated in both high-fat diets. Thus we hypothesize that, during the development of obesity, skeletal muscle becomes resistant to the effects of leptin, resulting in the accumulation of intramuscular TG. This may be an important initiating step in the development of insulin resistance common in obesity.


    ACKNOWLEDGEMENTS

We thank Shannon O'Donnell for expert technical assistance and Diana Philbrick for expertise with diet preparation. We also thank Amgen (Thousand Oaks, CA) for donating the leptin and R. P. Scherer (St. Petersburg, FL) for donating the fish oil used in this study.


    FOOTNOTES

These studies were funded by the Natural Sciences and Engineering Research Council of Canada.

Address for reprint requests and other correspondence: D. J. Dyck, Dept of Human Biology and Nutritional Sciences, Univ. of Guelph, Guelph, Ontario, Canada N1G 2W1 (E-mail: ddyck{at}uoguelph.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.

Received 7 April 2000; accepted in final form 7 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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