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Am J Physiol Endocrinol Metab 295: E179-E186, 2008. First published May 20, 2008; doi:10.1152/ajpendo.00282.2007
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Hormone-sensitive lipase is necessary for normal mobilization of lipids during submaximal exercise

Céline Fernandez,1,* Ola Hansson,2,* Pernilla Nevsten,3 Cecilia Holm,1 and Cecilia Klint1

1Department of Experimental Medical Science, Lund University, Lund; 2Department of Clinical Sciences, University Hospital Malmö, Lund University, Malmö; and 3National Center for High Resolution Electron Microscopy, Lund University, Lund, Sweden

Submitted 4 May 2007 ; accepted in final form 14 May 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
For the working muscle there are a number of fuels available for oxidative metabolism, including glycogen, glucose, and nonesterified fatty acids. Nonesterified fatty acids originate from lipolysis in white adipose tissue, hydrolysis of VLDL triglycerides, or hydrolysis of intramyocellular triglyceride stores. A key enzyme in the mobilization of fatty acids from intracellular lipid stores is hormone-sensitive lipase (HSL). The aim of the present study was to investigate the metabolic response of HSL-null mice challenged with exercise or fasting and to examine whether other lipases are able to fully compensate for the lack of HSL. The results showed that HSL-null mice have reduced capacity to perform aerobic exercise. The liver glycogen stores were more rapidly depleted in HSL-null mice during treadmill exercise, and HSL-null mice had reduced plasma concentrations of both glycerol and nonesterified fatty acids after exercise and fasting, respectively. The data support the hypothesis that in the absence of HSL, mice are not able to respond to an exercise challenge with increased mobilization of the lipid stores. Consequently, the impact of the lipid-sparing effect on liver glycogen is reduced in the HSL-null mice, resulting in faster depletion of this energy source, contributing to the decreased endurance during submaximal exercise.

skeletal muscle; treadmill exercise; lipolysis; glycogen

DURING HIGH-INTENSITY EXERCISE, creatine phosphate degradation and glycogen breakdown are the major pathways activated to meet the increased need for energy. However, during prolonged submaximal exercise, oxidative metabolism becomes the predominant mechanism to produce ATP (13). For the working muscle there are a number of available fuels for oxidative metabolism, including glycogen, glucose, and nonesterified fatty acids (NEFA). The relative reliance on these substrates is mainly determined by exercise intensity and duration. NEFA originate from lipolytic activity in white adipose tissue (WAT), from lipoprotein lipase-mediated hydrolysis of VLDL triglycerides, or from hydrolysis of intramyocellular triglyceride (IMTG) stores. The contribution of IMTG has been debated, but much of this controversy has been attributed to methodological difficulties in measuring the IMTG pool and differences in duration and intensity of the performed exercise (4, 45, 56). Studies using 1H-magnetic resonance spectroscopy (3, 22, 27, 32) or immunofluorescence microscopy (52) have supported the notion that the IMTG pool is an important source of energy during exercise, whereas other studies using muscle biopsies have suggested that IMTG contribute only to a minor degree (2, 26, 48, 49). Glycogen stores are normally utilized during exercise to uphold the plasma glucose concentration, and it is noteworthy that in rodents, in contrast to humans, a large proportion of peripheral glucose uptake is insulin independent (39). Glycogen is mainly stored in muscle and liver, with the relative proportion of glycogen stored in muscle being larger in humans than in rodents. Consequently, muscle glycogen in humans is more important to be able to sustain long-term exercise than it is in rodents (20, 23). In mice, it has been shown that an increase in muscle glycogen storage is not essential for maintaining strenuous exercise and that the absence of muscle glycogen does not lead to a decreased ability to perform such exercise (40). The impact of muscle glycogen on endurance capacity in rodents is, however, not completely clear. Indeed, mice lacking the type 1 protein phosphatase glycogen-targeting subunit RGL have a marked reduction in muscle glycogen storage and exhibit a reduced ability to perform strenuous exercise (1). However, mice overexpressing RGL have increased muscle glycogen stores but no increased ability to perform strenuous exercise (1). Another mouse model, the peroxisome proliferator-activated receptor-{alpha} (PPAR-{alpha})-null mouse model, is reported to have a reduced exercise capacity and to have fully depleted liver glycogen stores after exercise, whereas the muscle glycogen stores were only depleted by ~50% (37). This implies that one determining factor for the endurance capacity in mice could be the amount of glycogen stored in liver, rather than that stored in muscle.

It is well known that increased fat utilization during endurance exercise enables athletes to improve endurance capacity by a sparing effect on the glycogen stores, and this effect also has been shown in rodents (42). A key enzyme in the mobilization of fatty acids from lipid stores in adipocytes is hormone-sensitive lipase (HSL). Expression of HSL has been demonstrated in various tissues, including WAT (16), brown adipose tissue (17), skeletal muscle (16), pancreatic β-cells (35), and testis (18), with the highest level found in WAT. In response to lipolytic stimuli, in adipocytes, HSL is phosphorylated and translocates to the lipid droplet, where it is believed to catalyze key steps in lipolysis (6, 15). Activation of HSL in skeletal muscle has been demonstrated in response to both adrenaline (30) and contraction (29), and it also has been reported that this activation is, at least partially, additive (28). HSL translocation to IMTG droplets also has been shown in skeletal muscle in response to both these stimuli (41). Endurance training studies performed in rats have demonstrated that training does not change HSL activity or protein expression in skeletal muscle (5). However, both the sensitivity to adrenaline stimulation and the expression of HSL are increased in WAT after exercise training (5). Furthermore, a transient activation of HSL in human skeletal muscle during exercise has been reported by several groups (44, 54, 55). The impact of this activation on the overall metabolic response to exercise together with some contradictory findings remains to be clarified.

In recent years several HSL-null mouse models have been created (9, 36, 38, 53). One unexpected feature of these mice is that they do not become obese but, on the contrary, are protected against diet-induced obesity (7, 12). It also has been shown that catecholamine-induced lipolysis is abrogated, whereas basal lipolysis is retained, in isolated adipocytes from these mice. Together, these observations challenged the view of HSL as a rate-limiting enzyme in lipolysis and suggested the presence of other lipases. Recently, one such candidate has been identified in the adipose triglyceride lipase (ATGL; also called desnutrin or calcium-independent phospholipase A2{zeta}) (21, 57, 58). The role and importance of ATGL in the regulation of lipolysis has, however, been debated (31, 33). On one hand, it has been suggested that ATGL only participates in basal lipolysis as a triglyceride lipase and that HSL is the major lipase catalyzing the rate-limiting step in catecholamine-stimulated lipolysis, at least in humans (31). On the other hand, it has been proposed that ATGL plays a regulatory role by working in concert with HSL to hydrolyze stored triglycerides (46). The aim of the present study was to investigate the metabolic response of HSL-null mice challenged with exercise or fasting and to examine whether other lipases are able to fully compensate for the lack of HSL.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. HSL-null mice were generated by targeted homologous recombination of the HSL gene in 129SV-derived embryonic stem cells as described elsewhere (8). Male mice were used throughout this study. Animals in the different groups were littermates and had a mixed genetic background from the inbred strains C57BL/6J and SV129 (36). Unless otherwise stated, the mice had free access to normal chow diet. After mice were killed, tissues were rapidly dissected, snap frozen, and stored at –80°C before analysis. The study was reviewed and approved by the Ethical Committees in Lund and Gothenburg, Sweden, and is in accordance with the Council of Europe Convention (ETS 123).

Endurance capacity. Mice were exercised on a motorized treadmill (Columbus Instruments, Columbus, OH). Before the experiments, all mice were acclimatized to the treadmill during a 3-day period with 5 min of rest and 5 min of running at 10 m/min and 0° inclination each day. An incremental protocol with increasing workloads was then used. One wild-type and one HSL-null mouse placed in individual treadmill lanes at room temperature (23°C) ran in parallel each time. The test started with a fixed 20° inclination and 10 m/min belt speed. The speed was increased to 14 m/min after 5 min and to 18 m/min after an additional 15 min. The mice continued to run at 18 m/min for another 5 min (25 min of exercise) or until exhaustion. Exhaustion was defined as the inability to continue regular treadmill running despite the repeated tapping on the back of the mouse. The purpose of such a running design was to favor aerobic metabolism. One HSL-null mouse that stopped running after only 8 min was excluded from the study since it did not run until exhaustion, together with an outlier from the wild-type group that stopped running after 28 min and thus deviated by 19 min from the median running time until exhaustion of the group (47 ± 0.51 min).

Blood parameters. Blood samples were drawn from isoflurane-anaesthetized mice, using EDTA as an anticoagulant. Four different pools of mice, from 11 to 23 wk of age, were investigated. In all of them, blood was collected at 11:00 AM after a 4-h fast. One pool of animals was then further fasted for 12 h until the second blood sampling (16-h-fasted mice). Another pool was given free access to food until 8:00 AM the following morning (fed mice). The third and fourth pools were submitted to physical activity as stated above (25 min of exercise and exercised until exhaustion). The plasma concentrations of NEFA, glucose, triglycerides, lactate, and adrenaline were determined using commercially available kits (Wako Chemicals, Thermo Trace, Trinity Biotech, and Demeditec Diagnostics). Plasma glycerol was determined using an enzymatic method according to Hellmer et al. (14).

Determination of glycogen content. Mice were killed by cervical dislocation, and gastrocnemius muscle and liver were dissected and snap frozen in liquid nitrogen. Tissue samples were digested in 30% KOH for 20 min at 100°C with a ratio of 4 µl of 30% KOH per milligram of tissue. After this cooled to room temperature, a one-fourth volume of 8% Na2SO4 and three volumes of absolute ethanol were added. The samples were incubated overnight at –20°C. Precipitated macromolecules were centrifuged at 600 g for 5 min at room temperature and washed twice with 66% ethanol. Samples were dried and dissolved in water. To determine glycogen content, we used amyloglucosidase (Roche) to degrade glycogen (24) and measured the glucose formed via the glucose oxidase method (Thermo Trace).

Thin-layer chromatography of neutral lipids. After death, WAT and gastrocnemius muscle were dissected and snap frozen. Before analysis, the tissue was homogenized in sucrose medium (0.25 M sucrose, 1 mM EDTA, 1 mM dithiothreitol, 20 mg/l leupeptin, 10 mg/l antipain, and 1.0 mg/l pepstatin, pH 7.2), followed by twice-repeated extraction of total lipids in chloroform-methanol (2:1). Extracted lipids were dissolved in chloroform and analyzed for neutral lipid composition by thin-layer chromatography (TLC) on silica gel 60 plates (Merck) using a mobile phase consisting of 78.3% heptane, 17.4% diethylether, 2.6% methanol, and 1.7% acetic acid. For identification of lipid species, a mix of mono-, di-, and triglycerides (Supelco) and purified cholesterol oleate were used as standards. After completion, migration plates were dried and stained with iodine.

Biochemical measurement of tri- and diglyceride content. Acylglycerols were measured enzymatically using the Thermo Trace kit for triglycerides. The amount of lipids was calculated from a standard curve of known amounts of triolein or diolein.

Transmission electron microscopy. Extensor digitorum longus (EDL) muscles and livers from three fed mice of each genotype (i.e., HSL-null and wild-type mice) were dissected and fixated in 3% glutaraldehyde in PBS buffer overnight. After washing in cacodylate buffer (0.2 M), postfixation was performed in osmium tetraoxide (1% in 0.1 M cacodylate buffer) on ice, followed by washing three times in cacodylate buffer (0.1 M) and two times in Milli-Q water. The tissue pieces were stained en bloc with 1% uranyl acetate on ice for 1 h, followed by washing with Milli-Q water for 1 h. Chemical dehydration was performed in 2,2-dimethoxypropane for 30 min, followed by rinsing with acetone twice for 15 min. Spurr's resin was infiltrated gradually and then cured at 70°C for 16 h. Thin sections (50 nm) from two different tissue parts from each animal were cut with a diamond knife with an ultramicrotome (Leica UCT) and collected on Pioloform-filmed copper grids. Images from the sections were taken with a Philips CM 120 Biotwin instrument at 120 kV and analyzed using a systematic random procedure. Quantification of the relative areas of cellular structures was performed according to the method of Mayhew et al. (34).

Statistics. Data are means ± SE, and differences between the two genotypes were analyzed using nonparametric Mann-Whitney U-tests. Differences between the different states examined (i.e., fed, 4-h fasted, 16-h fasted, and exercised) within the same genotype were evaluated using the nonparametric Kruskal-Wallis test with Dunn's post hoc test for selected pairs. The log-rank test was used to analyze the endurance capacity of the two genotypes investigated. In all tests, P < 0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
HSL-null mice have a decreased ability to sustain aerobic exercise. The median running time until exhaustion for the HSL-null mice was significantly shorter than for that for wild-type mice (39.5 vs. 47.0 min; P < 0.01, Fig. 1). After 38 min of exercise, half of the HSL-null mice had already stopped running, whereas all the wild-type mice were still able to pursue the treadmill exercise. Wild-type mice were able to sustain their physical activity for an additional 9 min, when three out of five reached exhaustion. The results shown in Fig. 1 also indicate a larger variation in the individual running time before exhaustion in the HSL-null mice compared with the wild-type mice; i.e., HSL-null mice had reached exhaustion within a time interval of 10 min, whereas control mice stopped their individual runs within a time span of 3 min.


Figure 1
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  		Fig. 1. Endurance capacity of 11- to 16-wk-old male wild-type and hormone-sensitive lipase (HSL)-null mice during treadmill running. The test started with a 20° incline and 10 m/min belt speed. The speed was increased to 14 m/min after 5 min and to 18 m/min after an additional 15-min running period. The mice continued to run at 18 m/min until exhaustion. Results are shown as a Kaplan-Meier survival curve (n = 4–5), and comparison of the two groups' performance was evaluated using the log-rank test. P < 0.01.

 
HSL-null mice have reduced plasma NEFA, glycerol, and triglyceride concentrations. In the fed state, plasma NEFA levels were similar in the two genotypes (Fig. 2A). In the fasted state, mean plasma NEFA concentrations were ~23% lower in HSL-null mice compared with wild-type mice (P < 0.05 in the 4-h-fasted mice, P = 0.065 in the 16-h-fasted animals). Plasma NEFA levels were significantly increased by fasting in both wild-type and HSL-null mice (P < 0.01). The mean plasma NEFA concentrations in wild-type mice were significantly increased during exercise (~50%, P < 0.05) but were not changed in HSL-null mice (Fig. 2B). After exercise until exhaustion, the NEFA concentration in the HSL-null mice was only 50% of the wild-type concentration level (P < 0.05).


Figure 2
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  		Fig. 2. Plasma nonesterified fatty acid (NEFA) concentrations in the fed and 4-h or 16-h fasted states (A) and before, during, and after treadmill exercise until exhaustion (B) in 11- to 23-wk-old male wild-type (open bars) and HSL-null mice (filled bars). Plasma glycerol concentrations in the fed and fasted states are shown in C and plasma glycerol concentrations before, during, and after treadmill running until exhaustion in D for the same groups of animals. E: plasma NEFA-to-glycerol ratios. Values are means ± SE; n = 4–7 except for 4-h fasted animals (n = 7–12) and before exercise (n = 11). Differences between the genotypes were analyzed using Mann-Whitney U-tests. *P < 0.05; **P < 0.01; ***P < 0.001.

 
In agreement with the NEFA results in the fasted and exercised states, the plasma glycerol concentrations in HSL-null mice were 30–50% of the level in wild-type mice (P < 0.05, Fig. 2, C and D). The plasma glycerol concentration in wild-type mice was not significantly changed by fasting, whereas plasma glycerol concentration was significantly higher in fasted HSL-null mice compared with the fed state (P < 0.01). No significant change in plasma glycerol concentration was observed in either genotype in response to the exercise challenge (Fig. 2D).

HSL-null mice had a significantly higher NEFA-to-glycerol ratio than wild-type mice in all tested groups except for mice exercised until exhaustion: 3.1 vs. 0.8 in fed mice, 3 vs. 1.8 in 4-h-fasted mice, 3.3 vs. 2.2 in 16-h-fasted mice, 3.8 vs. 2.4 after 25 min of exercise, and 2.5 vs. 2.4 in mice exercised until exhaustion (Fig. 2E).

Plasma triglycerides were significantly reduced in HSL-null mice before (0.56 ± 0.035 vs. 0.31 ± 0.040 g/l, P < 0.01) and after 25 min of exercise (0.46 ± 0.024 vs. 0.21 ± 0.021 g/l, P < 0.01). The reduction in triglyceride concentration in the two genotypes before compared with after 25 min of exercise was similar (0.10 vs. 0.10 g/l), but the relative decrease was larger in the HSL-null mice (18 vs. 32%).

No significant differences in plasma glucose, lactate, or adrenaline concentrations were observed between HSL-null and wild-type mice. In both HSL-null mice and wild-type mice there was a tendency toward reduced plasma glucose levels after exercise, but the reduction was not significant (Table 1). After the 16-h fasting, however, plasma glucose levels were significantly decreased in both wild-type and HSL-null mice. Plasma lactate was not significantly different in HSL-null compared with wild-type mice in all conditions tested (Table 1). In addition, exercised as well as fed mice had similar plasma lactate values, indicating that the mice were exercised under aerobic conditions. Plasma adrenaline was unchanged in HSL-null mice compared with wild-type mice (Table 1). Similar adrenaline levels after exercise between the two genotypes indicated that the sympathetic nervous system was performing to the same extent in both mouse strains and thus was not the underlying cause for the observed difference in plasma NEFA and glycerol levels. Exercise had a tendency to increase plasma adrenaline for both wild-type and HSL-null mice.


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  		Table 1. Plasma parameters of HSL-null and wild-type littermates

 
HSL-null mice deplete their liver glycogen stores faster when subjected to exercise. In the fed state, the liver glycogen content was similar in the two genotypes (Fig. 3). After 16 h of fasting, the liver glycogen stores were equally depleted, indicating a similar response to an extensive fasting challenge in both HSL-null and wild-type mice. Before treadmill exercise, liver glycogen content was shown to be similar in both genotypes (Fig. 3). At 25 min of exercise, a significantly lower content of liver glycogen in HSL-null mice compared with wild-type mice was observed (P < 0.05). After mice ran until exhaustion, which occurred 7.5 min sooner in HSL-null mice than in wild-type mice, the liver glycogen content was close to depletion in both genotypes (Fig. 3).


Figure 3
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  		Fig. 3. Liver glycogen content in 11- to 23-wk-old male wild-type (open bars) and HSL-null mice (filled bars). Liver glycogen content is shown in the fed state and after a 16-h fast as well as before, during, and after a treadmill exercise until exhaustion. Values are means ± SE; n = 4–7. Differences between the genotypes were analyzed using Mann-Whitney U-tests. *P < 0.05.

 
Measurement of intramuscular energy stores. Images of EDL muscles from unexercised fed mice obtained by transmission electron microscopy demonstrated the low abundance of IMTG droplets in this muscle type. The heterogeneity between images within the same individual prevented any quantitative comparisons to be made between the two genotypes. However, no obvious qualitative differences between the two genotypes could be detected (Supplemental Fig. S1 and Table S1). (Supplemental data for this article is available online at the American Journal of Physiology-Endocrinology and Metabolism website.) To obtain quantitative data on intramuscular lipid levels, we next employed a biochemical determination. This approach suffers from the drawback that even a small contamination by adipocytes could greatly influence the result. To minimize the problem, we used large pieces of gastrocnemius muscle, because they could be carefully dissected and the pieces closest to the tendons, where most adipocytes are found, could be discarded. In view of the observed diglyceride accumulation in several tissues, including skeletal muscle, made in another strain of HSL-null mice (9), we separated the extracted lipids by TLC and quantified triglycerides and diglycerides separately. The TLC analysis showed an increase in the proportion of diglyceride species in gastrocnemius muscle samples from all HSL-null mice compared with wild-type mice after exercise (Fig. 4A), which was confirmed using enzymatic determination (Fig. 4B). No differences between the two genotypes was observed in gastrocnemius muscle triglyceride content using either TLC analysis or enzymatic determination.


Figure 4
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  		Fig. 4. Analysis of intramuscular energy stores in gastrocnemius muscle from HSL-null and wild-type mice. A: qualitative lipid analysis of white adipose tissue (WAT) and gastrocnemius muscle by thin-layer chromatography in 18- to 22-wk-old male wild-type (+/+) and HSL-null (–/–) mice was measured after 25 min of treadmill exercise (n = 5). Lipids were extracted, and equal amounts of total lipids were analyzed and visualized with iodine. One representative sample per genotype and tissue is displayed. B: quantification of triglyceride (TG) and diglyceride (DG) content by an enzymatic method in gastrocnemius muscle of HSL-null and wild-type mice after 25 min of treadmill exercise. DG content (inset shows an enlargement of the DG bars) in the muscle from HSL-null mice was significantly higher than in wild-type mice, suggesting that HSL plays a role in the breakdown of DG in skeletal muscle but that TG can be readily hydrolyzed. **P < 0.01. C: quantitative measurement of glycogen content in gastrocnemius muscle in HSL-null and wild-type mice before as well as after 25 min of exercise (n = 6–7). No significant difference in glycogen content could be found between the mouse strains in any of these conditions. MG, monoglycerides.

 
As a control, TLC analysis of WAT samples was performed showing that in adipocytes, the presence of a larger proportion of diglyceride species in HSL-null mice than in wild-type mice was as pronounced as in the muscle samples (Fig. 4A). However, monoglycerides were not detected in WAT as opposed to gastrocnemius muscle. No significant differences were found in gastrocnemius muscle glycogen content between the two genotypes before or after exercise (Fig. 4C).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrates that HSL-null mice have a reduced endurance capacity during aerobic treadmill running, a finding suggesting that other lipases are not able to fully compensate for the lack of HSL when the lipid mobilization machinery is challenged. In the absence of HSL, mice are not able to respond to the exercise challenge with an increased mobilization of the lipid stores in WAT, i.e., with liberation of NEFA and glycerol to the circulation, to the same extent as wild-type mice. Consequently, the described lipid-sparing effect of the liver glycogen stores (42) that in wild-type mice will decrease glycogen utilization and increase lipid utilization to meet the raised demand for energy substrates during the exercise is compromised in HSL-null mice. This metabolic inflexibility of HSL-null mice thus leads to a faster depletion of carbohydrate energy stores and reduced endurance capacity in this mouse strain.

It was earlier demonstrated that HSL-null mice have an impaired capacity to mobilize fatty acids from WAT in response to adrenergic stimuli both in vitro (33) and in vivo (6). Consistent with these earlier studies, we have shown in this study that the in vivo response in WAT to adrenergic stimuli during exercise is impaired in mice lacking HSL, evidenced as significantly lower levels of plasma NEFA and glycerol. The impaired response to adrenergic stimulus was not due to differences in catecholamine release, since adrenaline levels were equal in both mouse strains at rest as well as during the exercise challenge. The significantly higher rate of depletion of liver glycogen stores in HSL-null mice compared with wild-type mice is in support of our proposed mechanism that a forced higher degree of utilization of carbohydrate energy stores is the cause for the decreased endurance capacity in HSL-null mice. Further support for the proposed mechanism comes from a study performed in normal healthy individuals showing that reduced plasma concentrations of NEFA affect utilization of other available energy sources (51). In contrast to the liver glycogen stores, which were reduced by 85% after 25 min of exercise in the wild-type mice, the intramyocellular glycogen stores were not significantly reduced after 25 min of exercise and were not lower in HSL-null mice compared with wild-type mice either before or during exercise (P = 0.13, Fig. 4C). Thus liver glycogen seems to be the major carbohydrate energy source during this type of submaximal exercise in both wild-type and HSL-null mice and is extensively utilized during exercise even under the influence of adrenergic mobilization of fatty acids from WAT, whereas the intramuscular stores are spared. In response to the impairment of lipid utilization, such as in the absence of HSL, the liver glycogen stores are depleted even faster. A similar adaptation to impaired lipid utilization during exercise was earlier demonstrated in PPAR-{alpha} knockout mice (37). Plasma glucose levels did not decrease in either HSL-null or wild-type mice, which is in agreement with the notion that hypoglycemia does not develop until the liver glycogen stores have been completely emptied. The observation that the intramuscular stores of glycogen did not decrease significantly during exercise, together with the modest increase in plasma lactate levels with no difference between the genotypes, argues against the mice discontinuing the exercise due to muscle fatigue. The modest increase in plasma lactate levels and the lack of difference in lactate and adrenaline levels between the mouse strains also strongly argue against the possibility that impaired heart function and/or reduced vascularization underlies the reduced capacity for endurance exercise in HSL-null mice.

In agreement with what has been reported previously for fasted animals of another strain of HSL-null mice (9), plasma triglyceride levels were reduced in HSL-null mice. This was true both before and after exercise. In the previous report (9), the reduction in fasted levels of plasma triglycerides was shown to be accounted for by a reduction in VLDL triglycerides, which in turn was the result of reduced synthesis and increased clearance of VLDL. The reduced synthesis of VLDL is presumably a result of decreased delivery of NEFA to the liver, whereas the increased clearance is likely caused by the observed upregulation of lipoprotein lipase (LPL) activity in skeletal muscle, cardiac muscle, and WAT. The increased VLDL clearance contributes to compensate for the decreased availability of WAT-derived NEFA in the circulation. Thus both plasma NEFA and VLDL triglycerides, which represent two of the three major sources of lipid fuel for skeletal muscle, are reduced in HSL-null mice. The third source of lipid fuel in skeletal muscle, the intramyocellular lipid stores, was more difficult to assess in the current study and could not be distinguished from a possible fourth source, NEFA liberated from intramuscular adipocytes. No differences in intramuscular lipid content could be established between the two mouse strains after exercise, and there were no signs of the lipid stores being exhausted. Diglycerides accumulated in skeletal muscle of HSL-null mice, in agreement with the emerging notion that the role of HSL is predominantly that of a diglyceride lipase, whereas ATGL accounts for the hydrolysis of triglycerides (46). In humans it has been shown that IMTG are utilized during exercise to a major extent, but only in type 1 muscle fibers (25, 49, 50), a fiber type with a demonstrated two- to threefold higher lipid content than type 2 muscle fibers (19, 47). Assessment of IMTG utilization is hence more difficult in a muscle with mixed fiber types such as the gastrocnemius muscle. However, the intracellular lipid content in the type 1 fiber type soleus muscle was earlier shown to be increased in HSL-null mice compared with wild-type mice (11), suggesting a perturbation of the mobilization of this energy store in soleus muscle. Taking all data into account and also considering that studies in humans have indicated that the energy provided by intramuscular lipids during exercise constitutes a relatively small fraction (25), we can conclude that impaired mobilization of intramuscular lipids is not the major mechanism underlying the decreased endurance capacity observed in HSL-null mice.

The respiratory quotient (RQ), an indirect measure of the relative contribution of carbohydrate and lipid substrates to oxidation, was previously investigated in resting HSL-null and wild-type mice (7). In that study, no differences was observed in RQ between the two mouse strains in either the fed or the fasted state. From the results in the present study, a difference in RQ between HSL-null and wild-type mice would be expected. However, at rest, it is likely that both carbohydrate and lipid substrates, through the activity of ATGL, would be available to a degree sufficient to sustain normal metabolism. During fasting, lipid mobilization is increased, but the turnover rate of the substrate is comparatively low, and again, the constitutive release of NEFA from WAT via ATGL could be sufficient to meet the demands during the nonexercise state.

In line with findings in other HSL-null mouse strains, plasma concentrations of NEFA (Fig. 2A) and glycerol (Fig. 2C) were lower compared with those of wild-type mice after both a 4-h and a 16-h fasting challenge (10, 38, 43). An interesting observation was that the NEFA-to-glycerol ratio in the HSL-null mice was close to 3 in all conditions tested (Fig. 2E). The reason for this is not known, but it is possible that it reflects that in the absence of HSL, triglyceride hydrolysis by LPL, which is upregulated in HSL-null mice (10) as mentioned above, is the major mechanism involved in upholding plasma NEFA concentrations. This would imply a bypassing of the intracellular adipocyte NEFA-triglyceride flux in the HSL-null mouse strain, which would contribute to the metabolic inflexibility of HSL-null mice. The findings also are in agreement with the proposed hypothesis that HSL works in concert with ATGL in the regulation of lipolysis in rodents (46). Whereas hypoglycemia clearly developed in both genotypes following the 16-h fasting, significantly reduced plasma glucose levels were not detected at the end of the exercise period in either wild-type or HSL-null mice, although both genotypes exhibited a tendency toward lowered plasma glucose levels. In view of the fact that the liver glycogen stores in both genotypes were depleted to the same extent as in the 16-h-fasted mice, this is somewhat unexpected. However, it could be due to the exercise protocol employed, where mice were tapped repeatedly on the back when approaching exhaustion. Thus mice did not stop running suddenly, which could allow for some recovery of plasma glucose levels before the blood sampling was performed.

In conclusion, the present study is the first to investigate the response of an HSL-null mouse strain to an exercise challenge, revealing that HSL-null mice have a reduced ability to perform aerobic exercise due to a reduction of the lipid-sparing effect on liver glycogen stores. Thus HSL plays an important role in the mobilization of lipids during aerobic exercise, and other acylglycerol lipases cannot fully compensate for the lack of HSL in this respect.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Financial support was provided by the Swedish Research Council (Project No 112 84 to C. Holm and Project 621-2003-300 to Reine Wallenberg, The National Center for High-Resolution Electron Microscopy, Lund University), the European Union (Eurodia LSHM-CT-2006-518153), the Swedish Diabetes Association, and the following foundations: Novo Nordisk, A. Påhlsson, Salubrin/Druvan, and Torsten and Ragnar Söderberg. O. Hansson was supported by the Cell Factory for Functional Genomics, a program funded by the Swedish Foundation for Strategic Research, the Novo Nordisk Foundation, and University Hospital Malmö funds. C. Fernandez was supported by the Swedish Research School of Genomics and Bioinformatics.


   ACKNOWLEDGMENTS
 
We thank Ann-Helen Thorén-Fischer and Gunnel Karlsson for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Holm, Dept. of Experimental Medical Science, Lund Univ., BMC C11, SE-221 84 Lund, Sweden (e-mail: cecilia.holm{at}med.lu.se)

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.

* C. Fernandez and O. Hansson contributed equally to this work. Back


   REFERENCES
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 MATERIALS AND METHODS
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 DISCUSSION
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