| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Departments of Surgery (S.E., N.P., B.I., J.P.), Medicine (E.H.-T., J.B., P.A.), and Orthopedics (J.N.), Clinical Research Center at Karolinska Institutet (K.H.), and Department of Medical Laboratory Sciences and Technology (R.W.), the Karolinska University Hospital Huddinge, S-141 86 Stockholm, Sweden; and Department of Cell and Molecular Biology (C.H.), Lund University, 22100 Lund, Sweden
Address all correspondence and requests for reprints to: Staffan Enoksson, M.D., Ph.D., Department of Vascular Surgery, Center for Surgical Sciences, M81, Karolinska University Hospital Huddinge, S-141 86 Stockholm, Sweden. E-mail: staffan.enoksson{at}cfss.ki.se.
 |
Abstract |
|---|
 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
0.4). Biochemical experiments with hormone-sensitive lipase (HSL) showed that most enzyme activity was both in the cytosol and mitochondrial fraction and that it constituted the common long and active form of the protein. Electron microscopy studies in rat skeletal muscle using labeled highly specific HSL antibodies verified the cytosolic location of HSL and, furthermore, indicated an accumulation of HSL-adjoining mitochondria. These results suggest that FFA produced in myocytes during catecholamine-induced lipolysis are retained by the muscle and, therefore by inference, reused. It is conceivable that efficient hydrolysis of acylglycerol by HSL located in the cytosol as well as near the mitochondria may facilitate mitochondrial FFA oxidation. In addition, muscle lipolysis activity increases during aging and may be independent of total body fat. |
Introduction |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Little is known about the regulation of triglyceride metabolism in skeletal muscle. Regarding lipolysis, the presence of two major lipases, lipoprotein lipase and hormone-sensitive lipase (HSL), has been unequivocally demonstrated (6). According to the same review (6), the former lipase is responsible for hydrolysis of extracellular triglycerides because it is transported to the capillary bed, whereas HSL is responsible for the intracellular lipolysis in skeletal muscle. As discussed in detail (7), intramuscular HSL is activated by catecholamines through ß-adrenergic mechanisms. A critical role of intramuscular HSL in regulating the local triglyceride content has recently been demonstrated in HSL knockout mice (8).
The aim of this study was to investigate the regulation of intramuscular lipolysis in man, focusing on ß-adrenergic mechanisms and HSL. For this purpose, we developed an in vitro technique based on a method to study glucose transport in human muscle strips (9). The release of glycerol and free fatty acids (FFA), the two end products of lipolysis, was determined. In addition, we investigated the biochemical properties and intramyocyte distribution of lipids and HSL by means of enzymatic analysis, Western blot, and electron microscopy. Indications of a marked reutilization of intramyocytal FFA is demonstrated, suggesting a tissue-specific use of FFA liberated during intramuscular lipolysis.
|
Subjects and Methods |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
There were 35 participants in this study; 23 were men, and 12 were women [median age, 65 yr; range, 35–87 yr; body mass index (BMI), 26 ± 1]. The patients were admitted to the Huddinge University Hospital with a malignant (n = 22) or nonmalignant (n = 13) diagnosis. Patients who reported weight loss in excess of 4 kg in the preceding 6 months were excluded from participation in the study. There was no significant difference in age or BMI between malignant and nonmalignant subjects. Human skeletal muscle was obtained during elective abdominal surgery. The patients had fasted overnight, and only saline was given iv until skeletal muscle tissue was removed through a surgical incision initializing the surgical procedure. The study was explained in detail to the patients, and informed consent was obtained. In some experiments, male Sprague Dawley rats were used. They were fed standard laboratory food and fluids until reaching 300–350 g in body weight, and they were then used in the experiments. The Ethics Committee of the hospital approved all animal and human studies.
Human muscle-strip preparation
A human muscle preparation was adapted after the description by Dohm et al. (9). Immediately after the surgical incision, muscle strips (abdominal rectus muscle) were mounted at in vivo length by using one set of clamps, which was 2.5-cm wide and constructed from two pairs of hemostats. The clamp was placed by the surgeon on the muscle, and a 0.5–1 g muscle piece was clamped, excised, and immediately transferred to oxygenated Krebs-Henseleit’s bicarbonate buffer supplemented with HEPES for dispatch to the laboratory, which took 5–6 min. Muscle fiber strips, weighing between 20 and 50 mg, were dissected free from the mounted specimen, secured with a small Plexiglas support, and cut free. Particular care was taken to remove all visible fat. From one muscle piece, it was possible to obtain five to 10 muscle strips. After preparation, the muscle strips were washed for 30 min in a solution containing Krebs-Henseleit’s bicarbonate buffer supplemented with HEPES, 38 mM mannitol, 2 mM pyruvate, and 10 mg/liter of BSA. The viability of muscle was investigated in methodological experiments exactly as described by Andréasson et al. (10) by performing studies on 3–0-methylglucose transport. Insulin stimulated glucose transport in a concentration-dependent fashion; the maximum effect was about 2-fold stimulation of basal glucose transport. The latter rate was about 1 µmol/ml·h, which is in the same range as that reported by Andréasson et al. (10).
Lipolysis studies
In studies of glycerol release, muscle strips were incubated for indicated periods of time (usually 90 min) in 2 ml of Krebs-Ringer bicarbonate buffer supplemented with 20 mg/ml BSA (pH 7.4, BSA fraction V; Sigma, St. Louis, MO), 1 mg/ml glucose, and 0.1 mg/ml ascorbic acid in a shaking water bath (37 C), with air as gas phase. After incubation, an aliquot of the medium was removed for analysis of glycerol using an ultrasensitive bioluminescent method (11). Strips were incubated in duplicate or triplicate in the absence (basal) or presence of various concentrations of isoprenaline, which is a nonselective ß-adrenoceptor agonist. The muscle strip was removed from the medium and frozen in liquid nitrogen. It was then removed from the clamps and weighed. Lipolysis was expressed as millimole of glycerol per total incubation medium per time unit per milligram of muscle strip.
FFA studies
In studies of FFA release, muscle strips were incubated and analyzed exactly as described for lipolysis, except that 20 mg/ml of fatty acid-free BSA (Sigma) was used as the protein component in the incubation buffer, the number of strips in each type of incubation varied between five and 10, and FFA was analyzed by an ultrasensitive chemiluminescence method (12).
Measurement of HSL activity and protein
Frozen samples of adipose or muscle tissue (0.5–1 g) were homogenized in 3 ml of homogenization buffer (0.25 M sucrose, 1 mM EDTA, 1 mM dithioerythritol, 20 µg/ml pepstatin, and 1 mM benzamidin, pH 7.0) using a knife homogenizer. Fat-depleted infranatants were obtained after centrifugation at 10,000 x g at 4 C for 20 min. Pellets were resuspended in homogenization buffer to the same volume as the corresponding infranatant. The HSL activity measurements were performed as described (13) using the diolein analog 1(3)-mono[3H]oleoyl-2-oleylglycerol as substrate. Because the phosphorylated and unphosphorylated forms of enzyme have the same activity against this substrate, this assay is a measure of the total enzyme concentration. In Western blot analysis, aliquots of the infranatants, corresponding to 0.35 mU of diacylglycerol lipase activity, were separated by SDS-PAGE (8% polyacrylamide in the separation gel) and electroblotted to nitrocellulose membranes (Hybond C extra; Amersham, Buckinghamshire, UK). Membranes were blocked in Tris-buffered saline (20 mM Tris-HCl and 137 mM NaCl, pH 7.6) containing 5% dry skimmed milk, and all washes were done in Tris-buffered saline supplemented with 0.25% Tween 20. Affinity-purified rabbit antihuman HSL antibodies were used as primary antibodies, and horseradish peroxidase-conjugated antirabbit IgG (Amersham) were used as secondary antibody. Enhanced chemiluminescence was used as detection system.
Ultrastructural immunohistochemistry
These experiments were performed on Sprague Dawley rats. The preparation of tissues has been described previously (14, 15). Instant fixation of the tissues was accomplished by whole-animal vascular perfusion (through proximal aortic cannulation) with 3% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer. The soleus muscles were dissected, frozen under cryoprotection (2.3 M sucrose) in liquid nitrogen, and subjected to ultracryotomy at –90 C. Ultrathin sections were cut, thawed at room temperature, placed on formvar-coated nickel grids, and subjected to immunohistochemistry. PBS containing BSA was used for dilution and rinsing. Nonspecific binding was blocked by incubation with 0.1 M glycine followed by BSA in phosphate buffer. Sections were incubated overnight with primary polyclonal antibodies against rat HSL raised in rabbits (16) at a dilution of 1:300. Antibodies were detected by exposing the tissues to protein A coated with 10 nm colloidal gold. Specificity was confirmed using nonlabeled protein A in excess preceding the addition of protein A-gold complex. Finally, sections were contrasted with uranyl acetate. Tissues were explored by use of a Philips 400 electron microscope (Philips, Stockholm, Sweden) at 100 kV. Micrographs were taken randomly and analyzed at a final magnification of x39,000.
Morphology
Sprague Dawley rats were used in the experiments. Nordahl et al. (16) described the tissue preparation protocol previously. Fixation was achieved by vascular perfusion with 3% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer. Tissues were then immersion-fixed in sodium cacodylate-buffered 2% glutaraldehyde containing sucrose, followed by postfixation in OsO4. Dehydration was performed with ethanol at a gradual lowering of temperature to finally –40 C. Tissues were embedded in epoxy resin LX-112 (Ladd, Williston, VT). Subsequently, ultrathin sections were cut and placed on formvar-coated grids. Contrast was enhanced by uranyl acetate and lead citrate. Microdroplets of fat were detected intracellularly by use of micrographs taken at the ultrastructural level at a magnification of x12,600.
Isolation of mitochondria
Mitochondria were isolated from the human muscle biopsy samples using standard procedures (17). The samples (in average, 70 mg) were dissected free from visible fat and connective tissue on a glass plate that was cooled over ice. The minced muscle was homogenized in ice-cold buffer consisting of 100 mM KCl, 50 mM Tris (hydroxymethyl) amino methane, 5 mM MgCl2, 1.8 mM ATP, and 1 mM EDTA (pH 7.2) with HCl. After centrifugation for 3 min at 650 x g, the pellet was discarded, and the supernatant was centrifuged for another 3 min at 15,000 x g. The new pellet that was formed, containing the mitochondria, was washed in homogenizing buffer, recentrifuged, and resuspended in a solution containing 250 mM sucrose, 15 mM K2HPO4, 2 mM MgAc2, 0.5 mM EDTA, and 0.5 g/liter human serum albumin (pH 7.2) with KOH, to a final concentration of approximately 2 g protein/liter. The isolated mitochondria were stored frozen at –80 C.
Statistical methods
Values are given as mean ± SEM. They were compared using the Student’s paired and unpaired t test, ANOVA, or Wilcoxon’s paired test. All tests were two sided. P = 0.05 or better was considered to be statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
The time course for glycerol release from skeletal muscle strips is shown in Fig. 1
. The release was apparently linear with time for at least 90 min; thereafter, there was slightly less of an increase in glycerol output for at least another 90 min. Similar findings were obtained in the basal state and with isoprenaline (10 µM), but the rate of glycerol release was increased about two-fold in the presence of the ß-adrenergic agonist. On the basis of these results, a 90-min incubation period was used in all further experiments.
|
depicts the results with concentration-response experiments using isoprenaline (0–1 µM). A concentration-dependent increase in glycerol release was demonstrated with a doubling of the rate at 1 µM of isoprenaline. This concentration was considered the maximum effective concentration because no additional effect was found with 10 µM (i.e. the results of experiments presented in Fig. 1
). Therefore, isoprenaline was used at 1 µM in all subsequent experiments.
|
). No significant differences between groups regarding basal or isoprenaline-stimulated glycerol release were recorded. The same was true when men and women were compared (data not shown). There was no correlation between BMI and rate of glycerol release (Table 2). However, basal and isoprenaline-stimulated glycerol release showed a positive correlation with the age of the subjects (r = 0.5, P < 0.05; Table 2).
|
|
The release of FFA and glycerol was determined in the same incubation medium (Fig. 3). Whereas isoprenaline doubled the rate of glycerol release, no effect of the drug was observed on FFA release. In the basal state, glycerol and FFA were released at approximately equimolar rates (0.3 nmol/mg muscle strips·90 min). Assuming that most of lipolysis is related to breakdown of triglycerides and that hydrolysis of one triglyceride molecule forms three FFAs and one glycerol molecule, this implies a considerable basal reuptake and, conceivably, reutilization of FFA. However, no attempts were made to calculate FFA reutilization, bearing in mind that the amount of glycerol metabolism and the degree of formation of mono- or diglycerides in these experiments were unknown.
|
In two subjects, a biochemical investigation of HSL was performed in sc fat and skeletal muscle. The diacylglycerol lipase activity was about 3 times higher in the cytosolic fraction than in the pellet fraction in both tissues. Cytosol diacylglycerol lipase activity varied between 2 and 20 mU/mg protein in fat but was rather constant in muscle (between 1.2 and 1.3 mU/mg protein; Table 3). Western blot experiments revealed that the molecular mass of HSL was similar in both tissues (
88 kDa), which corresponds to the long form of the lipase (Fig 4).
|
|
Immunohistochemical analysis of rat soleus muscle at the ultrastructural level showed specific intramyocellular labeling for HSL. Markers for the enzyme were evenly distributed in the cytoplasm, but interestingly, some were also found in close association with mitochondria (Fig. 5). Although the gold stain, which marks the intracellular location of HSL, shown in Fig. 5A appears less distinctive, the absence of stain on the nucleus is a strong and widely accepted sign of specific binding because the nucleus usually demonstrates a high degree of unspecific affinity. Moreover, semiquantitative estimates of concentrations of gold stain in these areas also show increased density of gold stain in association with mitochondria. In an effort to further elucidate whether HSL antibodies incorrectly could have bound to mitochondria (not HSL associated), mitochondria were isolated from two subjects and enzyme activity was measured in both mitochondria preparation and in supernatant representing the cytoplasm. We found an equal distribution of HSL activity in the two fractions (Table 3), and thus, we were unable to corroborate or exclude a tight HSL-mitochondria association. It is conceivable that a better separation between mitochondria and cytoplasm than we were able to achieve in our preparation is needed. Tissue preparations for morphological assessment revealed the presence of microdroplets of fat immediately adjacent to mitochondria (Fig. 6).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
We used an incubation system, which was previously developed for studies of glucose transport in human skeletal muscle. Methodological investigations showed that this muscle preparation was valid for lipolysis studies. The strips are free from all visible white fat cells. First, we obtained expected results with basal and insulin-stimulated glucose uptake, indicating a viable tissue. Second, release of glycerol from the tissue to the medium was linear over time both under basal and isoprenaline-stimulated conditions, showing that the lipolysis process was not saturated during the present incubation conditions.
Isoprenaline, a nonselective ß-adrenergic agonist, caused a concentration-dependent stimulation of glycerol release. At the maximum effective concentration (1 µM), a 2-fold stimulation was obtained. This might be an underestimation of true ß-adrenoceptor-mediated lipolysis stimulation because some glycerol is reused by the muscle tissue (18). The results with isoprenaline are expected because in vivo infusion of isoprenaline to skeletal muscle by means of microdialysis markedly elevates interstitial glycerol levels in humans (19). When, on the other hand, the release of fatty acids was determined, the results were much different from those with glycerol. Skeletal muscle released significant amounts of fatty acids in the basal state. However, isoprenaline at 1 µM failed to stimulate fatty acid release, although glycerol release was markedly augmented. In this experimental setting, it is impossible to study the ultimate fate of liberated fatty acids, which may be reesterification and/or storage (extra- or intracellular), as well as oxidation. However, our findings suggest that, during lipolysis stimulation, all fatty acids produced above the basal level are immediately reused. The observation of almost equimolar rates of FFA and glycerol release in the basal state indicate that, even during resting conditions, human skeletal muscle immediately reuses most of FFA formed through lipolysis. Unfortunately, the small amount of tissue that could be obtained from our patients did not allow for a detailed investigation of fatty acid metabolism.
The findings with FFA are in marked contrast to those obtained with human fat cells incubated under almost identical conditions (20, 21). Catecholamine stimulates the release of FFA and glycerol release to almost the same extent in human fat cells of different origin (i.e. sc or visceral), indicating that there is no or minimal reutilization of fatty acids when lipolysis is activated.
Another unexpected finding concerns the distribution of triglycerides and HSL within the muscle cells. As in fat cells, the muscle HSL was predominantly localized in the cytosolic fraction, although ultracentrifugation experiments showed that HSL was present both in the mitochondria and the cytosolic fraction. As in fat cells, the protein constituted the long form of HSL. A short inactive form is also sometimes present in human tissues (22). However, the results with electron microscopy revealed that triglycerides formed multiple droplets in the cytosol and that some HSL-protein and lipid droplets were adjoining mitochondria. This is different from the situation in fat cells, as reviewed previously (23, 24). Adipocyte triglycerides form a single fat droplet, and HSL is, in the basal state, freely distributed in the cytosol and transported to the fat droplet after catecholamine stimulation.
The electron microscope results were very clear in the rodent experiments. However, we were unable to demonstrate the same distinct features in human muscle cells (data not shown). This may be explained by methodological and ultrastructural differences. First, although the use of tissue fixation by vascular perfusion, as in the rat experiments, is considered the most efficient technique to preserve immunogenic properties of a tissue, it cannot be used in humans. Second, there is a species-related difference in intracellular composition (e.g. in comparison to rat muscle, the cytoplasm in human muscle is very scarce and contains less mitochondria).
When the results with fatty acid release and intracellular localization of lipids and HSL are put together, it is tempting to speculate that muscle lipolysis, at least to some extent, occurs near mitochondria, and therefore, some or most of the fatty acids produced during hydrolysis of triglycerides may immediately undergo oxidation so that only minor release from the muscle cells occurs. This efficient local energy consumption might explain the rapid decrease in intramyocytal triglycerides that, according to others (2, 3), occurs after exercise. Clearly, our hypothesis must undergo further evaluation by detailed investigations of HSL and mitochondria in human muscle.
A third surprising finding concerns the clinical data on lipolysis in skeletal muscle. The rate of glycerol release was independent of BMI. However, basal and isoprenaline-stimulated glycerol release increased significantly by increasing age. This is in contrast to findings with human fat cells, in which catecholamine-stimulated glycerol release decreases with increasing age or BMI (25, 26). The reason for this tissue-specific discrepancy may be explained, in part, by an age-related increase in muscle triglycerides (27), but it also points to a tissue-specific regulation of lipolysis in skeletal muscle. In addition, in vivo evidence for a tissue-specific regulation of lipolysis in man is present. For example, insulin infusions iv inhibit glycerol output from sc adipose tissue but has either no effect or increases glycerol output from leg skeletal muscle (28, 29).
Because of difficulties in recruiting subjects for this study, a very heterogeneous population was studied, including male and female patients and malignant and nonmalignant subjects. However, there was no apparent effect of gender or malignancy on the present findings. It should be pointed out, though, that no attempt was made to study the influence of pathophysiological states in this first in vitro study of lipolysis regulation of skeletal muscle lipolysis. It is quite possible that conditions such as obesity, diabetes, and dyslipidemia influence lipolysis in muscle; indeed, rat experiments suggest that lipolysis in skeletal muscle is influenced by high-fat feeding (30).
It is very unlikely that the results are influenced to an important extent by lipolysis in fat cells interspersed within the muscle tissue. The electron microscopy studies revealed no fat cells between muscle cells, and all visible fat was removed from the muscle strip preparations. Furthermore, fat cells within the muscle would be expected to behave as fat cells elsewhere (i.e. show stimulation of FFA release after isoprenaline exposure as well as decreased lipolysis rates by aging).
In summary, this study demonstrates several features related to lipolysis regulation that are specific to skeletal muscle tissue, suggesting a unique role for skeletal muscle in lipolysis and FFA homeostasis. Most important is the indication of marked reutilization of FFA in the basal state and after catecholamine stimulation, which may be due to a unique localization of the lipolysis process near the mitochondria, allowing rapid and efficient fatty acid oxidation.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
First Published Online November 23, 2004
Abbreviations: BMI, Body mass index; FFA, free fatty acid; HSL, hormone-sensitive lipase.
Received June 7, 2004.
Accepted November 12, 2004.
|
References |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
-3 fatty acids in muscle phospholipid. Diabetes 40:280–289[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  M. G. Cree and R. R. Wolfe Postburn trauma insulin resistance and fat metabolism Am J Physiol Endocrinol Metab, January 1, 2008; 294(1): E1 - E9. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Tarnopolsky, C. D. Rennie, H. A. Robertshaw, S. N. Fedak-Tarnopolsky, M. C. Devries, and M. J. Hamadeh Influence of endurance exercise training and sex on intramyocellular lipid and mitochondrial ultrastructure, substrate use, and mitochondrial enzyme activity Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2007; 292(3): R1271 - R1278. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
