1 Department of Medicine, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden
2 Department of Pathology, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden
3 Department of Rheumatology, Karolinska Hospital, Stockholm, Sweden
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Another important aspect of lipolysis is whether its activity is subject to regional variations. Such variations do exist for adipose tissue (17). Lipolysis is more easily stimulated and less readily inhibited in the visceral, as compared with the subcutaneous, region. It is not known whether lipolysis also differs between muscle groups. Human skeletal muscle is a heterogeneous tissue with respect to both function and fiber composition (i.e., containing slow-twitch type 1 and fast-twitch type 2 fibers). It has been shown in rats that the rates of TG synthesis and hydrolysis differ markedly among muscle groups (18,19).
The aim of the present study was to determine whether lipolytic activity in humans differs among various muscle groups at rest, and if any differences could be related to TG content and/or fiber composition of the muscle. The deltoid, vastus lateralis, and gastrocnemius muscle groups were chosen to reflect a range of function. Local lipolytic activity was estimated by combining arterial and tissue measurements of glycerol with estimations of muscle blood flow using the microdialysis and Xe-washout techniques, respectively (8). We found marked differences in net glycerol release among the muscle groups, which were likely related to fiber composition.
![]() |
RESEARCH DESIGN AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protocols
Microdialysis.
In all subjects, glycerol concentrations in the intercellular space of the three different skeletal muscle tissue regions were determined using microdialysis, as described in detail elsewhere (8,20). Briefly, a double lumen catheter, with a semipermeable membrane glued to its end, was inserted percutaneously in the tissue and perfused with a sterile solution. An exchange of molecules will take place over the membrane, and the composition of the outflow solution will reflect that of the extracellular space. Only small substances will pass across the membrane, so the samples are therefore protected from enzymatic degradation. Optimization of the catheter length and perfusion speed gives an almost complete recovery, whereby the outgoing concentration of glycerol equals the true interstitial tissue level.
The subjects reported to the laboratory at 8:00 A.M. after an overnight fast. Teflon catheters (Venflon) were inserted in a retrograde manner in the dorsal hand vein for blood sampling. The hand was placed in an air-heated box (63°C) to ensure arterialization of the venous blood (21). Microdialysis catheters (0.3 x 30 mm, 20-kDa molecular weight cutoff; CMA Microdialysis AB, Solna, Sweden) were inserted in the medial part of the gastrocnemius muscle, mid-part of the vastus lateralis muscle, and posterior part of the deltoid muscle, respectively, after topical anesthesia (EMLA, Astra, Sweden). The thickness of the subcutaneous tissue at the site of insertion was determined by ultrasound (515 mm) to ensure intramuscular location of the microdialysis device. In addition, the penetration of the muscular fascia was in most cases easily recognized, and the intramuscular location was confirmed by the development of muscular twitches during insertion.
Immediately after insertion, the microdialysis catheters were perfused with Ringers solution (147 mmol/l sodium, 4 mmol/l potassium, 2.3 mmol/l calcium, 156 mmol/l chloride; Apoteksbolaget, Umeå, Sweden) using a flow rate of 0.3 µl/min. This rate has previously been shown to give near-complete recovery of glycerol in skeletal muscle tissue (22); hence, the dialysate glycerol concentration acquired is very close to the glycerol concentration in the tissue extracellular space. After an equilibration period of 120 min, the dialysates were sampled in 15-min time fractions during a 90-min period. Plasma samples were drawn in the middle of each dialysate fraction. It has been previously demonstrated that the muscle tissue level of glycerol is at steady state 2 h after catheter insertion (22).
To study local uptake of glycerol, six of the subjects (three women, three men) were investigated with a similar microdialysis protocol on a second occasion. After a baseline sampling period, the circulating glycerol concentration was raised by means of a 3-h infusion of triacylglycerol emulsion (Intralipid 200 mg/ml; Pharmacia) at 1.85 ml · kg-1 · h-1. Blood sampling for the analysis of glycerol and TGs was carried out throughout the experiment. Microdialysis catheters were inserted as described above in the three muscle regions and, in addition, in abdominal adipose tissue in the periumbilical region for the analysis of dialysate glycerol.
Blood flow.
For practical reasons, in the first study tissue blood flow had to be registered on a separate occasion using the 133Xe-washout method (23), as previously described in detail (8). The subjects came to the laboratory in the morning after an overnight fast. 133Xe (0.3 MBq in 0.1 ml saline; Mallinckrodt, Petten, the Netherlands) was injected in the respective skeletal muscle tissue region. Residual activities were recorded with a scintillation detector (Mediscint; Oakfield Instruments, Oxford, U.K.). Recordings were started 5 min after the injection and continued for 30 min. Because the decay curve becomes multiexponential over time (24), only the first 10 min of the skeletal muscle decay curve were used for calculations. We recently demonstrated that muscle tissue blood flow measured in this way is stable over time. The coefficient of variation for determinations on two separate occasions is 20.6% (25). Therefore, blood flow was considered to be the same as during microdialysis.
For the glycerol-infusion study, skeletal muscle blood flow was determined with the same method. 133Xe was injected as described above in the three skeletal muscle regions, opposite to the microdialysis site. Recordings were performed during the last 30 min of the baseline period. Because of changes in the decay curve, the injection had to be iterated and recordings again performed during the last 30 min of the infusion period. For determination of adipose tissue blood flow, Xe (1 MBq in 0.1 ml saline) was injected into the subcutaneous abdominal tissue contralaterally to the microdialysis catheter 30 min before the start of the baseline period. Recordings were performed throughout the experiment, and the blood flow was determined for the last 30-min period of baseline and infusion, respectively.
Muscle biopsies.
Percutaneous muscle biopsies were carried out in two of the three investigated regions: the vastus lateralis and gastrocnemius muscle. All subjects were asked to undergo biopsies, but only half of the group (three men and three women) were willing to do so. Because of practical reasons, it was not possible to perform the biopsies concomitantly with either the microdialysis or blood flow experiments. The biopsies were always performed after the microdialysis experiment. With the patient in the supine position, under sterile conditions, the skin and fascia were penetrated with a 1-cm cut after local anesthesia. Using a Weil-Blakesly nasal cutting forceps 100 mg muscle tissue were cut out from the middle region of the vastus lateralis muscle and the medial part of the gastrocnemius muscle. The specimens were trimmed from adipose and connective tissue and then frozen at -70°C in liquid nitrogen for biochemical analyses. For histological examination, the biopsies were snap frozen in isopentane and chilled with dry ice. Then 8- µm-thick cryostat sections were stained with hematoxylin-eosin (H-E), oil red O, sudan black, and ATPase with preincubation at pH 4.3 and 9.6.
Protein isolation and Western blot.
Total protein lysates were obtained from frozen subcutaneous adipose tissue or muscle tissue and then crushed and lysed in protein lysis buffer (1% Triton-X100, Tris-HCL [pH 7.6], and 150 mmol/l NaCl) supplemented with protease inhibitors (1 mmol/l phenylmethylsulfonyl fluoride and Complete [Roche, Basel, Switzerland]), as previously described (26). The protein content in each sample was determined using a kit of reagents from Pierce (Rockford, IL). Next, 50 (adipose tissue) or 300 µg (muscle tissue) of total protein were loaded on 12% polyacrylamide gels and separated by standard SDS-PAGE. To control for differences in gel migration, exposure time, and antibody incubation, among others, samples were run on the same gels and transferred to the same polyvinylidine fluoride membranes (Amersham Pharmacia Biotech, Little Chalfont, U.K.). After a standard blocking procedure, blots were incubated in the presence of either polyclonal antibodies directed against human hormone sensitive lipase (kindly provided by Dr. Cecilia Holm, Lund University, Sweden) or human perilipin (Progen Biotechnik GmBh, Heidelberg, Germany). Proteins were detected by chemiluminescence using Super Signal (Pierce Chemical, Rochford, IL). Films were scanned and quantified by NIH Image software (National Institutes of Health, Bethesda, MD).
Analytical procedures.
Microdialysate glycerol was determined with an enzymatic fluorometric method, using a tissue sample analyzer (CMA/60; CMA Microdialysis, Stockholm, Sweden) (16). Plasma glycerol was determined by bioluminescence (27). Muscle triacylglycerol content was determined after extraction of total lipids, as previously described (28). Extracts were dried and then saponified to FFAs and glycerol with KOH, as previously described (28). Glycerol, which is stoichiometrically equivalent to the TG concentration, was then determined by bioluminescence (27). Myofibrillar ATPase staining was used to identify muscle fiber types (29).
Calculations.
Tissue blood flow (TBF) was calculated according to the following formula:
![]() |
Glycerol release from the tissues was calculated according to Ficks principle, as follows:
![]() |
![]() |
Although glycerol is perfectly water soluble, it uses transport systems in human red cells (32). These transporters probably belong to the "aquaporin" gene family (33). An aquaporin with a high affinity for glycerol (aquaporin-3) is present in human erythrocytes (34). Therefore, a correction for hematocrit was used in the present and previous microdialysis studies by us and others when Ficks principle is applied to the calculation of net release of glycerol from adipose tissue and muscle. However, for comparison, the net glycerol release was also calculated without the correction for hematocrit.
Turnover rates of the tissue TG pool were calculated in the six subjects undergoing biopsies using the following parameters: wet weight of tissue (A), moles of TG in the tissue (B; for muscle, is the amount of glycerol after lipid extraction and hydrolysis), and the rate of glycerol mobilization from tissue per unit of tissue weight (C). The turnover rate, expressed as percent per 24 h, was calculated as B divided by A divided by C.
Statistics.
Data are means ± SE. Comparisons among the muscle groups were performed using Students paired or unpaired t test or Wilcoxons signed-rank test, where applicable. ANOVA was used for comparisons of parameters over time. Statistical calculations were made using a statistical software package (Statview II; Abacus Concepts, Berkeley, CA).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
The triacylglycerol infusion resulted in a 9-fold increase in plasma glycerol and a 13-fold increase in plasma TG (Fig. 3A). The dialysate concentrations of glycerol for adipose tissue and the muscle regions increased in parallel, and steady-state conditions were reached for all compartments during the last hour of infusion (NS for ANOVA repeated measures). Thus this period was used for calculations. The IA glycerol difference during infusion is shown in Fig. 3B. There was a negative IA glycerol difference in the three skeletal muscle regions during infusion, indicating uptake of glycerol, whereas there was a positive IA glycerol difference in adipose tissue. The IA glycerol difference did not differ among the skeletal muscle regions. Blood flow did not change from basal levels when triacylglycerol was infused. The net glycerol release was calculated as described above, and during glycerol infusion these "release" values were negative (indicating net glycerol uptake) for all skeletal muscle regions, with no significant interregional differences (Fig. 3C).
|
|
|
|
Turnover of triglyceride.
The turnover of muscle TG after an overnight fast was calculated in the gastrocnemius muscle in the set of subjects undergoing muscle biopsy (n = 6). The gastrocnemius muscle was chosen as it was the only muscle group that showed significant glycerol release (3.3 ± 1.4% over 24 h). Using data from our previously published study on healthy nonobese men and women (8), we also calculated the turnover of subcutaneous adipose tissue TG after an overnight fast. The following assumptions were made: 1) 70% of adipose tissue content is lipids, 2) >95% of adipose tissue lipids are TG, and 3) triolein represents the average molecular weight of adipose TG. Glycerol release from the abdominal subcutaneous adipose tissue of lean subjects after an overnight fast was 0.2 ± 0.04 µmol · 100 g-1 · min-1 (8), which corresponds to a TG turnover of 0.38 ± 0.07% over 24 h. The latter value is 10 times lower than the presently calculated value for muscle TG turnover (P < 0.01).
Contribution of intramuscular lipolysis to fatty acid oxidation at rest in gastrocnemius muscle.
Fatty acid production by the muscle was calculated using the following: 1) one molecule of glycerol formed equals three molecules of fatty acids formed; 2) the degree of glycerol metabolized was determined to be 0.28 from data in Fig. 3 during steady-state infusion of triacylglycerol according to the formula: ( plasma glycerol -
tissue glycerol)/
plasma glycerol, where delta refers to the difference between glycerol during infusion and during baseline; and 3) total glycerol produced by muscle equals net glycerol release multiplied by the correction factor for "true" glycerol production, 1.28.
Estimated fatty acid production at rest was calculated using values for the glycerol release: 0.36 x 3 x 1.28 = 1.4 µmol · kg-1 · min-1 or 0.58 x 3 x 1.28 = 2.3 µmol · kg-1 · min-1, depeding on if correction for hematocrit was made or not when using Ficks principle. FFA oxidation at rest in relation to exercise (at 50% of VO2max) was calculated as follows using data by Schrauwen et al. (37): plasma FFA oxidation at rest/at exercise in healthy nonobese volunteers: 233/972 = 0.24. Muscle oxidation during exercise at 45% of VO2max in healthy nonobese males is reported by Guo et al. (38) to be 8.2 + 8.4 = 16.6 µmol · kg-1 · min-1. Thus, fatty acid oxidation in muscle at rest can be estimated to 0.24 x 16.6 = 4.0 µmol · kg-1 · min-1.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
No methods are yet available to enable a mechanistic study of the cause of variation in lipolytic activity among human muscle groups. It is possible that differences are related to fiber composition, as the gastrocnemius muscle has a high content of type 1 fibers plus a high rate of lipolysis. It is tempting to speculate that muscles designed for endurance performance (slow-twitch type 1 fibers) may need an endogenous source of FFAs to a greater extent than do other muscles having a relatively high content of fast-twitch type 2 fibers. It is less likely, though, that the rate of lipolysis is determined by the size of the muscle TG pool. Despite major differences in net glycerol release, the gastrocnemius and vastus lateralis had similar TG contents.
It has been previously established that resting skeletal muscle TG content is largely dependent on the skeletal muscle fiber type, with the highest lipid content being found in type 1 fibers (10,41). We were not able to detect any difference in lipid content between the muscle types with higher and lower portions of type 1 fibers. However, large variations in the triglyceride content from vastus lateralis biopsies have been shown in healthy subjects (42). In our study, the lipid content varied from 10 to 36 µmol/g in the vastus lateralis muscle and from 13 to 42 µmol/g in the gastrocnemius muscle. Of the six subjects studied, only one showed a clearly higher value in the gastrocnemius muscle. The fiber type composition is also reported to be largely variable (43,44), at least in the vastus lateralis muscle. The proportion we found is in agreement with previous findings in healthy subjects (44).
Using present techniques, it is not possible to establish the exact origin of tissue glycerol. Glycerol can be released from intramuscular TG (intracellular), from TG in adipocytes marbled between the myocytes (extracellular), or from local breakdown of TG delivered by the blood stream through the activity of lipoprotein lipase (LPL). In exercising muscle, there is evidence of a fiber type-dependent utilization of intracellular TG, so that the slow- twitch (type 1) fibers are capable of a larger turnover of TG (45). If this also holds true for resting muscle, then the gastrocnemius muscle should release more glycerol from intramuscular TG because of the relative dominance of type 1 fibers. Unfortunately, the present sample size was too small to allow correlation statistics to be performed between fiber type and glycerol release. LPL is mainly activated by decreased intracellular TG to replenish intracellular TG stores from extracellular TG after, for example, exercise (46). It is also possible that it contributes to glycerol release in the fasted state. TG from adipocytes within the muscle is the third alternative source of glycerol. However, it seems unlikely that the glycerol recovered in microdialysis experiments of skeletal muscle originates from adipocytes. First, there was no important difference in local TG content between two of the different muscle groups studied (vastus lateralis and gastrocnemius). Second, the histological examination of extramyocytic TG showed very small amounts of adipocyte-derived TG staining. Third, only diminutive amounts of the adipocyte-specific proteins perilipin in the muscles were investigated, with no differences found between the two regions. If adipocytes were responsible for the substantial contrasts in net glycerol release, adipocyte-specific proteins would be present in protein lysates from muscle in amounts correlating to the quantity of contaminating adipose tissue. Fourth, there is no reason to believe that muscle fat cells have a much higher turnover rate than do adipose tissue fat cells.
Another reason for the interregional differences in glycerol release could be differences in glycerol uptake in the respective muscles. However, this seems less likely as the glycerol fractional extraction (IA difference) as well as the calculated glycerol uptake was similar in the three regions when circulating glycerol was artificially increased well above tissue levels. Moreover, because the tissue blood flow was constant during the infusion experiment, an estimate of the uptake of infused glycerol can be performed using the formula: ( plasma glycerol -
tissue glycerol)/
plasma glycerol, where delta refers to the difference between glycerol during infusion and baseline. It can be assumed that during circumstances with markedly increased circulating glycerol, the skeletal muscle tissue glycerol uptake is still at most 2030% of the infused glycerol. Furthermore, there were no differences among the regions. Differences in glycerol uptake is therefore not considered to explain the large difference (
400%) in glycerol release found in different skeletal muscle regions. Direct reutilization within the myocyte of glycerol derived from intramuscular lipolysis could theoretically be subject to regional variations that could not be evaluated in the present study.
When calculating glycerol release, we used a constant permeability surface product area (PS value) of 5 ml · 100 g-1 · min-1. As extensively reviewed by Crone and Levitt (47), it has been experimentally shown that the PS values for low-molecular substances (e.g., glycerol) are comparable in most organs with continuous capillaries, including skeletal muscle and adipose tissue. Thus we do not believe that skeletal muscle regional differences in the PS values could be of importance for the findings. Moreover, to abolish the regional differences found, the PS value for the gastrocnemius muscle has to be <3 ml · 100 g-1 · min-1 or increased >100-fold in the gastrocnemius region.
Present and previous data (current study; 8,25) indicate that there are major differences in the regulation of lipolysis in muscle and adipose tissue. The action of the most important hormones, insulin and catecholamines, differ. Some muscle groups may have no or little lipolytic activity at rest, whereas others may have lipolysis rates far exceeding those seen in adipose tissue, as estimated from TG turnover data. These differences among and within tissues might be important not only for normal regulation of energy homeostasis but also for the development of insulin-resistant conditions.
We found higher glycerol levels in muscle than in blood at rest (22) than did Rosdahl et al. (48), who used similar microdialysis techniques. The present results most likely explain the divergence in these reports. We investigated the gastrocnemius muscle, whereas Rosdahl and colleagues studied the vastus lateralis muscle. It is apparent that regional differences in metabolic activity must be considered when interpreting data from a single muscle group.
It would be of interest to investigate the extent to which intracellular triglyceride lipolysis contributes to fatty acid oxidation in skeletal muscle at rest. We calculated that in gastrocnemius muscle, the production of fatty acids by intramuscular lipolysis was 1.42.3 µmol · kg-1 · min-1, depending on the mode of calculation. Furthermore, we calculated that fatty acid oxidation in skeletal muscle at rest is 4 µmol · kg-1 · min-1. If we assume that after an overnight fast essentially all fatty acids produced in resting muscle are oxidized, then muscle lipolysis contributes 3350% of the fatty acid oxidation in gastrocnemius muscle. Interestingly, Guo et al. (38) found that during exercise, half of the fatty acids oxidized derived from intramuscular lipolysis. The data of Guo and colleagues probably represent several muscle groups, and we do not know if there are also differences among muscle groups in local lipolysis during exercise. Because we found no evidence of significant net glycerol release in any muscle group except gastrocnemius, it is likely that local lipolysis is important to the energy supply in the resting state only in this muscle group.
In summary, the findings of this study indicated that there are significant differences in resting lipolytic activity among different skeletal muscle groups in healthy nonobese humans. Net glycerol release, indicative of lipolytic activity, was observed from only the gastrocnemius muscle, which is mainly composed of type 1 muscle fibers. This suggests that local lipolysis is most important for endurance muscle activity.
![]() |
ACKNOWLEDGMENTS |
---|
The excellent technical assistance of Lisa Dungner, Eva Sjölin, Britt-Marie Leijonhufvud, and Katarina Hertel is acknowledged.
![]() |
FOOTNOTES |
---|
Received for publication 24 August 2001 and accepted in revised form 26 August 2002.
FFA, free fatty acid; H-E, hematoxylin-eosin; HSL, hormone-sensitive lipase; IA, interstitial-arterial; LPL, lipoprotein lipase; PS value, permeability surface product area; TBF, tissue blood flow; TG, triglyceride.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|