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Departments of Medicine (V.Q., E.H.-T., E.M., P.A., J.B.) and Vascular Surgery (S.E.), Karolinska University Hospital–Huddinge, Karolinska Institutet, SE-141 86 Stockholm, Sweden
Address all correspondence and requests for reprints to: Jan Bolinder, M.D., Ph.D., Professor, Department of Medicine, M54 Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden. E-mail: jan.bolinder{at}medhs.ki.se.
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Abstract |
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Objective: The objective of the study was to investigate the effect of endogenous catecholamines on TG lipolysis in human SM in vivo. Adipose tissue (AT) was studied for comparison.
Design and Main Outcome Measures: Glycerol levels (index of lipolysis) were measured using microdialysis in the gastrocnemius muscle and abdominal sc adipose tissue during a hyperinsulinemic, hypoglycemic clamp (n = 13) and in response to in situ perfusion of epinephrine and norepinephrine (10–10 to 10–5 M) (n = 12). Local tissue blood flow was monitored with the ethanol perfusion technique.
Setting: This was an experimental study.
Participants: The study population consisted of healthy subjects.
Results: Plasma epinephrine increased 10-fold and plasma norepinephrine 2-fold in response to insulin-induced hypoglycemia. In parallel, the fractional glycerol release (difference between tissue and arterial glycerol) increased 2-fold in both tissues (P < 0.0001). No changes in AT and SM blood flow were registered. When the catecholamines were perfused in situ, tissue glycerol increased significantly at 10–7 M of either epinephrine and norepinephrine (P < 0.0001) in AT. The maximum stimulation was seen at 10–6 M norepinephrine (2-fold increase) and 10–5 M epinephrine (3-fold increase). In SM, tissue glycerol increased at 10–7 M epinephrine and 10–6 M norepinephrine, respectively (P < 0.0001); the maximum increase of glycerol values (at 10–6 M) was 2.5 times for epinephrine and 1.6 times for norepinephrine, respectively (P < 0.01).
Conclusions: The lipolytic activity of SM is increased by endogenous catecholamines in vivo and appears to be more responsive to epinephrine than norepinephrine stimulation.
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Introduction |
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Despite the potential significance of skeletal muscle lipolysis, its regulation is not fully elucidated. In adipose tissue, the lipolysis rate is acutely inhibited by insulin and accelerated in response to catecholamine stimulation (8). Findings in earlier studies, in which interstitial glycerol levels were used as a measure of the lipolytic activity in vivo, suggested similar effects of the hormones also in skeletal muscle (2, 3, 9, 10, 11). However, these results have not been corroborated in later investigations in which instead the fractional tissue release of glycerol was assessed by calculating the difference between interstitial and arterial concentrations of glycerol (I-A glycerol difference). When using this approach, insulin had no antilipolytic effect in the skeletal muscle in contrast to adipose tissue (4, 12, 13, 14). Moreover, increased sympathetic activity did not influence the lipolytic activity in skeletal muscle (15), which may suggest that norepinephrine possesses no stimulatory effect on skeletal muscle lipolysis in humans. On the other hand, in situ perfusion of the ß2-agonist terbutaline increase lipolysis in human skeletal muscle (9, 11). The ß2-adrenoreceptor, which is the predominating ß-adrenoreceptor subtype in skeletal muscle (16, 17), has a greater affinity for epinephrine than for norepinephrine (18). Thus, it is possible that epinephrine, rather than norepinephrine, is the main adrenergic-stimulating catecholamine on skeletal muscle lipolysis. This thesis is supported by recent data that show that epinephrine stimulates lipolysis and hormone-sensitive lipase activity in rat skeletal muscle (19, 20). Whether the latter in vitro data in animal models are relevant for humans remains to be established. In the present study, we investigated the in vivo catecholamine-mediated regulation of lipolysis by monitoring the I-A glycerol difference in skeletal muscle and adipose tissue with microdialysis during a hyperinsulinemic hypoglycemic clamp because hypoglycemia is the most powerful elicitor of endogenous epinephrine release (21). Moreover, to compare the effects of the two endogenous catecholamines, the lipolytic activity was measured in both tissues in response to in situ perfusion of epinephrine and norepinephrine. Local tissue blood flow was measured with the ethanol perfusion technique.
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Subjects and Methods |
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Twenty-five healthy, drug-free volunteers were investigated (13 women and 12 men, age 28 ± 1 yr, range 22–55 yr, body mass index 23.0 ± 0.5 kg/m2). Each subject participated in only one of the two studies. All of them performed regular physical activity but not at an athletic level. The Ethics Committee of the Karolinska Institutet approved the study. The subjects were given a detailed description of the study before their informed consent was obtained.
Microdialysis
The principles of microdialysis have been described in detail previously (22). The microdialysis catheter (CMA/60, CMA Microdialysis AB, Stockholm, Sweden) consists of a semipermeable membrane (30 x 0.62 mm, molecular mass cut-off of 20 kDa) that is glued to the end of a double-lumen polyurethane tube. The probe is connected to a microinfusion pump (CMA/100 microinjection pump, CMA Microdialysis AB) and is continuously perfused with a sterile solution. An exchange of metabolites takes place over the microdialysis membrane and the composition of the outflow solution reflects the extracellular fluid. Qualitative estimates of variations in local tissue blood flow can be made by the ethanol perfusion technique (23). Ethanol, which is not locally degraded and does not affect tissue metabolism, is added to the perfusate as a flow marker. Changes in the ethanol concentration ratio (out- vs. in-going ethanol concentration) hence reflect changes in the local blood flow.
Study protocol
All subjects were investigated in the supine position after an overnight fast. The experiments started at 0730 h.
Hyperinsulinemic hypoglycemic clamp (n = 13). A Teflon catheter (Venflon) was inserted in a dorsal hand vein. The hand was placed in a heated box (63 C) for sampling of arterialized venous plasma. In the cubital vein of the contralateral arm, a second Teflon catheter was placed for infusion of insulin and glucose. After superficial skin anesthesia (EMLA, Astra, Södertälje, Sweden), two microdialysis catheters were inserted into the periumbical sc adipose tissue and two catheters into the medial part of the gastrocnemius muscle. One muscle catheter and one fat catheter were perfused at 0.3 µl/min with Ringer’s solution (Apoteksbolaget, Umeå, Sweden) (147 mmol/liter Na, 4 mmol/liter K, 2.3 mmol/liter Ca, and 156 mmol/liter Cl) alone. This flow rate has been shown to give almost complete recovery (>95%) of glycerol in skeletal muscle and adipose tissue (2). One muscle catheter and one fat catheter were perfused at 2.0 µl/min with Ringer’s solution supplemented with ethanol (50 mM) for estimation of tissue blood flow variations. The perfusate from each microdialysis catheter was collected in 15-min fractions for analysis of glycerol or ethanol. After a 120-min equilibration phase, and after 45 min of basal sampling, an iv insulin infusion (0.15 U/kg body weight; Actrapid, NovoNordisk, Copenhagen, Denmark) was given over 60 min. After 30 min, the concentration of arterialized venous blood glucose had fallen to approximately 2.5 mmol/liter and a variable glucose infusion (200 mg/ml) was started to maintain the blood glucose at 2.5 mmol/liter for 30 min. The insulin infusion was then terminated and blood glucose was allowed to recover gradually to the fasting level during the following 120 min. Blood glucose was monitored bedside for adjustments of the glucose infusion rate (Hemocue, Ängelholm, Sweden). Plasma samples were drawn in the middle of each dialysate sampling period for analysis of plasma glycerol. Plasma catecholamine and insulin concentrations were determined at regular intervals during the procedure, as indicated in Results. We previously reported interstitial skeletal muscle and adipose tissue glycerol and glucose data from subsets of these experiments (2, 9, 24).
In situ catecholamine perfusion (n = 12). Two microdialysis catheters, one for epinephrine and one for norepinephrine perfusion, were inserted into the abdominal sc tissue and the medial part of the gastrocnemius muscle, respectively. All four probes were continuously perfused with ethanol (50 mM) in Ringer’s solution. The flow rates were 2.0 µl/min in adipose tissue and 5 µl/min in the skeletal muscle. These flow rates have been shown to give similar relative recoveries in the two tissues (14). The dialysate samples for analysis of glycerol and ethanol were collected in 15-min fractions.
After 60 min of baseline sampling, increasing concentrations of norepinephrine and epinephrine (10–10 to 10–5 M), respectively, were added to the perfusate of the microdialysis catheters in the adipose tissue and the skeletal muscle. The catecholamine concentrations were increased stepwise with 60-min intervals. A pharmacological evaluation of the effect of the catecholamines in situ on glycerol release was made as follows: lipolytic sensitivity was defined as the lowest concentration given a significant increase in tissue glycerol. Lipolytic responsiveness was defined as the increase in tissue glycerol at the maximum effective catecholamine concentration.
Biochemical analysis
Dialysate glycerol was measured with an enzymatic fluorometric method with an automatic tissue dialysate sample analyzer (CMA/600). Dialysate ethanol was determined with an enzymatic spectrophotometric method (25). Plasma glycerol was determined by bioluminescence (26). Plasma catecholamines were measured with high-performance chromatography with electrochemical detection (27). Insulin in plasma was analyzed with a commercial RIA kit (Pharmacia, Uppsala, Sweden).
Statistics
Data are presented as means ± SEM. Variations over time in the same individual were evaluated with one-factor ANOVA, corrected for repeated measurements. Comparison over time between norepinephrine vs. epinephrine perfusion in situ was analyzed with two-factor repeated-measurement ANOVA. Factorial ANOVA was used for comparison between groups not involving time. Post hoc analyses were performed by Fischer’s protected least significant difference. Student’s paired t test was used to compare different time points. A value of P < 0.05 was considered statistically significant.
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Results |
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Glucose and insulin in plasma are shown in Fig. 1
(top panel). Plasma glucose concentrations were 5.1 ± 0.1 mmol/liter during basal sampling and gradually decreased during the first 30 min of insulin infusion to 2.4 ± 0.1 mmol/liter. This glucose level was then maintained for the remaining 30 min of the infusion. Plasma glucose then successively returned to initial values. Plasma insulin increased 19 times, from 43.6 ± 4.0 to 835 ± 44.9 pmol/liter, during the insulin infusion. After termination of the insulin infusion, plasma insulin levels gradually decreased, and at 1 h thereafter they had returned to baseline values. The plasma catecholamine responses are presented in Fig. 1 (bottom panel). The peak values of both endogenous catecholamines occurred 50–70 min after the insulin infusion was started. The epinephrine concentrations increased more than 10-fold above the baseline, and they were still significantly increased 1 h after the end of the insulin infusion (P < 0.01, Student’s paired t test). For norepinephrine the increase was barely 2-fold (P < 0.001, Student’s paired t test), and the values had returned to baseline 1 h after the insulin infusion was stopped. The I-A glycerol concentration differences in adipose tissue and skeletal muscle are shown in Fig. 2. In the basal state, the I-A glycerol difference was five times higher in adipose tissue than skeletal muscle (P < 0.0001, ANOVA). In response to iv insulin, the I-A glycerol difference was reduced by 60% in the adipose tissue (P < 0.0001, one-way ANOVA for repeated measurements), whereas it did not change significantly in skeletal muscle. In response to hypoglycemia, the I-A glycerol difference increased significantly in both tissues (P < 0.0001, one-way ANOVA for repeated measurements); the rise in the I-A glycerol differences coincided with the peak values of the catecholamines. In adipose tissue, the I-A glycerol concentration difference gradually returned to basal levels. In skeletal muscle tissue, the I-A glycerol difference was doubled at 35 min after the hypoglycemic nadir and then decreased toward initial values.
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Catecholamine administration in situ
Adipose tissue dialysate glycerol was about four times higher than skeletal muscle dialysate glycerol during basal sampling (P < 0.0001, ANOVA). In adipose tissue there was a concentration-dependent increase in glycerol levels after in situ administration of either catecholamine. Dialysate glycerol increased significantly above basal levels at 10–7 M of either epinephrine and norepinephrine (P < 0.0001, one-way ANOVA repeated measurements) (Fig. 3, top panel). In other words, the lipolytic sensitivity was similar for the two catecholamines. At the highest epinephrine concentration (10–5 M), a 3-fold rise in the glycerol values was registered. With norepinephrine the maximal stimulation was about 2-fold, which was seen at 10–7 M norepinephrine. In skeletal muscle there was also a concentration-dependent effect of either catecholamine on tissue glycerol. A significant elevation of dialysate glycerol was registered at 10–7 M epinephrine but first at 10–6 M norepinephrine (P < 0.0001, one-way ANOVA repeated measurements) (Fig. 3, bottom panel). Thus, the lipolytic sensitivity was 10-fold higher for epinephrine than norepinephrine. The maximum increase in glycerol was seen at 10–6 M of both catecholamines. The dialysate glycerol levels were then 2.5 and 1.6 times higher, compared with basal values, respectively. In both adipose tissue and skeletal muscle, the maximum stimulating effect on tissue glycerol was significantly higher with epinephrine than norepinephrine (P < 0.01, two-factor ANOVA repeated measurements). Thus, the lipolytic responsiveness was higher for epinephrine than for norepinephrine in both tissues.
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0.45–0.46) in adipose tissue and skeletal muscle In adipose tissue, a significant decrease in ethanol ratio, indicating an increase in local blood flow, was seen when low concentrations (10–9 to 10–8 M) of both epinephrine and norepinephrine were administrated (P < 0.05, one-way ANOVA, repeated measurements). After the nadir at 10–8 M of epinephrine and norepinephrine, the ratio returned to basal levels. In the skeletal muscle, a discrete successive elevation of the ethanol outflow to inflow ratio, i.e. a decrease in blood flow, started at 10–5 M epinephrine and the ratio was increased by 20% at 10–5 M epinephrine (P = 0.0001 by one-way ANOVA, repeated measurements). The ethanol ratio was not significantly changed when norepinephrine was administrated to the muscle. |
Discussion |
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The latter finding is in accordance with recent studies (4, 12, 14), showing that insulin possesses no appreciable antilipolytic effect in skeletal muscle. More important, in response to hypoglycemia and the endogenous catecholamine release, significant increases in I-A glycerol difference were seen in adipose tissue as well as in skeletal muscle, demonstrating a stimulation of the lipolytic activity in both tissues. In skeletal muscle, the I-A glycerol difference was approximately doubled at 35 min after the hypoglycemic nadir, which coincided with the peak in plasma catecholamine levels. It then gradually declined toward basal values although the circulating catecholamine concentrations were still significantly increased. Whether this transient increase in fractional glycerol release was the result of adrenoceptor tachyphylaxis (28, 29, 30) or due to the waning of the plasma catecholamine levels (or both) is not known. In adipose tissue, a rapid increase in the I-A glycerol difference was found in response to hypoglycemia, although plasma insulin concentrations were still elevated at levels known to maximally suppress lipolysis rates (13, 31, 32, 33). In keeping with previous reports (11, 34), this finding is probably best explained by a catecholamine-mediated stimulation of the adipose tissue lipolytic activity that overrides the antilipolytic effect of insulin.
Whereas epinephrine mainly originates from the adrenal medulla and acts via the circulation on target tissues, most norepinephrine is synthesized and released locally from sympathetic nerves. Moreover, the tissue concentration of norepinephrine is influenced by neuronal reuptake and spillover to the circulation (35). In the present study, as expected (21), the rise in circulating epinephrine in response to hypoglycemia was markedly more pronounced (>5-fold) than that in norepinephrine levels. However, in a previous study Maggs et al. (36), who used a similar hypoglycemic clamp protocol and showed almost identical increases in circulating plasma catecholamine levels as in our study, reported comparable relative increments in epinephrine and norepinephrine levels in skeletal muscle microdialysate, indicating local tissue release of norepinephrine. Therefore, in the present study, in an attempt to better differentiate the lipolytic effectiveness of the catecholamines, a wide range of epinephrine and norepinephrine concentrations were perfused in situ in skeletal muscle and adipose tissue to determine lipolytic sensitivity and responsiveness of either catecholamine. In the latter experiments, different microdialysis perfusion flow rates were used in the two tissues, to achieve similar relative recovery rates, and, hence, comparable rates of local catecholamine administration (14).
In adipose tissue, epinephrine mediated a concentration-dependent rise in glycerol levels, and the maximum response was significantly higher with epinephrine than norepinephrine; the latter being consistent with earlier reports (28, 30). However, the lowest concentration of the two catecholamines causing a significant increase in adipose tissue glycerol was the same with norepinephrine as with epinephrine, which may suggest similar lipolytic sensitivity to either catecholamine in adipose tissue. This finding is in keeping with that reported by Millet et al. (37), who observed similar lipolytic sensitivities to epinephrine and norepinephrine in sc adipose tissue in vivo. More importantly, in the present study, the lipolytic effectiveness in skeletal muscle was clearly more pronounced with epinephrine than with norepinephrine, both in terms of sensitivity and responsiveness. Thus, the increase in skeletal muscle glycerol was significantly greater and occurred at lower concentrations of epinephrine than of norepinephrine. At the highest epinephrine concentration tested, there was also a reversal of the lipolytic effect, which may have been the consequence of adrenoceptor tachyphylaxis (28, 29, 30). Hence, considered together, our data definitely show that catecholamines accelerate the lipolysis rate in skeletal muscle as in adipose tissue in vivo. Moreover, it appears that skeletal muscle lipolysis is more responsive to epinephrine than norepinephrine stimulation. This is supported by the findings that the ß2-adrenoceptor subtype predominates in skeletal muscle (16, 17) that has the greatest affinity to epinephrine (18). Our results may thus explain why increased sympathetic nerve activity and local tissue norepinephrine release induced by lower body negative pressure failed to enhance skeletal muscle lipolysis, as was reported by Navegantes et al. (15). The findings are also in keeping with our recent demonstration that systemic norepinephrine infusion had no appreciable influence on the lipolytic activity in skeletal muscle (38).
The local blood flow is also a determinant of the interstitial concentrations of glycerol (39). With regard to catecholamine stimulation, the nutritive blood flow in adipose tissue is increased by ß-adrenergic and decreased by 
-adrenergic activation (40), whereas their effects (38) on the nutritive skeletal muscle blood flow are less clear. Studies have reported both vasodilatation and vasoconstrictive reactions by the catecholamines (40, 41, 42), and the ß2-adrenoreceptor is believed to be the main adrenoreceptor subtype mediating vasodilatation (9). To measure the local blood flow, we used the ethanol perfusion technique. This method detects marked changes (>50%) in blood flow (43), and consequently minor blood flow changes might have been overlooked. Nevertheless, no major variations in the local blood flow were registered during the clamp. Hence, the observed fractional glycerol values should mainly have reflected the local lipolytic activity and not have been influenced by major variations in the local blood flow. In the in situ experiments, a transient increase in local blood flow was seen in adipose tissue in response to both epinephrine and norepinephrine. This may indicate a tachyphylactic effect of the hormones also in the vascular bed in adipose tissue. At the highest concentration, however, epinephrine mediated a decrease in local adipose tissue blood flow, which may have been the result of a transition from ß-adrenoceptor to -adrenoceptor stimulation (40). In the skeletal muscle, a discrete decrease in local blood flow by epinephrine was observed (increase of ethanol ratio). The maximal lipolytic stimulation of epinephrine in situ, however, was seen at 10–6 M of epinephrine, whereas the maximal blood flow effect was registered at higher concentrations of epinephrine. The observed increase in muscle glycerol levels could therefore not be explained by a vasoconstrictive effect of epinephrine.
The stimulatory effect of the catecholamines on the interstitial glycerol could, in theory, also reflect a decrease in glycerol reuptake by the myocyte. The enzyme that regulates this process, glycerokinase, is present in the skeletal muscle but not adipose tissue (44). Studies in rodents have shown reuse of glycerol by the myocytes (45). We indeed recently demonstrated that there is glycerol reuptake also in the human skeletal muscle, which amounted to about 40% of the corresponding release of glycerol (14). The pronounced elevation of interstitial glycerol values by catecholamine stimulation observed in this study (doubled values during hypoglycemia) can thus not possibly be explained by decreased or even eliminated glycerol reuptake. It might also be argued that the glycerol recovered by microdialysis in skeletal muscle originates from adipocytes dispersed within the muscle tissue. However, histological findings together with documented lack of adipocyte-specific proteins (46) renders this possibility unlikely. Moreover, the calculated turnover rate of triglycerides in skeletal muscle was much higher than that encountered in adipose tissue (46).
In conclusion, the results of this study have clearly demonstrated that the lipolytic activity in skeletal muscle is accelerated by hypoglycemia-induced catecholamine release. Our data also suggest that lipolysis in skeletal muscle is more responsive to epinephrine than norepinephrine stimulation.
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Acknowledgments |
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Footnotes |
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The authors have no conflict of interest.
First Published Online November 22, 2005
Abbreviations: FFA, Free fatty acid; I-A, difference between interstitial and arterial concentrations of glycerol; TG, triglyceride.
Received April 20, 2005.
Accepted November 11, 2005.
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