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Lundberg Laboratory for Diabetes Research, Departments of Internal Medicine (M.S., S.G., L.S., P.L.) and Neurophysiology (M.E.), Sahlgrenska University Hospital, Göteborg, S-413 45 Sweden; and Department of Physiology (L.C.C.N.), School of Medicine of Ribeirão Preto, University of São Paulo, 14049-900 São Paulo, Brazil
Address all correspondence and requests for reprints to: Peter Lönnroth, M.D., Ph.D., Lundberg Laboratory for Diabetes Research, Department of Internal Medicine, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden. E-mail: peter.lonnroth{at}vgregion.se.
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Abstract |
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50%, P < 0.05). Muscle glycerol was not changed by either insulin or LBNP. Glucose infusion rate during the clamp was significantly decreased during LBNP (7.82 ± 0.88 vs. 8.67 ± 1.1 ml/kg·min, P < 0.05). We conclude that the sympathetic nervous activation by LBNP results in an increased lipolysis rate in adipose tissue both in the postabsorptive phase and during insulin infusion. On the other hand, muscle glycerol output was not affected by either LBNP or insulin. The data suggest that 1) lipolysis is regulated differently in muscle and adipose tissue, 2) postabsorptive lipolysis is mainly regulated by insulin, and 3) sympathetic nervous activation effectively inhibits the antilipolytic action of insulin by inducing insulin resistance. |
Introduction |
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To further clarify the control of lipolysis in vivo, standardized models of activation and inhibition of lipolysis rate should be explored. In most such investigations, lipolysis was activated through lipolytic agents administered generally or locally or after activation by stress or exercise or after oral glucose or during an insulin infusion. Recent data from our laboratory, comparing healthy subjects with patients with cervical spinal cord injury (with a decentralized sympathetic nervous system), suggest that lipolysis in the postabsorptive state is regulated by insulin only (8, 9). In an attempt to standardize sympathetic stimulation of white adipose tissue we recently introduced intraneural electrical stimulation of the lateral cutaneous femoral nerve combined with microdialysis measurements in the sc adipose tissue innervated by the stimulated nerve fascicle, as a model to investigate neural control of lipolysis. We found that such stimulation induced a clear activation of lipolysis (70% increase) (10). Although these findings indicate a lipolytic effect of sympathetic neural activation, the possibility that intraneural electrical stimulation elicits nonsympathetic effects (i.e. via antedromic activation of afferent nerve fibers) cannot be excluded.
The primary aim of the present study was to validate the findings of the above-mentioned intraneural stimulation study using the well-established lower body negative pressure (LBNP) model for cardiopulmonary receptor-mediated sympathetic activation (11). The second aim of the study was to evaluate the effect of sympathetic activation (via LBNP) at normal and high insulin levels to gain further insight into the complex inter- and counterregulatory systems involve, and the extent to which sympathetic activation influences insulin sensitivity. A third aim was to compare the data obtained in sc adipose tissue with those in muscle during LBNP-induced sympathetic activation. Muscle cells also contain triglycerides, in amounts related to insulin resistance (12, 13). We have recently shown and it was previously shown that the interstitial concentration of glycerol in muscle, in contrast to that in adipose tissue, is not regulated by insulin (14) but does increases during local infusion of ß-adrenergic agents by means indicating a lipolytic response of microdialysis (145). However, lipolysis in the muscle may be regulated differently than in adipose tissue because the phosphodiesterase subtypes are different (15), and, furthermore, insulin does not affect muscle lipolysis (16). In the present study, we wanted to examine the effect of sympathetic neural activation on muscle glycerol.
To meet these three aims, microdialysis measurements were taken in adipose tissue and muscle, and rates of lipolysis and glucose uptake were assessed during LBNP and insulin infusion. The data show that insulin inhibits lipolysis in the postabsorptive state but does not inhibit the activation exerted by sympathetic neural activation.
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Subjects and Methods |
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Eleven lean healthy individuals (six women and five men; age range 23–54 yr; body mass index 24.2 ± 0.9 kg/m-2) participated in the study. Six of the subjects (three women and three men; age range 23–54 yr; body mass index 24.4 ± 1.4 kg/m-2) were reexamined during a euglycemic hyperinsulinemic clamp. The subjects did not take any regular medication and had abstained from smoking and alcohol for 1 and 3 d, respectively, before the experiment. The study was approved by the ethical committee of the University of Göteborg, and all participants gave their written informed consent.
LBNP during postabsorptive state
The studies began at 0800 h in a quiet room with a constant temperature of 25 C. All subjects had fasted overnight. By use of local anesthesia, a blood sampling cannula was inserted into the radial artery. Two microdialysis catheters were inserted in the abdominal periumbilical sc tissue, and two additional catheters were inserted into the brachioradialis muscle in the forearm. The subjects were placed in a supine position in the LBNP-box about 1 h before the start of the experiment. The LBNP consisted of a clear polycarbonate cylinder enclosing the legs and lower abdomen (from the iliac crests) of the subjects and a plastic foil waist seal. After a basal period of 1 h, reflex activation of the sympathetic nervous system was elicited by application of -20 mm Hg LBNP over 30 min. This was followed by a 30-min poststimulus period. Negative pressure was produced with a standard vacuum cleaner adjusted by a variable transformer. Heart rate and continuous noninvasive blood pressure were continually monitored with the volume clamp technique (Finapress, Ohmeda Monitoring Systems, Englewood, CO). Arterial blood samples were collected twice during the basal period and every 10 min after the application of LBNP until the end of the experiment. Blood samples were immediately centrifuged at 4 C, and the plasma was stored at -20 C. Plasma was analyzed for glucose, glycerol, insulin, and FFAs.
LBNP during euglycemic-hyperinsulinemic clamp
In six of the subjects, LBNP was combined with a euglycemic-hyperinsulinemic clamp. Examinations were performed as described by DeFronzo et al. (17) and modified according to the following description. A 10-min primed insulin infusion was followed by a constant insulin infusion at a rate of 60 mU/m-2·min-1 for 120 min, paralleled with glucose infusion to maintain euglycemia. Potassium chloride (0.1 mmol/liter) was infused at a rate of 10 mmol/h during the clamp to prevent hypokalemia. One hour after initiation of the clamp, blood and microdialysate sampling was conducted over a period of 1 h. LBNP was then performed in the postabsorptive state as described above.
Microdialysis
Microdialysis probes were inserted and an equilibration period of 30–45 min was allowed. The principle of microdialysis has been earlier described in detail (16, 18, 19). In the present study, catheters of single dialysis tubing (30 x 0.3 mm, Gambro, Cuprophane, 3000-molecular-weight cutoff, CMA Microdialysis, Stockholm, Sweden) were glued to nylon tubing (standardized length of 50 mm) with cyanoacrylate and sterilized and then inserted in the abdominal periumbilical sc tissue. Commercially available custom-made microdialysis catheters were used in muscle tissue (16 x 0.5 mm, 20-kDa molecular cut-off in musculi brachioradialis: CMA/10 spec. 70/16 PC, CMA Microdialysis). The inlet for the catheters was connected to a microinjection pump (CMA 100, CMA Microdialysis), and the perfusion fluid consisted of 1.5 mmol/liter glucose and 25 µmol/liter glycerol added to isotonic saline. The flow rate was 2.5 µl/min. Dialysates for glycerol and glucose measurements were collected at 10-min intervals before (two samples), during (three samples), and after (three samples) LBNP application. Calibration of the microdialysis probes was carried out using urea as an internal reference (21).
Blood flow measurements
Blood flow in the forearm was measured by venous occlusion plethysmography using a Whitney strain gauge (22). Adipose tissue blood flow was measured by the 133Xe-washout method (23). Briefly, 6–9 MBq 133Xe (Mallinchrodt, Petten, The Netherlands) in 0.1 ml sterile saline was injected into the sc adipose tissue, with one deposit in the left side and one in the right side of the umbilical region. The deposit was injected slowly over a 2-min period at least 5 mm under the skin at an angle of 45 degrees. After a 60-min decay period, radioactivity was monitored continuously with a registration every 30 sec. Adipose tissue blood flow was calculated for 30 min before, during, and after LBNP stimulation.
Analyses
Glycerol, urea, and glucose in dialysates and blood samples were determined with enzymatic fluorometric methods, using a microdialysis sample analyzer (CMA/600, CMA Microdialysis). Glucose concentrations in plasma were determined enzymatically using 10-µl samples for analysis on a YSI 2700 select biochemical analyzer (Yellow Springs Instrument Co. Inc., Yellow Springs, OH). FFA concentrations were estimated enzymatically (Wako Chemicals, Neuss, Germany). Plasma insulin was determined with a double-antibody RIA (Pharmacia, Uppsala, Sweden).
Calculations
The interstitial glycerol concentration was estimated from the dialysate concentration and recovery factor. The recovery factor is the permeability of the microdialysis catheter and is calculated from the concentration in the dialysate relative to the concentration outside the catheter. To calibrate the catheters, urea was used as a reference (the endogenous reference technique) and the relative recovery of urea in vivo was estimated from urea concentration in plasma and dialysate. Recovery of glycerol was calculated from the recovery of glycerol relative to urea in vitro (21).
Insulin sensitivity was calculated as the glucose infusion rate per minute adjusted for body weight during the clamp before LBNP (90–120 min) and during LBNP (120–150 min).
Statistics
Results are expressed as means ± SEM. ANOVA with time (two control values, three peristimulus values, and three poststimulus values) as a repeated measure factor was performed, followed by a Student-Newman-Keuls post hoc test. P < 0.05 was considered statistically significant. The SigmaStat version 1.0 (Sigma, St. Louis, MO) and StatView ANOVA programs for Windows (SAS Institute, Cary, NC) were used for all analyses.
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Results |
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In healthy subjects, after an overnight fast, sc adipose tissue and muscle interstitial glycerol levels were significantly higher than plasma glycerol (Fig. 1A
, P < 0.05). The arterial glycerol concentration during the fasting state (Fig. 1A) was significantly higher than the arterial glycerol concentration during the euglycemic-hyperinsulinemic clamp (Fig. 1B, P < 0.05). Both sc adipose tissue and muscle interstitial glucose concentrations were significantly lower than arterial plasma glucose (Fig. 2A, P < 0.05).
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Plasma insulin at steady-state obtained during the last 30 min of insulin infusion before LBNP was 612 ± 54 pmol/liter. The corresponding mean arterial plasma glucose concentrations was 5.85 ± 0.08 mmol/liter. During the same period, the glucose infusion rate was 7.8 ± 0.9 mmol/kg and per minute. Induction of insulin infusion significantly decreased glycerol concentration in plasma (49 ± 8 vs. 24 ± 5 µmol/liter, P < 0.001).
The interstitial sc glycerol level was significantly reduced during the clamp (Fig. 1B
, P < 0.05) when compared with the fasting state (Fig. 1A). There was no difference in basal muscle interstitial glycerol concentrations between fasting subjects (Fig. 1A) and subjects during the clamp (Fig. 1B).
Effect of LBNP
In the fasting state, 30 min of LBNP elicited a significant (25%) increase in sc adipose tissue interstitial glycerol concentration (Fig. 1A), whereas no significant effect was observed in skeletal muscle (Fig. 1A). At steady-state clamping conditions, LBNP evoked a similar increase (30%) in sc interstitial glycerol levels (Fig. 1B) but not in skeletal muscle (Fig. 1B). In postabsorptive conditions, interstitial-arterial concentration difference (I-A) glycerol concentration difference in sc adipose tissue was also significantly increased by LBNP. Conversely, the I-A difference of muscle glycerol was not affected (Fig. 3). No change was observed in either sc or muscle interstitial glucose concentration during LBNP, regardless of whether LBNP was combined with the euglycemic-hyperinsulinemic clamp (Fig. 2). Neither the arterial glycerol (Fig. 1) nor the arterial insulin and glucose (Fig. 2) concentrations differed significantly between basal and stimulation periods. The glucose infusion rate was significantly decreased by LBNP (P < 0.05) (8.7 ± 1.0 vs. 7.8 ± 0.9 mg/min-1·kg-1, P < 0.05).
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Table 1 shows that heart rate was increased significantly by 17% during all periods of LBNP. Systolic and diastolic blood pressure remained unchanged during the LBNP maneuver (Table 1). Neither the forearm nor the sc blood flow was affected by LBNP in the postabsorptive state (Table 1) or during euglycemic-hyperinsulinemic clamp (data not shown).
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Compared with basal levels, plasma insulin concentrations were 30% higher after 30 min of stimulation (Table 1). Basal plasma FFA concentrations were not affected at the end of the stimulation period (Table 1). During the euglycemic-hyperinsulinemic clamp, LBNP did not affect plasma FFA levels (data not shown).
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Discussion |
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The unloading of cardiopulmonary baroreceptors with LBNP is a well-established procedure to elicit sympathoexcitation, including a marked increase in sympathetic nerve discharge to skeletal muscle (25, 26), whereas an effect on sympathetic nerve discharge to skin has been questioned (27). However, both vaso- and sudomotor sympathetic fibers in cutaneous nerves are subject to baroreflex modulation (28) and a significant activation of sympathetic nerves to adipose tissue as well as skeletal muscle in humans, resulting in local release of norepinephrine, was recently demonstrated during orthostasis and LBNP maneuvers (29, 30). Previous studies using microdialysis to study sc lipolysis during unloading of cardiopulmonary baroreceptors have yielded conflicting results. Henry et al. (31) found no significant change in sc lipolysis during a 75-min, 15 mm Hg LBNP in the postabsorptive state in seven healthy subjects but a significant lipolytic effect during glucose infusion. Millet et al. (32) reported a significant sc lipolytic effect induced by a 20-min active tilt procedure in 14 healthy subjects. In the present study, application of mild nonhypotensive LBNP (simulating orthostasis) resulted in a highly reproducible increase in the arterial/interstitial glycerol concentration difference in sc adipose tissue, whereas muscle interstitial glycerol remained unaffected. Adipose tissue and muscle blood flows were not significantly affected by the procedure. Consequently, glycerol production (an index of lipolysis) was significantly increased in adipose tissue, but not in skeletal muscle, during nonhypotensive LBNP. The approximately 50% increase in adipose tissue lipolysis was surprisingly similar to that seen after exercise (23) or during electrical stimulation of regional sympathetic nerves innervating the fat tissue investigated with microdialysis (10) and during exercise (24). It should, however, be noted that LBNP also resulted in a small but significant increase in plasma insulin, which, in turn, may have influenced the lipolytic rate negatively.
Both the temporal and quantitative aspects of the LBNP-induced lipolytic effect were comparable with those achieved by intraneural electrical stimulation of regional sympathetic nerves (10), suggesting a neurally evoked lipolysis, supporting the interpretation that the lipolytic effect is mediated via sympathetic neural activation. However, concomitant adrenal activation cannot be ruled out because adrenaline spillover has been found to increase significantly during nonhypotensive 15 mm Hg LBNP (33). Also, an inhibition of parasympathetic tone in all probability contributes to the recorded heart rate response. Interestingly, a cholinergic regulation of lipolysis has recently been demonstrated to mediate an anabolic effect in fat tissue (34). Consequently, the possibility that an LBNP-induced parasympathetic inhibition may contribute to the lipolytic effect demonstrated in our study cannot be ruled out.
In six of our 11 subjects, LBNP was also applied during an insulin infusion, resulting in plasma insulin levels considered to give a maximum antilipolytic effect (35). Insulin itself induced the expected antilipolytic effect, as demonstrated by a marked decrease in plasma glycerol. However, LBNP performed during the insulin infusion elicited an activation of lipolysis similar to that seen without hyperinsulinemia, indicating that the lipolytic effect of physiological sympathoexcitation overrides the antilipolytic effect of insulin. Although this is a predicted consequence of the ß-adrenergic effect on insulin signaling, it is to our knowledge the first demonstration of the robust nature of this sympathetic lipolytic effect, in the face of a concomitant insulin-mediated activation of cAMP phosphodiesterase. The finding that a nonhypotensive 20 mm Hg LBNP elicits such marked metabolic alterations clearly indicates that results obtained in the supine position cannot readily be extrapolated to the upright position. In fact, the major role for insulin in governing lipolysis in the postabsorptive state, illustrated in recent experiments in subjects with spinal cord injury who lacked normal neural control of major adipose tissue deposits (8, 9), may be limited to resting supine conditions.
In contrast to the results in adipose tissue, muscle interstitial glycerol did not change during LBNP. We have previously shown that the discovery of interstitial muscle glycerol concentrations higher than those in arterial plasma does not lead to any clear conclusions about the lipolysis rate in either plasma or muscle (16). In fact, the brisk turnover of muscle glycerol renders it inadequate as a quantitative index for lipolysis (36, 37). Furthermore, recent research has shown that glycerol turnover rate varies considerably in different muscle regions (38). Insulin does not exert a clear antilipolytic effect on the glycerol concentration in the muscle (16), whereas local application of lipolytic agents to muscle leads to increased muscle glycerol levels (14). The present data obtained during LBNP suggest that physiological activation of sympathetic nerves does not lead to local concentrations of norepinephrine high enough to increase muscle interstitial glycerol. Consequently, although the turnover of muscle triglycerides has been shown to be related to insulin resistance (12, 13), the value of muscle glycerol measurements is limited by the fact that this variable does not seem to be regulated by insulin or by sympathetic nerves. Further studies including the measurements of muscle glycerol turnover are therefore warranted.
LBNP applied in the postabsorptive state resulted in a small increase in plasma insulin, whereas the interstitial glucose levels in adipose tissue and muscle as well as in plasma remained unaltered. The regional blood flows in adipose tissue and muscle were not significantly affected by LBNP. Hence, glucose uptake was constant despite the small increase in plasma insulin, indicating that LBNP induced a small reduction in peripheral insulin sensitivity. During insulin infusion, LBNP resulted in a small decrease in glucose infusion rate, further suggesting that insulin resistance was induced. These data confirm previous investigations demonstrating that LBNP, even at mild levels, induces insulin resistance (39). The mechanisms underlying LBNP-induced insulin resistance may include multiple steps of action. ß- Adrenergic stimulation leads to a reduced number of insulin receptors in the plasma membrane (40), inhibition of the insulin receptor tyrosine kinase, and reduced activity of glucose transporters in the plasma membrane (6). Furthermore, the increased rate of lipolysis may result in increased lipid oxidation and a decreased glucose oxidation rate (42).
Limitations of the study
Highly reproducible data led us to limit the demanding hyperinsulinemic clamp phase of our protocol to six of our 11 subjects. The investigation was also limited to the study of short-term LBNP in the postabsorptive state and during an insulin infusion. The interaction between sympathetic activation and insulin should be further elucidated by studying the effect of insulin during long-term LBNP because long-term sympathetic activation and ß-adrenergic stimulation may down-regulate the lipolytic response (43).
In conclusion, the present study shows that physiological sympathoexcitation evoked by nonhypotensive LBNP results in increased lipolysis and resistance to the antilipolytic effect of insulin in sc adipose tissue. Conversely, muscle glycerol release remains unaffected.
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Footnotes |
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Abbreviations: FFA, Free fatty acid; I-A, interstitial-arterial concentration difference; LBNP, lower body negative pressure.
Received March 13, 2003.
Accepted July 28, 2003.
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) and interstitial glycerol concentrations in sc adipose tissue (
) and skeletal muscle (•) before, during, and after LBNP at -20 mm Hg for 30 min in 11 fasting subjects (A) and in six subjects during euglycemic-hyperinsulinemic clamp (B). **, P < 0.01; *, P < 0.05 LBNP vs. basal period. Black bars indicate stimulation period.
