0021-972X/01/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2001 by The Endocrine Society
Vol. 86, No. 5 Printed in U.S.A.
Physiological Levels of Glucagon Do Not Influence Lipolysis in Abdominal Adipose Tissue as Assessed by Microdialysis* CLAUS HØJBJERG GRAVHOLT, NIELS MØLLER, MICHAEL D. JENSEN, JENS SANDAHL CHRISTIANSEN, AND OLE SCHMITZ Department of Endocrinology M and Medical Research Laboratories (C.H.G., N.M., J.S.C., O.S.), Århus University Hospital, DK-8000 Århus C, Denmark; and Endocrine Research Unit (M.D.J.), Mayo Clinic, Rochester, Minnesota 55905 ABSTRACT To determine whether glucagon stimulates lipolysis in adipose tissue, seven healthy young male volunteers were studied, with indwelling microdialysis catheters placed sc in abdominal adipose tissue. Subjects were studied three times: 1) during euglucagonemia (EG; glucagon infusion rate, 0.5 ng/kg䡠min); 2) during hyperglucagonemia (HG; (glucagon infusion rate, 1.5 ng/kg䡠min); and 3) during EG and a concomitant glucose infusion mimicking the glucose profile from the day of HG (EG⫹G). Somatostatin (450 g/h) was infused to suppress hormonal secretion, and replacement doses of insulin and GH were administered. Sampling was done every 30 min for 420 min. Baseline circulating values of insulin, C-peptide, glucagon, GH, glyc-
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DISTURBED PANCREATIC islet function is the main pathophysiological feature of type I diabetes mellitus. In addition to insulin deficiency (absolute or relative), hypersecretion of glucagon is present. The glucagon excess is most pronounced in poorly controlled diabetes mellitus, but circulating levels are raised, even in well-controlled diabetes (1). In particular, the postprandial suppression of glucagon is reduced in diabetes mellitus (2, 3). Glucagon increases endogenous glucose release and, consequently, blood glucose. The glucoregulatory role of glucagon (modifying glycogenolysis, gluconeogenesis, and glycogen synthesis) is well established, and glucagon also increases amino acid degradation and inhibits protein synthesis (4). However, a possible role of glucagon on lipid metabolism in human physiology and pathophysiology still remains to be clarified. In in vitro experiments, glucagon has been shown to augment lipolysis in human adipose tissue (for review, see Ref. 5); and, in man, pharmacologic levels of glucagonemia have been shown to induce lipolysis (6 – 8). In contrast, human experiments using physiological hypergluReceived September 7, 2000. Revision received January 10, 2001. Accepted January 12, 2001. Address all correspondence and requests for reprints to: Claus Højbjerg Gravholt, M.D., Department of Endocrinology M, Århus Kommunehospital, Århus University Hospital, DK-8000 Århus C, Denmark. E-mail:
[email protected]. * This study was supported by a grant from the Danish Diabetes Association, Kong Christian den Tiender Fond, and by the Danish Health Research Council, Grant 9600822 (Århus University—Novo Nordisk Center for Research in Growth and Regeneration). The study was presented, in part, at the annual meeting of the European Association for the Study of Diabetes, Bruxelles, Belgium, September 28 –October 2, 1999.
erol, and free fatty acids were comparable in all three conditions. During EG and EG⫹G, plasma glucagon was maintained at fasting level (20 – 40 ng/L); whereas, during HG, it increased (110 –130 ng/L). Interstitial concentrations of glycerol were similar in the three conditions [30,870 ⫾ 5,946 (EG) vs. 31,074 ⫾ 7,092 (HG) vs. 29,451 ⫾ 6,217 (EG⫹G) mol/L䡠120 min, P ⫽ 0.98]. Plasma glycerol (ANOVA, P ⫽ 0.5) and free fatty acids (ANOVA, P ⫽ 0.3) were comparable during the different glucagon challenges. We conclude that HG per se does not increase interstitial glycerol (and thus lipolysis) in abdominal sc adipose tissue; nor does modest hyperglycemia, during basal insulinemia and glucagonemia, influence indices of abdominal sc lipolysis. (J Clin Endocrinol Metab 86: 2085–2089, 2001)
cagonemia (HG) have yielded conflicting results. Employing the pancreatic clamp technique Jensen et al. (9) found that glucagon infusion had no (or only minor) effects on palmitate fluxes in both healthy volunteers and in type 1 diabetic patients. This is, however, in contrast with a later study using a comparable design (10). It cannot be excluded that this discrepancy, at least in part, can be explained by differences in glycemia. It is of note, however, that the effects of glucagon on the lipid metabolism observed in vivo have been studied only in a compartment remote from adipose tissue, namely blood. Consequently, the present study was undertaken to examine the possible influence of physiological levels of glucagon on fat metabolism in the primary target tissue. This was done by using the recently introduced microdialysis technique, which allows measurement of interstitial levels of low-weight molecular compounds (such as glucose, glycerol, and lactate) by perfusion of a dialysis catheter in the tissue of interest (11). The method is useful for detecting metabolic changes after endogenous, as well as exogenous, stimuli. Healthy male volunteers were studied, during a pituitarypancreatic clamp, using different levels of glucagonemia and glycemia. Lipolysis was evaluated by measurement of glycerol with indwelling microdialysis catheters in abdominal adipose tissue. Materials and Methods Subjects A power analysis was performed to determine the sample size, based on the findings by Carlson et al. (10). They found that HG increased glycerol rate of appearance by approximately 40%. Based on an expected increase in the level of glycerol (index of lipolysis) of 30% above basal
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in adipose tissue [⬃200 ⫾ 40 mol/L (mean ⫾ sd)] during HG, we determined that the sample size should be seven individuals, with a power of more than 80% and ␣ ⫽ 0.05. Seven young, lean, healthy males [age, 26.7 ⫾ 1.1 yr (range, 24 –32; mean ⫾ se); weight, 80.4 ⫾ 3.6 kg (range, 65.0 –96.7); and body mass index, 24.7 ⫾ 1.0 kg/m2 (range, 19.8 –29.2)] gave their written informed consent after receiving oral and written information concerning the study according to the Declaration of Helsinki II. The study was approved by the Aarhus County Ethical Scientific Committee.
Experimental protocol Subjects were admitted to the Clinical Research Center in the morning, after an overnight fast (10 –12 h) without any caffeine consumption or cigarette smoking; only ingestion of tap water was allowed. Participants were asked not to perform major physical exercise and to consume a weight-maintaining carbohydrate-rich diet for the last 3 days before examination and to refrain from alcohol intake on the day before investigation. Participants were placed in the supine position in a bed, in light clothes, at room temperature (approximately 22–24 C), and they remained there throughout the study. One iv catheter (Viggo AB, Helsingborg, Sweden) was placed in an antecubital vein for infusion, and another in a vein draining a hand that was heated in a box with an air temperature of approximately 65 C to provide arterialized blood. Blood samples were drawn every 30 min throughout the study period, starting at t ⫽ ⫺150 min. Interstitial levels of metabolites were sampled, every 30 min, from the abdominal sc adipose tissue (see below). Each subject was studied three times; protocol I and II were performed in random order, with at least a 2-week interval. Protocol I (euglucagonemia, EG). Hormone levels were clamped with the infusion of somatostatin (450 g/h), insulin (0.06 mU/kg䡠min), and GH (2 ng/kg䡠min) from t ⫽ ⫺120 min and throughout the study period (t ⫽ 270 min). At t ⫽ 0 min, an infusion of glucagon (0.5 ng/kg䡠min) was started, and it continued until the end of the study. Protocol II (HG). This was identical to protocol I, apart from a higher glucagon infusion rate (1.5 ng/kg䡠min). Protocol III (EG with hyperglycemia, EG⫹G). Again, insulin and GH were clamped with the infusion of somatostatin (450 g/h), insulin (0.06 mU/kg䡠min), and GH (2 ng/kg䡠min) throughout the study period. At t ⫽ 0 min, an infusion of glucagon (0.5 ng/kg䡠min) was started, and it continued until the end of the study. Glucose was infused throughout the study period to simulate the glucose profile from the day of HG (protocol II). In all three protocols, the initial three subjects were infused with insulin (0.05 mU/kg䡠min) and glucagon (EG, 0.6 ng/kg䡠min; HG, 1.8 ng/kg䡠min); but because of a tendency for the glucose level to rise during the latter part of the study period during protocols I and II, the insulin dose was increased, and the glucagon dose was decreased (see description above). No untoward clinical events occurred.
Microdialysis A microdialysis catheter (CMA 60, CMA, Stockholm, Sweden) was placed in the abdominal sc adipose tissue after anesthetization of the skin with 0.05 mL lidocaine at the site of perforation of the skin. The microdialysis catheter used has a molecular cut-off of 20 kDa. Immediately after placement, perfusion of the catheters with physiological perfusion fluid (perfusion fluid T1, CMA; Na⫹, 147 mmol/L; K⫹, 4 mmol/L; Ca2⫹, 2.3 mmol/L; Cl⫺, 156 mmol/L, pH, 6; osmolality, 290 mosmol/kg), at a flow rate of 0.3 L/min, with the use of a portable pump (CMA 106, CMA), was accomplished. At this flow rate, the rate of recovery with the microdialysis catheter is almost 100% (12). The microdialysis catheter was placed at t ⫽ ⫺210 min. After an hour of calibration with perfusion of the microdialysis catheter, allowing local edema and hemorrhage to subside, sampling started at t ⫽ ⫺150 min and continued, every 30 min, until t ⫽ 270 min. The first sample was thus withdrawn at t ⫽ ⫺120 min. This sample reflects the integrated level of interstitial glucose during the preceding 30 min, and the sample was assigned the time t ⫽ ⫺135 min. This principle was used for all samples. The observed changes in interstitial glycerol concentration can be seen as an index of lipolysis (13–15). Baseline values for glycerol obtained in
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the present study correspond closely with the ones available in the literature (15–18).
Blood flow measurements The sc adipose tissue blood flow (ATBF) in the abdominal region in which microdialysis was performed was measured by the local 133Xe washout method (19). In short 3.7 MBq (0.1 mL) 133Xe was injected into the sc area of interest, equivalent to a whole-body radiation dose of 0.5 mSv. Disappearance of 133Xe was monitored with a 2 ⫻ 2- inch NaI detector (model 905) connected to a photomultiplier base (model 276) (EG&G Ortec, Wokingham, Berks, UK) covered by a cylindrical copper collimator and coupled to a multichannel AceMate (model 925) amplifier (EG&G Ortec). The system was connected to a computer for simultaneous sampling. The registration of the washout rate was started at least 30 min after the injection. ATBF was calculated as follows: ATBF ⫽ k䡠䡠100 (mL/100 g䡠min), where k is the rate constant of the washout curve and is the tissue to blood partition coefficient for 133Xe at equilibrium; counts were collected every minute, and a straight line was fitted through the experimental points in a semilogarithmic diagram as a function of time. Experimental values of k were determined as the slope of the regression line during a specified T, where T is the time frame (min). The time interval was at least 15 min. was calculated as follows: ⫽ 0.22䡠SFT ⫹ 2.99, where SFT is the skin-fold thickness of the abdominal adipose tissue (19, 20).
Assays Plasma glucose was measured immediately after sampling, in duplicate, on an autoanalyzer (Beckman Coulter, Inc., Palo Alto, CA), by the glucose oxidase method. The autoanalyzer was calibrated frequently, with known human plasma standards, as well as standards supplied by the company with the equipment; and the intraassay coefficient of variation (CV) was below 0.5%. GH was measured with a double monoclonal immunofluorometric assay (DELFIA, Wallac, Inc. Oy, Turku, Finland). The interassay CV in samples varied between 1.7 and 2.4%, the intraassay CV varied between 1.9 and 3.0% for GH concentrations of 12.08 and 0.27 g/L, and the detection limit was 0.01 g/L. Serum insulin was measured by enzyme-linked immunosorbent assay employing a two-site immunoassay (21). The intraassay CV was 2.0% (n ⫽ 75) at a serum level of 200 pm. Serum free fatty acids (FFA) was determined by a colorimetric method employing a commercial kit (Wako Pure Chemical Industries Ltd., Neuss, Germany). Blood samples were deproteinized with perchloric acid for determination of alanine, glycerol, 3-hydroxybutyrate, and lactate, and were assayed by an automated fluorometric method (22). Plasma glucagon was measured by an RIA (23). Glycerol, glucose, and lactate in the dialysate were measured by an automated spectrophotometric kinetic enzymatic analyzer (CMA 600, CMA).
Statistical analysis All statistical calculations were done with SPSS for Windows, version 8.0 (SPSS, Inc., Chicago, IL). Area under the curve for hormone and metabolites values was calculated according to the trapezoidal rule. Data were subsequently examined by one-way ANOVA. Post hoc analysis (Student-Newman-Keuls) was used. Results are expressed as mean ⫾ se. Significance levels under 5% were considered significant.
Results Circulating hormones (Fig. 1)
Before infusion of somatastatin levels of insulin (⬃45 pmol/L), C-peptide (⬃480 pmol/L), glucagon (⬃65 ng/L), and GH (⬃0.25 g/L) were comparable in the three situations. Endogenous insulin release, as assessed by serum Cpeptide, was suppressed to insignificant values till t ⫽ 210 min, after which a minor breakthrough occurred in the studies where plasma glucose rose. Of notice, even during the breakthrough, serum C-peptide is less than one third of basal values (⬃450 –500 pmol/L in all three protocols). A similar
EFFECTS OF GLUCAGON IN ADIPOSE TISSUE
FIG. 1. A, Glucagon vs. time during the study; B, insulin vs. time during the study; C, C-peptide vs. time during the study; D, GH vs. time during the study. Throughout, filled circles indicate EG, open circles indicate HG, and gray triangles indicate EG with hyperglycemia (EG⫹G). Error bars, SEM.
trend was observed for serum insulin. ANOVA did not reveal statistically significant differences, among the three conditions, for either C-peptide or insulin. Serum GH was maintained constant at baseline levels in all three protocols. During protocols I and III, plasma glucagon was maintained stable at basal level, whereas glucagon increased to levels of 110 –130 ng/L in the protocol with a high glucagon infusion rate. Plasma and interstitial glucose (Fig. 2)
Plasma glucose rose slowly from 4 to approximately 6 mmol/L in the latter part of the study in the EG condition. During HG and EG⫹G, plasma glucose reached levels of 11 mmol/L at t ⫽ 150 min, and then subsequently trailed off to levels around 9 mmol/L by the end of the study. The fasting steady-state interstitial glucose levels were approximately 30% lower than in plasma, whereas comparable peaks were
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FIG. 2. A, Plasma glucose vs. time; B, interstitial glucose vs. time. Throughout, filled circles indicate EG, open circles indicate HG, and gray triangles indicate EG with hyperglycemia (EG⫹G). Error bars, SEM.
reached in interstitial levels. The fluctuations observed in interstitial glucose closely paralleled the changes in plasma. However, there was a slight time delay in the dynamics, reflecting (in part) the fact that interstitial measurements are integrated over 30 min. Circulating and interstitial lipid intermediates (Fig. 3)
Baseline values of interstitial glycerol, plasma glycerol, and serum FFA were similar (data not shown). There was no change in levels of interstitial glycerol, plasma glycerol, and serum FFA during the period where only somatostatin, insulin, and GH were infused (t ⫽ ⫺120 to 0 min) (data not shown). During glucagon infusions, levels of plasma glycerol (for entire study period: ANOVA, P ⫽ 0.4; for t ⫽ 120 to 270 min: ANOVA, P ⫽ 0.5), and FFA (for entire study period: ANOVA, P ⫽ 0.1; for t ⫽ 120 to 270 min: ANOVA, P ⫽ 0.3) were comparable (Fig. 3). Furthermore, post hoc analysis failed to demonstrate any difference among the three situ-
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FIG. 3. A, Plasma glycerol vs. time; B, interstitial glycerol in vs. time; C, serum FFA vs. time. Throughout, filled circles indicate EG, open circles indicate HG, and gray triangles indicate EG with hyperglycemia (EG⫹G). Error bars, SEM.
ations. During the three protocols, levels of interstitial adipose glycerol were not different during the entire study period [64,856 ⫾ 22,461 (EG) vs. 63,452 ⫾ 28,575 (HG) vs. 58,018 ⫾ 15,423 (EG⫹G) mol/L䡠390 min, ANOVA, P ⫽ 0.9], or during the period of maximal stimulation by glucagon [30,870 ⫾ 5,946 (EG) vs. 31,074 ⫾ 7,092 (HG) vs. 29,451 ⫾ 6,217 (EG⫹G) mol/L䡠120 min, ANOVA, P ⫽ 0.98]. Abdominal ATBF
Abdominal tissue blood flow was identical in all three situations (results not shown). Discussion
The present study shows that physiological HG does not affect lipolysis as assessed either by serum FFA and glycerol or by interstitial glycerol in abdominal sc adipose tissue. The participants were examined under circumstances with high, as well as basal, physiological levels of glucagon; and, be-
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cause other hormones influence lipolysis, it was important to control insulin and GH, employing the pituitary-pancreatic clamp technique. Because hyperglycemia might possibly confound the result, it was also necessary to examine the subjects in a third protocol, mimicking the glucose profile from the protocol with high physiological levels of glucagon. In none of the protocols was there any trend toward an effect of glucagon per se on lipolysis, as assessed by interstitial glycerol. Microdialysis allows continuous monitoring of changes of fluxes of a variety of compounds, from interstitial fluid to the dialysate, and it has been used in a large number of tissues in the human body since it was first introduced (24). True equilibrium can be accomplished across the membrane when a very low flow rate is used (18). Thus, to the extent that tissue disposal rates are constant, the changes in interstitial glycerol concentration can be seen as an index of lipolysis, because glycerol is only produced and not taken up by adipose tissue (13–15). The observed baseline values for glycerol obtained in the present study correspond closely to the ones available in the literature (15–18). The results are in agreement with two previous studies performed in humans (9, 25); in both of these studies, the hyperglycemia caused by high physiological levels of glucagon could confound interpretations. In a more recent study exploring the effect of glucagon on lipolysis in humans, Carlson et al. (10), with an approach comparable with the two above-mentioned studies, found that physiological HG did, in fact, increase levels of FFA, palmitate, and rate of appearance of glycerol. Furthermore, they found that hypoglucagonemia did decrease the same lipolytic variables in comparison with normoglucagonemia. The results are clearly in contrast to ours, despite the fact that the experimental design of the present study and that by Carlson et al. are very similar. However, Carlson et al. used higher replacement doses of insulin (0.10 vs. 0.06 mU/kg䡠min in the present study) and GH (4 vs. 2 ng/kg䡠min in the present study), and there were minor differences in the infusion rate of glucagon (low glucagon, 0.65 vs. 0.50 ng/kg䡠min; HG, 1.3 vs. 1.5 ng/kg䡠min in the present study). We studied lipolysis in abdominal sc adipose tissue and whole-body circulating levels of lipolytic markers, whereas Carlson et al. studied whole-body turnover with tracers. Also, we performed a power analysis, based on the findings by Carlson et al., expecting to find at least a 30% difference in lipolysis, and thus studied seven individuals. Nevertheless, the profound differences in the conclusions of the two studies are difficult to reconcile. We noted, however, that during the time of HG in the Carlson study, the levels of insulin, a potent inhibitor of lipolysis, were indeed 10% lower than during the time of EG, perhaps explaining the difference in lipolysis. Likewise, a similar difference was noted between the hypoglucagonemia and the EG study (10). Recently, we demonstrated that even small changes in interstitial glycerol concentrations can be detected with microdialysis. We were able to show that a small bolus of GH (200 g) did, in fact, produce a persistent and robust lipolytic response (26). Likewise the lipolytic potential of catecholamines (27) has been demonstrated with the use of microdialysis. Thus, the methodology does allow for a dynamic study of the aspects of local lipolysis, even when the
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lipolytic agent is present only in low doses. Because of the small numbers studied, a type 2 error can, of course, not be excluded; and thus, glucagon may have a lipolytic effect, albeit small. Here, we also showed that hyperglycemia failed to affect the concentration of glycerol in abdominal adipose tissue and on a whole-body level, the latter in accordance with earlier studies (28). We do not suspect that the absence of lipolytic response was attributable to the use of somatostatin to suppress endogenous secretion of pituitary and pancreatic hormones, because previously we have shown that lipolysis is unaffected by somatostatin (29). In conclusion, we have shown that glucagon does not increase concentrations of lipid intermediates in abdominal adipose tissue or in the circulation. This seems compatible with the notion that glucagon is devoid of any effects on lipolysis. Acknowledgments We thank Anette Mengel for expert technical help. The microdialysis catheters and other utensils were generously supplied by Roche Diagnostics GmBH (Mannheim, Germany).
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