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Original Studies |
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
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: ch.gravholt{at}dadlnet.dk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 5); and, in man, pharmacologic levels of glucagonemia have been shown to induce lipolysis (6, 7, 8). In contrast, human experiments using physiological hyperglucagonemia (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 pituitary-pancreatic clamp, using different levels of glucagonemia and glycemia. Lipolysis was evaluated by measurement of glycerol with indwelling microdialysis catheters in abdominal adipose tissue.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 in adipose tissue
[
200 ± 40 µmol/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 µg/h), insulin (0.06 mU/kg·min), and GH (2 ng/kg·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·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·min).
Protocol III (EG with hyperglycemia, EG+G). Again, insulin and GH were clamped with the infusion of somatostatin (450 µg/h), insulin (0.06 mU/kg·min), and GH (2 ng/kg·min) throughout the study period. At t = 0 min, an infusion of glucagon (0.5 ng/kg·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·min) and glucagon (EG, 0.6 ng/kg·min; HG, 1.8 ng/kg·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 µL/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, 14, 15). Baseline values for glycerol obtained in the present study correspond closely with the ones available in the literature (15, 16, 17, 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 x 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·
·100 (mL/100
g·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·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 µg/L, and the detection limit was 0.01 µg/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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)
Before infusion of somatastatin levels of insulin (45 pmol/L),
C-peptide (480 pmol/L), glucagon (65 ng/L), and GH (0.25
µg/L) were comparable in the three situations. Endogenous insulin
release, as assessed by serum C-peptide, 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 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.
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)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 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.
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)
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 situations. 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) µmol/L·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) µmol/L·120 min, ANOVA,
P = 0.98].
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Abdominal tissue blood flow was identical in all three situations (results not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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, 14, 15). The observed baseline values for glycerol obtained in the present study correspond closely to the ones available in the literature (15, 16, 17, 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·min in the present study) and GH (4 vs. 2 ng/kg·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·min; HG, 1.3 vs. 1.5 ng/kg·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 µg) 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 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.
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Acknowledgments |
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Footnotes |
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Received September 7, 2000.
Revised January 10, 2001.
Accepted January 12, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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