1 Karolinska Institute, Department of Endocrinology and Diabetology, Karolinska Hospital and 2 Department of Medicine, Endocrinology Section, Stockholm Söder Hospital, Stockholm, Sweden
* Author to whom correspondence should be addressed at: Department of Endocrinology and Diabetology, Karolinska Hospital, 117 76 Stockholm, Sweden. E-mail: jan.calissendorff{at}ks.se
(Received 10 June 2003; in revised form 22 October 2003; accepted 13 January 2004)
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ABSTRACT |
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INTRODUCTION |
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SUBJECTS AND METHODS |
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Protocol
Each individual took part in two experiments (A and B) which were performed in a metabolic ward, in random order, 1 week apart.
Experiment A. At 07:30 hours a catheter was inserted into one of the antecubital veins and kept patent by a slow drip of normal saline. After an equilibration period of 30 min, basal blood samples were collected from the catheter. At 08:00 hours placebo was given orally. Then three identical 150-ml doses of alcohol were ingested at 09:00, 10:30 and 12:00 hours. Each dose contained 0.45 g ethanol/kg b.w. The alcohol concentration in the solutions ranged between 16.5 and 25.6%/vol. (mean ± SEM: 20.5 ± 1.2%/vol.). A second oral placebo dose was given at 12:00 hours.
Experiment B. In this experiment propranolol was substituted for placebo; 40 mg propranolol were ingested at 08:00 hours and an additional 20 mg at 12:00 hours. In all other details, experiment A and B were identical.
In both experiment A and B, pulse rates were recorded and serum concentrations of insulin, IGF-1 and leptin determined immediately before the first dose of placebo/propranolol (08:00 hours), and subsequently at 09:00, 11:00, 13:00 and 15:00 hours. Serum ethanol levels were analysed at intervals, as shown in Fig. 1. Adrenaline and noradrenaline excretion by the urine over a period of 7 h (08:0015.00 hours) was also determined.
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Statistical analysis and calculations
To test whether placeboalcohol would have different effect than that of propranololalcohol on pulse rates and serum levels of ethanol, insulin, IGF-1 and leptin, we used a two-way ANOVA for repeated measures followed by Turkey's post-hoc test.
Percentage changes of pulse rates and leptin concentrations from baselines were also determined (Figs 1,4, left panels). These changes measured over time were used to calculate areas under the curve (AUC). AUC obtained in experiment A (AUC-A), were combined as means, and compared with those obtained in experiment B (AUC-B). The means ± SEM are shown in Figs 1 and 4 (right panels). Serum ethanol increments over time were also calculated and presented as AUC (Fig. 1, right panel). However, the ethanol AUC were based on absolute serum ethanol changes over time, not on percentage changes. To determine whether AUC-A differed significantly from AUC-B, Student's t-test was applied (paired differences). This test was also used to compare CA excretion values of experiment A and experiment B. P-values of <0.05 were considered significant.
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RESULTS |
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Pulse
When alcohol was consumed after administration of placebo (experiment A) the pulse rate increased significantly (P < 0.02 at 11:00 hours and P < 0.001 at 15:00 hours). The pulse-AUC-A0815 (reflecting the percentage change of the pulse rate over time) was 103.5 ± 39.1.
Propranolol, given alone in the initial part of experiment B, lowered the pulse rate by 14 ± 4% at 09:00 hours (from 68 ± 4 beats/min at 08:00 hours to 58 ± 3 beats/min at 09:00 hours; P < 0.05). When alcohol was ingested after propranolol priming, the pulse rate did not increase as in experiment A; it tended to stay low for at least 4 h and did not reach levels above basal until the end of the experimental period (Fig. 1, left panel). This was reflected by the percentage change of the pulse rate AUC-B0815 which appeared below the baseline (Fig. 1, right panel). It differed significantly from the corresponding area in experiment A (52.8 ± 25.9 vs. 103.5 ± 39.1; P < 0.01; Fig. 1, right panel).
Serum ethanol
In experiment A the serum ethanol level increased from 0 mmol/l at 09:00 hours to a maximum of 28.1 ± 0.8 mmol/l at 15:00 hours. The ethanol concentration curve was very similar in experiment B (Fig. 1, left panel). Consequently, the ethanol-AUC0915 did not differ significantly in the two experiments (Fig. 1, right panel).
Urinary adrenaline
The urine excretion of adrenaline between 08:00 and 15:00 hours was 27.4 ± 5.0 nmol/7 h in experiment A and 28.2 ± 7.4 nmol/7 h in experiment B. These values did not differ significantly (Fig. 2).
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Serum insulin
Basal serum insulin concentrations were similar in experiments A and B (5.3 ± 1.2 and 4.6 ± 1.0 mU/l, respectively). The small subsequent changes in insulin level were not significantly different in the two experiments (Fig. 3).
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Serum leptin
Both experiments presented similar serum leptin concentrations at 08:00 hours (7.1 ± 2.0 µg/l for A, 6.5 ± 1.5 µg/l for B). Also, at 09:00 hours similar serum leptin concentrations were recorded in the two experiments (6.8 ± 2.0 and 6.3 ± 1.5 µg/l, respectively). After ingestion of alcohol at 09:00 hours, the leptin level declined significantly in both experiments. The percentage leptin decline between 09:00 and 15:00 hours was 28.6 ± 5.4% in experiment A and 29.0 ± 2.9% in experiment B (Fig. 4, left panel). When the total leptin decline over a 6-h period (09:0015:00 hours) was expressed by a percentage decremental area under the curve (AUC0915), the AUC obtained in the two experiments were not significantly different, as shown in Fig. 4 (right panel).
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DISCUSSION |
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Leptin, like many other hormones, is secreted rhythmically throughout the day. High serum leptin concentrations prevail in the early morning and low serum leptin concentrations prevail around mid-day (Saad et al., 1998). This means that the declining leptin level, which was found in the present investigation after ingestion of alcohol, could reflect a normal secretory profile of leptin, rather than an alcohol-induced leptin inhibition. However, this appears unlikely for the following reason. We recently studied healthy subjects of both sexes who, in almost all respects, were comparable to those included in the present investigation (Röjdmark et al., 2001
). When these individuals were given three oral doses of water in the morning their serum leptin levels declined by 22.7 ± 3.3%. After ingestion of three oral doses of alcohol the corresponding leptin decline was 30.3 ± 3.9% (P < 0.05). This leptin decline after alcohol was in close accordance with that found in both experiments A and B (a leptin decline of
29%). For that reason we believe that alcohol has an inhibitory effect on human leptin secretion. This assumption is further supported by the fact that alcohol also appears to inhibit leptin secretion during the night (Röjdmark et al., 2001
), when serum leptin levels normally tend to increase (Sinha et al., 1996
).
If CA can be excluded from the list of plausible mediators of the alcohol effect, several other should be considered. Insulin, cortisol, testosterone and IGF-1, are all plausible mediators, as all have the potential of influencing leptin secretion (Williams et al.; Malmström et al., 1996; Larsson and Ahrén, 1996
; Wabitsch et al., 1997
; Dagogo-Jack et al., 1998
; Nyomba et al., 1999
). However, these plausible mediators were scrutinized in our previous study and none was found to mediate the leptin-inhibitory effect of alcohol (Röjdmark et al., 2001
). Insulin and IGF-1 levels were also determined in the present investigation. Neither of them changed noticeably after ingestion of alcohol. This finding thus supports our previous observations.
It may be said that our results are at variance with those reported by Nicolas et al. (2001). They found increased serum leptin levels in chronic alcoholics, regardless of nutritional status or presence of liver cirrhosis. The discrepancy between their findings and ours is unexplained, but different disposal of leptin has to be considered. It has been maintained that the splanchnic organs and the kidneys cooperate in the disposal of leptin. By use of an arteriovenous technique, Garibotto et al. (1998)
were able to determine not only the fractional splanchnic extraction, but also the fractional renal extraction of leptin in nonobese subjects. The splanchnic extraction was 16% and the corresponding renal extraction 9.5%, but only small quantities of native leptin were found in the urine. Although this implies that leptin is metabolized within the kidneys, Garibotto's findings suggest that more leptin may be metabolized in the normal liver than in the kidneys. In patients with liver insufficiency, this may not be the case, as such patients probably metabolize less leptin in the liver than do healthy subjects. If so, this could, at least in part, explain why chronic alcoholics appear to have higher serum leptin levels than healthy individuals.
Ghrelin is also of interest in this context, as this hormone stimulates NPY (Toogood and Thorner, 2001; Cowley et al., 2003
), and increases the intake of food (Wren et al., 2000
). It is not known whether alcohol stimulates the secretion of ghrelin. If so, it still remains to be investigated how such alcohol-induced ghrelin secretion affects human adipocytes. Until such studies have been performed, we are left with the fact that alcohol decreases serum leptin levels. This effect does not appear to be indirect. It may be caused by direct inhibition of adipocytes, but changed hepatic and/or renal disposal of leptin are other possibilities that require further investigation.
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ACKNOWLEDGEMENTS |
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