INTRODUCTION

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THE PRO-GLUCAGON-DERIVED glucagon-like peptide-(736) amide (GLP-1) is a gastrointestinal hormone that is released in response to food intake from the distal small intestine (3, 8). Its biological effects include a glucose-dependent insulinotropic effect on the pancreatic B cell and inhibition of gastric emptying. The last effect can be interpreted as being part of the "ileal break mechanism," an endocrine feedback loop that is activated by nutrients in the ileum (8, 9). It is well accepted that nutrients in the ileum not only inhibit upper gastrointestinal functions, but also affect appetite and food intake (16, 17). Along these lines of investigation, GLP-1 has been proposed to play a physiological regulatory role in controlling appetite and energy intake in humans (4, 7) and animals (14, 15). Because of its various biological effects, GLP-1 is currently being considered as a therapeutic agent in the treatment of the hyperglycemia of diabetes mellitus type 2 (1, 6, 10). Because many patients with diabetes mellitus type 2 are also overweight, we wondered whether intravenous GLP-1 could also inhibit energy intake in these patients. On the basis of this information, the present study was designed to investigate in a randomized, double-blind, crossover fashion the effects on appetite perception and energy intake in an ad libitum meal of GLP-1 infused intravenously at a rate that effectively reduces hyperglycemia in diabetes mellitus type 2 (11).

	METHODS AND PARTICIPANTS

TOP ABSTRACT INTRODUCTION METHODS AND PARTICIPANTS RESULTS DISCUSSION REFERENCES

Twelve male patients were recruited for the study who had diabetes mellitus type 2 for at least 1 year. Patients were admitted to the research unit after giving written informed consent and after the experimental protocol was explained to them. The mean age of the patients was 55 ± 2 yr; their mean HbA₁C was 8.1 ± 0.4, and their mean body mass index was 29.4 ± 1.2. Seven patients were on oral antidiabetics; the other five patients were on diet only. The participants were tested on two different days in a placebo-controlled, randomized, double-blind, crossover study. The two test days were separated by at least 1 wk and by no more than 3 wk. The study was approved by the local Ethics Committee in accordance with the Helsinki II declaration.

Experimental protocol. On each test day, patients arrived in the research unit in the morning after an overnight fast. A standardized, fixed energy breakfast (2 scrambled eggs served on 2 slices of toast, 100 ml orange juice, 200 ml skimmed milk; energy content 360 kcal) was then served, and it had to be completed before 8:30 AM. After breakfast, patients were able to read and listen to music. At 11:00 AM, two venflon catheters were inserted into the anticubital veins of each arm, one for infusion, the other one for blood drawing. At 11:30 AM, a prestudy blood sample was taken and infusion was started (either GLP-1 at a dose of 1.5 pmol · kg¹ · min¹ or saline as placebo) and continued for the next 2 h. Infusions were delivered by ambulatory infusion pumps. Sixty minutes after the start of the respective infusion, a test meal was presented, and the participants were invited to eat and drink as much as they liked.

Beginning with the infusions, participants scored their subjective feelings of hunger and fullness in 15-min intervals throughout the experiments using visual analog scales (VAS) of 0-10 with text expressing the most positive and most negative rating anchored at each end. VAS were used to assess hunger, fullness, and prospective food consumption using scales and scores that were previously designed and described (2, 16, 17).

Each subject was free to eat and drink as much as he wished until "comfortably satisfied" of a standard meal that consisted of 1) orange juice as an appetizer (480 kcal/l); 2) ham sandwiches (60 g bread, 10 g butter, 25 g ham: 266 kcal/sandwich) with more orange juice or with water; 3) strawberry mousse (100 kcal/100 g); 4) coffee with cream (coffee could be sweetened with aspartame if desired; both cream and aspartame were optional; 12 g cream = 20 kcal). No additional food or fluid was allowed during the study. In addition, the order of food intake had to follow the above schedule. To reduce participants' awareness of the amount of food being provided, food was served in excess. The quantity of food eaten and the quantity of fluid drunk were measured. From these observations, the total energy intake could be calculated. In the premeal period and after eating, blood samples were taken in regular intervals (intervals: 0, 20, 40, 60, 80, and 100 min) for glucose and hormone determinations. Adverse events were assessed by the attending physician through close observation of each patient; in addition, each participant was questioned after each experiment and after he had completed the two tests whether he had experienced any adverse events.

Infusions. For the GLP-1 infusions, commercially available synthetic human GLP-1-(736) amide was purchased from Bachem, Bubendorf, Switzerland. The peptide was dissolved in 0.9% saline solution containing 0.5% human serum albumin and prepared under aseptic conditions by the University of Basel Hospital Pharmacy. Aliquots of 50 µg/5 ml were stored at 20°C. Infusion solutions were prepared by diluting appropriate amounts of GLP-1 with 0.9% saline containing 0.1% human serum albumin. Control solutions contained albumin in 0.9% saline alone; they were indistinguishable in appearance from GLP-1 infusions. The solutions were prepared by a pharmacist of the Kantonsspital Aarau who was not involved in the study. The physician in charge of the experiment was therefore not aware of the respective treatment, thereby making it possible to conduct treatments in a double-blind fashion.

Laboratory analyses. Blood was drawn through the indwelling anticubital cannula into syringes on ice. These contained EDTA (6 µmol/l) and aprotinine (1,000 kIU/l). After centrifugation, plasma hormone analyses were kept frozen at 20°C. Plasma glucose was analyzed by the glucose oxidase method. Insulin and glucagon concentrations in plasma were analyzed by commercially available radioimmunoassay kits (Biermann, Bad Nauheim, Germany). Immunoreactive GLP-1 was measured using the specific polyclonal antibody GA 1178 (Affinity Research, Nottingham, UK) as previously described (13). Immunoreactivities were extracted from plasma samples on C₁₈ cartridges using acetonitrile for elution of samples. The detection limit of the assay was 0.25 pmol/l. Intra- and interassay coefficients of variation were 3.8 and 10.9%, respectively.

Statistical analysis. The amount of food eaten and the amount of fluid drunk, as well as the corresponding energy intake, were compared between the two treatments by Wilcoxon signed ranks test using the SAS software package. The same statistical procedure was used to analyze the results of GLP-1-induced changes in plasma hormone concentrations using the area under the curve (AUC) analysis. Scores for hunger and fullness were compared by calculating the  score from baseline (0 min) to 60 min of infusion using the Wilcoxon signed ranks test. Differences were considered significant if P was <0.05.

	RESULTS

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Effect of GLP-1 on food intake. Intravenous infusion of synthetic GLP-1 dramatically reduced the amount of food eaten and the amount of fluid consumption (P = 0.034 and P = 0.011, respectively; Table 1). The maximal reduction in food consumption with GLP-1 amounted to 29%, resulting in a decrease in calorie intake of 27% (P = 0.034; Table 1).

None of the participants reported any abdominal discomfort or side effects during any infusion of GLP-1. Furthermore, when questioned at the end of each experiment, none of them experienced or reported any adverse reaction.

Effect of GLP-1 on eating behavior. The GLP-1 infusion significantly influenced the mean VAS (Fig. 1). Subjects experienced a reduced degree of hunger and a concomitant increased feeling of fullness in the premeal period with GLP-1 infusion. When we compared baseline scores with the 60-min values, the difference reached statistical significance (P = 0.028 for fullness and P = 0.026 for hunger). Subjects felt less hungry and fuller with GLP-1 infusion compared with saline administration.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Appetite scores (hunger and satiation) during glucagon-like peptide-1 (GLP-1; ) and saline () infusions in 12 patients with diabetes type 2. Data are means ± SE for both parameters. Significant changes compared with baseline during GLP-1 infusion (P = 0.028 for fullness and P = 0.026 for hunger).

Effect of GLP-1 on hormone levels. Figure 2 shows blood glucose, plasma insulin, and glucagon levels. Prestudy blood glucose concentrations were slightly hyperglycemic, but similar in both experiments. There was a continuous increase in blood glucose in response to meal intake in the control experiment. With GLP-1, blood glucose levels were significantly lower (AUC = 1,197 ± 101 with saline vs. 765 ± 65 mmol · min¹ · l¹ with GLP-1; P < 0.0001) for the duration of the experiment (Fig. 2). Insulin concentrations were significantly increased (AUC = 1,775 ± 357 for saline vs. 2,920 ± 516 U · min¹ · l¹ with GLP-1; P < 0.0001) with GLP-1 compared with saline infusion, whereas the glucagon response was significantly reduced (P < 0.006) with GLP-1. The GLP-1 infusion increased the plasma concentrations of GLP-1 (Fig. 2).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Plasma glucose, plasma glucagon, and plasma GLP-1 concentrations, as well as insulinogenic index during GLP-1 () or saline (), in 12 patients with diabetes type 2. Data are means ± SE. Significant treatment effects (P < 0.003-0.0001) for all parameters.

	DISCUSSION

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This investigation has shown that peripherally administered GLP-1 significantly reduces energy intake by inhibiting hunger sensations in patients with diabetes mellitus type 2. The marked effect on appetite is clearly illustrated by each of the food parameters and accompanied with lower fasting as well as meal-stimulated plasma glucose concentrations. These data extend previous findings in healthy volunteers that exogenous GLP-1 acts as a physiological regulator of food intake and appetite sensations (4, 7). The reduction in energy consumption along with the well-defined biological effects of GLP-1 lead to the question of whether a clinical application can be envisaged for the peptide for this indication.

Several groups, together with support from medical companies, are trying to convert GLP-1 into a useful therapeutic agent for different aspects of diabetes mellitus type 2. Treatment of hyperglycemia of diabetes mellitus type 2 with GLP-1 has received the major share of interest (1, 5, 10), but additional therapeutic indications are possible. Many patients with diabetes mellitus type 2 are overweight or even obese, and it would be extremely helpful if peripherally administered GLP-1 were able to reduce energy consumption by promoting early satiety. The present data indicate that this can be achieved, at least in the short term. The mean body mass index of our patient group was 29 (range 25-36), suggesting that the majority was clearly overweight. Given the fact that obesity aggravates diabetes mellitus type 2, our observations of the food intake-reducing effects of GLP-1 in these patients are of great clinical interest.

Several problems with GLP-1 as a therapeutic agent are apparent. First, as a peptide, GLP-1 cannot be taken orally. In addition, its pharmacokinetic properties (short half-life due to rapid degradation) (12) make it unsuitable for broad clinical use. Orally active analogs with an increased half-life would be necessary to overcome this problem. Second, we have shown that acute administration of GLP-1 promotes early satiety and reduces energy intake; whether repetitive administration would induce similar effects has not been studied in detail yet. Preliminary data recently reported in abstract form (M. B. Toft-Nielsen, unpublished data) indicate that a 48-h continuous subcutaneous infusion of GLP-1 1) lowered fasting and meal-stimulated glucose concentrations and 2) reduced appetite sensations. Furthermore, it is known that intravenously administered GLP-1 retains its blood glucose-lowering potency during continuous infusion for up to 7 days (J. Larsen, unpublished data). On the basis of these observations, we suggest that the appetite-reducing properties of GLP-1 should be further investigated. At the present stage, it is premature to conclude that the effect of GLP-1 on food intake will not exhibit tachyphylaxis given the complexity of neurohormonal signals that control food intake and satiety in man. Our findings suggest and imply, however, that GLP-1 therapy could be effective in diabetes mellitus type 2 patients and that it is worthwhile to pursue this line of investigation.

Perspectives

Two recent studies in humans showed that peripherally administered GLP-1 significantly reduces energy intake and modulates subjective appetite sensations in healthy volunteers (4, 7). Along this line, the study illustrates that GLP-1 exerts the same effects in patients with diabetes mellitus type 2, supporting a physiological role for the peptide in regulating appetite. GLP 1 may therefore be one of the physiological signals arising from the gastrointestinal tract that regulate food intake. Many questions remain, however, unanswered. Does GLP-1 interact with other gastrointestinal hormones released in response to meal intake that have been proposed to regulate food intake (cholecystokinin, gastrin-releasing peptide, and peptide YY)? What is the mechanism of action? GLP-1 dose-dependently inhibits gastric emptying; does inhibition of gastric emptying in itself reduce food intake, perhaps in association with distension of the stomach? The precise physiological role of endogenous GLP-1 in the regulation of food intake can only be determined when a specific and sufficiently potent antagonist of GLP-1 becomes available for human use.