Introduction

Obesity is associated with increased risks of cardiovascular diseases, impaired glucose tolerance, and insulin resistance (1,2). Two of the factors that have been suggested to be related to the development of obesity are increased energy intake and decreased energy expenditure (3,4). Energy intake is partly controlled by neural and humoral signals that are generated by a biological system that senses and processes food (5). There is evidence that glucagon-like peptide 1 (GLP-1)¹ is one of the mediators involved in the postmeal satiety response (6). Together with glucose-dependent insulinotropic polypeptide, GLP-1 acts as an incretin and has synergistic effects on insulin release after food ingestion.

GLP-1 is a 30 amino acid peptide hormone secreted from intestinal L-cells after intake of a mixed meal (7,8) and has been shown to affect appetite ratings and food intake. Peripheral GLP-1 administration reduced food intake and suppressed appetite in normal weight subjects (9). Intravenous GLP-1 infusion in obese subjects led to significantly lower hunger ratings, compared with a saline infusion, and reduced ad libitum energy intake (8).

Basal GLP-1 concentrations and postprandial GLP-1 release seem to be attenuated in obese subjects, although statistical significance is unclear (10,11). It has been suggested that this is related to increased concentrations of nonesterified fatty acids, which are associated with obesity (12). Only a few studies have been investigating the effect of weight reduction on GLP-1 concentrations and have found an increase in meal-induced GLP-1 response to a level between that of obese and lean subjects (11), effects of gastric pacing, or effects of orlistat (13,14).

The purpose of this study was to further investigate the effect of weight loss on GLP-1 release in overweight/obese subjects.

Research Methods and Procedures

Subjects

Forty subjects were recruited by means of advertisements in local newspapers. Thirty-two obese (BMI, 30.1 2.6 kg/m²) subjects (23 women and 9 men), between 20 and 60 years of age, participated in the study. Selection criteria included being in good health, not taking any medications except for birth control, and no history of diabetes or chronic disease.

Informed written consent was obtained, and the study was approved by the Medical Ethics Committee of Maastricht University.

Experimental Design

A repeated measurement design (two visits, T1 and T2) was applied to the 32 selected subjects before (T1) and after a 6-week weight loss period (T2) with a very-low-energy diet (VLED). Subjects came to the laboratory in the morning in a fasted state. They were instructed to fast from 10:00 PM the night before the test day. After filling in the questionnaires, collecting urine samples, and measurement of resting energy expenditure (REE), an in-dwelling cannulae (Baxter BV, Utrecht, The Netherlands) was inserted in an antecubital vein. After 20 minutes of rest, subjects received a standard breakfast. The breakfast (1.9 MJ) had an energy density of 3.9 kJ/g and consisted of two slices of brown bread (100 grams), a baked egg (85 grams), and 300 mL skim milk. The distribution of energy was 48.8% energy from carbohydrates, 28.5% energy from protein, and 22.6 percent energy from fat.

Blood samples were taken for a total of 2 hours, every one-half hour after ingestion of the breakfast, for determining GLP-1, insulin, glucose, and free fatty acids.

To determine the appetite profile, subjects rated their subjective feelings of hunger and satiety on anchored 100-mm visual analog scales before each blood sample (15).

After the baseline measurements, a VLED intervention followed for 6 weeks to produce weight loss in the subjects. The VLED (Optifast; Novartis Consumer Health, Osthofen, The Netherlands) was supplied in three packets per day, which were dissolved in water to obtain a milkshake, pudding, or soup. Three packets provided 2540 kJ/d, consisting of 52.5 grams of protein (35% energy), 13.5 grams of fat (20% energy), and 67.5 grams of carbohydrate (45% energy). Two hundred grams of vegetables or fruit was allowed in addition to the VLED.

After the 6 weeks of the VLED, all measurements were repeated.

Anthropometry

For all subjects, body weight (BW) was measured on a digital balance (Seca, Hamburg, Germany), with subjects in underwear, in a fasted state, and after voiding their bladders. Height was measured using a wall-mounted stadiometer (Seca). The BMI was calculated as BW per height squared. Systolic and diastolic blood pressures were measured with an automatic blood pressure monitor (705 CP; Omron Healthcare, Hamburg, Germany).

The distribution of fat was determined by measuring the waist and hip circumferences and calculation of the waist-to-hip ratio (WHR). The waist circumference was measured at the site of the smallest circumference between the rib cage and the ileac crest, with the subjects in standing position. The hip circumference was measured at the side of the largest circumference between the waist and the thighs. The WHR was calculated by dividing the waist circumference by the hip circumference.

Body composition was measured using the deuterium (²H₂O) dilution technique (16). The dilution of the deuterium isotope is a measure for total body water (TBW) (17). The evening before the 2 test days, subjects drank a deuterium dilution (70 grams of water with an enrichment of 5 atom% excess ²H) after voiding. Deuterium enrichment was measured in the urine from the second voiding of the following morning. ²H concentrations in the urine samples were measured using an isotope ratio mass spectrometer (Micromass Optima, Manchester, UK). TBW was determined by dividing the measured ²H dilution space by 1.04 (16).

Fat-free mass (FFM) was calculated by dividing TBW by the hydration factor (0.73). By subtracting FFM from body weight, fat mass was obtained. Percentage of body fat was calculated according to the equation of Siri (18).

REE and Substrate Oxidation

REE was measured by means of an open-circuit ventilated hood system. Subjects came to the laboratory in the morning by car or by bus to minimize the amount of physical activity before the test. REE was measured at the beginning of each of the 2 test days with subjects in a fasted state while lying supine for 30 minutes. Gas analyses were performed by a paramagnetic O₂ analyzer (Servomex type 500A; Servomex Controls, Crowborough, Sussex, UK) and an IR CO₂ analyzer (Servomex type 500A), similar to the analysis system described by Schoffelen et al. (19). Calculation of REE was based on the formula of Weir (20). Respiratory quotient was calculated as CO₂ produced/O₂ consumed.

Fat oxidation was calculated using the following equation (21):

Insulin Resistance

Measures for insulin resistance were obtained from fasting plasma insulin and glucose concentrations using the homeostasis model assessment. It is assumed that normal weight subjects under 35 years of age have an insulin resistance of 1. Based on that assumption, resistance can be calculated according to the following equation (22):

Attitude Toward Eating

Eating behavior was analyzed at the beginning of each test day using a validated Dutch translation of the Three-Factor Eating Questionnaire (TFEQ) (23,24). Cognitive restrained and unrestrained eating behavior (factor 1), emotional eating and disinhibition (factor 2), and the subjective feeling of hunger (factor 3) were scored.

Blood Parameters

Blood samples for GLP-1 were taken in iced syringes and mixed with EDTA and 40 L of DPP-IV inhibitor to prevent degradation (Linco Research, St. Charles, MO). Blood samples for other blood parameters were mixed with EDTA to prevent clotting. Plasma was obtained by centrifugation for 10 minutes at 2800g at 4 °C. Plasma was collected, frozen in liquid nitrogen, and stored at –20 °C for analysis.

GLP-1 concentrations were measured using an ELISA kit (EGLP-35K; Linco Research) for nonradioactive quantification of biologically active forms of GLP. The assay has an intraassay coefficient of variation of 8% or less and an interassay coefficient of variation of 12% or less. Sensitivity of the analysis is 2 pM (25).

Plasma glucose concentrations were determined using the hexokinase method (Glucose HK 125 kit; ABX diagnostics, Montepellier, France). The WAKO NEFA C-kit (Wako Chemicals, Neuss, Germany) was used to determine free fatty acid concentrations.

Insulin concentrations were measured using a radioimmunoassay kit (Insulin RIA-100; Pharmacia, Uppsala, Sweden).

Statistical Procedures

Data are presented as mean SE or as mean SD. Differences for blood parameters, appetite profile, and anthropometric data between T1 and T2 were determined by ANOVA for repeated measures and Scheffe-F posthoc tests (Statview; SE Graphics). Area under the curve (AUC) was calculated as incremental area under the curve over time (2 hours). Pearson correlation coefficients, r, were calculated to determine the relationship between FFM and REE. The level of significance was set at p < 0.05.

Results

During the 6-week weight loss period, subjects lost a modest amount of weight (p < 0.05), reducing their BMI as well as waist and hip circumferences. Cognitive restraint (factor 1, TFEQ) was significantly increased after weight loss, and general hunger scores (factor 3, TFEQ) were decreased (Table 1).

Table 1. - Subject characteristics before and after weight loss.

Full table

Respiratory quotient was significantly lower after weight loss compared with before weight loss because of increased fat oxidation [4.16 1.31 (SD) g/h before weight loss compared with 5.49 1.73 g/h after weight loss; p < 0.05]. REE was a function of FFM before (r = 0.81; p < 0.05) and after weight loss (r = 0.43; p < 0.05; Figure 1). Anthropometric data for T1 and T2 are shown in Table 1.

Figure 1.

REE before (circles, dashed line) and after (squares, solid line) weight loss as a function of FFM. Regression equation before weight loss is REE (MJ/d) = 0.08 FFM (kg) + 2.3 (p < 0.05; r = 0.81). Regression equation after weight loss is REE (MJ/d) = 0.05 FFM (kg) + 4.3 (p < 0.05; r = 0.43).

Full figure and legend (72K)

Comparison of GLP-1 concentrations before (T1) and after (T2) weight loss showed the following. Although only approaching significance, fasting GLP-1 concentrations at T2 tended to be lower compared with T1 (p = 0.07). A similar outcome was observed when GLP-1 concentrations at time-point t = 0 were calculated as changes within every individual (p = 0.08).

After weight loss, stimulated GLP-1 concentrations were on a level below that of T1. GLP-1 concentrations were significantly different between T1 and T2 at 30 [F(1,31) = 5.39; p < 0.05] and 60 minutes [F(1,31) = 8.27; p < 0.05]. The AUC for GLP-1 concentration at T1 was significantly higher compared with T2 (Figure 2A). While meal-induced GLP-1 release was significantly different from fasting concentrations at 30 [F(1,31) = 10.19; p < 0.05], 60 [F(1,31) = 23.05; p < 0.05], and 120 minutes at T1 [F(1,31) = 16.07; p < 0.05], GLP-1 concentrations measured at T2 did not differ significantly from fasting concentrations at T2 at any time (Figure 2A).

Figure 2.

Plasma GLP-1 (A), insulin (B), glucose (C), and free fatty acid (D) concentrations at baseline and postprandially after ingestion of a standard breakfast before and after a 6-week weight loss period. Data are presented as means SE. (Left) Plasma concentrations before weight loss () and after weight loss (). *Significantly different at p < 0.05 (ANOVA for repeated measurement). (Right) AUC (average 2 hours) before (left bar) and after (right bar) weight loss. *Significantly different at p < 0.05 (ANOVA for repeated measurement).

Full figure and legend (107K)

After ingestion of the breakfast, insulin concentrations increased, peaking at 60 minutes at T1 and T2. There were no differences in baseline or postprandial insulin concentrations between T1 and T2 (Figure 2B). Insulin resistance, calculated using the homeostasis model assessment, was not different between T1 and T2 (2.54 0.25 at T1 compared with 2.53 0.26 at T2).

Glucose concentrations increased after ingestion of the breakfast and peaked at 60 minutes at T1 and T2. In contrast to the insulin results, glucose concentrations at T2 were always below the concentrations at T1, with significant differences between T1 and T2 at baseline [F(1,27) = 14.44; p < 0.05] and 30 minutes [F(1,26) = 9.46; p < 0.05; Figure 2C).

Free fatty acid concentrations decreased over time after breakfast. At every blood draw, concentrations were increased after weight loss compared with before weight loss. Differences between T1 and T2 were significant at baseline [F(1,26) = 5.24; p < 0.05] and postprandially at 30 [F(1,26) = 6.07; p < 0.05], 60 [F(1,26) = 11.22; p < 0.05], 90 [F(1,26) = 6.27; p < 0.05], and 120 minutes [F(1,26) = 9.87; p < 0.05; Figure 2D].

Baseline satiety ratings were not different between T1 and T2. Postprandial satiety ratings were significantly increased after the weight loss period compared with T1 at 120 minutes [F(1,22) = 5; p < 0.05; Figure 3A]. Hunger ratings were not different at baseline comparing T1 and T2 but were decreased after weight loss compared with before weight loss at 90 [F(1,21) = 4.52; p < 0.05] and 120 minutes [F(1,22) = 4.64; p < 0.05; Figure 3B]. Satiety and hunger ratings were not related to the increase in GLP-1 release.

Figure 3.

Subjective satiety (A) and hunger (B) ratings before () and after () a 6-week weight loss period. Data are presented as means SE. *Significantly different at p < 0.05 (ANOVA for repeated measurement).

Full figure and legend (47K)

Discussion

This study shows decreased GLP-1 concentrations with weight loss in a group of overweight/obese subjects. Not only were fasting GLP-1 concentrations decreased, but it seems that secretion caused by nutrient ingestion is abolished after weight loss as well. This result is similar to a study reporting decreased levels of GLP-1 after weight loss in severely obese subjects (13), but partly different from a study showing a marginal increase in GLP-1 concentrations after weight loss in obese subjects (11). However, incremental GLP-1 concentrations were not improved after weight loss (11,26). In contrast to these studies, this study investigated subjects with a BMI of 30 2.68 kg/m² compared with an average BMI of 38 kg/m² (11) or an average BMI of 44 kg/m² (26) in the other studies. This may partly explain the inconsistent results. Because, in this study, subjects lost less weight than in other studies (11,13,26) and the weight loss period was shorter, our results should be described as effects of the first phase of weight loss on GLP-1 concentrations, likely being induced by a strong negative energy balance. Free fatty acid concentrations were increased during weight loss, as has been shown before (27).

GLP-1 secretion may be inhibited by circulating nonesterified fatty acids (10,12,28), especially during the first phase of weight loss, when energy balance is negative. It has been suggested that the postprandial fall in plasma nonesterified fatty acids is an important mechanism by which GLP-1 release is stimulated and that an impaired postprandial suppression of nonesterified fatty acids may, in part, be responsible for the impaired GLP-1 secretion in obesity (12) This mechanism might have played a role in this study. An alternative explanation for decreased GLP-1 concentrations with weight loss may be an alteration in the autonomic nervous system caused by obesity or weight loss. There is no consensus yet on how the autonomic nervous system is changed and/or possibly contributes to obesity (29). Findings of decreased levels of vagally mediated gastrointestinal hormones after weight loss suggest that the effect of weight loss on vagal tone in the sense of either depression or hyperstimulation might play an important role in decreased GLP-1 concentrations after weight loss (13). Although GLP-1 concentrations were decreased after weight loss, overall GLP-1 concentrations in our subjects were still higher than what has been reported from other studies in normal weight subjects (30,31). Fasted average GLP-1 concentrations of 4.71 pM before weight loss and 2.6 pM after weight loss are higher compared with fasted concentrations, which ranged between 0.4 and 1.4 pM (30) or between 0.4 and 2 pM (31) found in normal weight subjects. That comparison shows that subjects in this study do not support the assumption of lower GLP-1 concentrations in overweight/obese subjects compared with normal weight subjects.

The decrease of GLP-1 concentrations during weight loss might be a response to a proceeding negative energy balance, thereby playing a role as a neuroendocrine factor signaling energy deficiency as part of a neuroendocrine response to starvation (32).

After weight loss, satiety ratings were increased, and hunger ratings were decreased compared with before weight loss. After 2 hours, subjects felt more satiated and less hungry, which may be explained by less energy requirements caused by weight loss, although the test meal provided the same energy content as before weight loss. No relationship was found between GLP-1 and satiety ratings before or after weight loss. Previously, a positive relationship between satiety and GLP-1 release in normal weight subjects was shown by Flint et al. (9). Näslund et al. (8,33) showed a relationship between satiety and GLP-1 in obese men when GLP-1 was infused. In a previous study, we showed an increase in GLP-1 release in obese subjects when stimulated with a galactose and guar gum solution. However, a corresponding increase in satiety did not occur (unpublished data). We suggest, given the results of this study, that GLP-1 is a satiety regulator in the short term and that a possible relationship between satiety and GLP-1 release is weaker in obese subjects compared with normal weight subjects.

It has been suggested that GLP-1 secretion normalizes gradually when overweight is reduced, as concluded based on increased fasting concentrations after weight loss. However, incremental AUC does not seem to be normalized after weight loss (11). More research needs to be done to study how the reduced GLP-1 concentrations after weight loss change during weight maintenance and regain. It is likely that, during the first phase of weight loss, when energy balance is very negative, GLP-1 decreases and that it normalizes subsequently. Similar changes in other hormones during different phases of weight loss have been shown before (13).

As expected, fasting and postprandial glucose concentrations were lower, and free fatty acid concentrations were increased after weight loss (27,34). Insulin sensitivity was not different after weight loss compared with before weight loss. Although many weight loss studies have found improvement of insulin sensitivity even after a 6-week weight loss period, there are others that have not found changes (35). That no differences occurred in this study might be caused by a rather modest weight loss. REE as a function of FFM decreased because of weight loss, and restraint score increased significantly during weight loss as expected. Subjects lost a little less weight than expected based on the three packets per day. Weight loss was 6 kg on average. This means that subjects consumed an average energy of 4.2 MJ/d, providing evidence for a very negative energy balance, but also showing that subjects did not stick to the VLED completely. The difference of 1.6 MJ/d cannot be explained by the additional intake of 200 grams of fruit or vegetables.

In conclusion, this study shows that, in the presence of a very negative energy balance, fasting GLP-1 release decreases in modestly obese subjects. Even the ability of nutrients to stimulate GLP-1 release seems to be abolished. The question remains how GLP-1 concentrations develop when subjects are back in energy balance.

Notes

¹ Nonstandard abbreviations: GLP-1, glucagon-like peptide 1; VLED, very-low-energy diet; REE, resting energy expenditure; BW, body weight; WHR, waist-to-hip ratio; TBW, total body water; FFM, fat-free mass; TFEQ, Three-Factor Eating Questionnaire; AUC, area under the curve.

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Acknowledgments

The authors thank Manuela Lejeune and Kathleen Melanson for expert assistance. This research was supported by Novartis, Consumer Health, Nyon.