1 Laboratory of Obesity Research, Center for Clinical and Basic Research, Ballerup, Denmark
2 Neuroendocrine Pharmacology
3 Pharmacological Research 2, and
4 Molecular Pharmacology, Novo Nordisk A/S, Copenhagen, Denmark
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ABSTRACT |
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INTRODUCTION |
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Central administration of 13 µg of GLP-1 specifically inhibits food intake in rats via a hypothalamic site that is sensitive to neonatal monosodium glutamate (MSG) lesioning (12). However, central administration of slightly higher doses of GLP-1 leads to taste aversion, but because this latter effect is unaffected by MSG treatment, it further stresses the specificity of the central GLP-1induced anorexia (12). In addition, it is possible to elicit anorexia without concomitant taste aversion if GLP-1 is injected directly into the hypothalamic paraventricular nucleus (13). Acute injections of both GLP-1 and NN2211 exerts profound adipsia and diuresis. These effects on body water homeostasis could potentially hamper long-term treatment of patients with type 2 diabetes with GLP-1 agonists, and the potential anorectic effects of these agonists may be accompanied with debilitating affects on body water homeostasis.
Given that peripheral administration of GLP-1 affects food intake in humans, we decided to study further the anorectic potential of this peptide in the laboratory rat. Dose-response studies investigating the effect of intravenously administered GLP-1(7-37) or a novel long-acting acylated GLP-1 derivative NN2211 (14) on food intake were performed. In continuation of acute pharmacological studies, we examined the effect on food intake and body weight of twice daily subcutaneous administration of NN2211 for 10 days followed by a 5-day recovery period. To study the impact of 7 days of NN2211 treatment on energy expenditure (EE) and body composition, we studied normal rats by indirect calorimetry and subsequently subjected them to dual energy X-ray absorptiometry (DEXA) scanning. Before, during, and after treatment, blood biochemical markers of energy and fluid homeostasis metabolic state were monitored.
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RESEARCH DESIGN AND METHODS |
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Formulation of GLP-1(7-37), GLP-1(1-37), exendin(9-39), and NN2211.
GLP-1(7-37) and GLP-1(1-37) were obtained from L. Thim (Novo Nordisk); exendin(9-39) was purchased from Bachem (Bissendorf Biochemicals, Hamburg, Germany). High-performance liquid chromatography analyses of all peptides claim >90% purity. Peptides were dissolved in sterile isotonic saline to which 1% bovine serum albumin (BSA) was added (Fraction V, #735 086; Boehringer Mannheim, Mannheim, Germany). The GLP-1 derivative NN2211 (Arg34, Lys26-[N-(
-Glu[N-
-hexadecanoyl])]-GLP-1[7-37]) was synthesized according to a previously described procedure (14). The compound NN2211 is a member of a group of pharmacologically active GLP-1 derivatives that have long plasma half-lives (14). Thus, NN2211 is an acylated GLP-1 derivative with a plasma half-life of
14 h in pigs. Pharmacokinetic data from rats show that t1/2 = 4 h probably as a result of considerably higher endogenous DPP-IV activity in this species (L.B.K., unpublished observations). NN2211 was dissolved in sterile phosphate-buffered saline (50 mmol/l, pH 7.4) to a final concentration of either 0.1 or 1.0 mg/ml. Solutions were always made fresh
1 h before use and stored at 4°C in sterile tubes. Material from several batches of NN2211 was used, and corrections for impurity were always performed. Subcutaneous injections were administered using standard 1-ml syringes equipped with 25-G needles.
Experiment 1: single dose of GLP-1.
Sixteen adult male Wistar rats were equipped with jugular intravenous catheters (Department of Pharmacology, The Panum Institute, University of Copenhagen). Catheters were implanted under Avertin (tribromoethanol, 200 mg/kg) anesthesia in the right jugular vein with the tip aiming at the right atrium. After placement, the catheter was externalized via subcutaneous tunneling to the interscapular area. Catheter patency was secured by instillation of heparinized (1,000 IU/l) isotonic sterile saline into the catheter before closure with a metal rod. After 7 days of postoperative recovery, animals were housed individually in standard metabolic cages (Techniplast, Gazzoda, Italy) and acclimatized over a period of 7 days to a 5-h restricted feeding scheme with access to food from 8:00 A.M. to 1 P.M. and water ad libitum.
Seven days after initiation of the restricted feeding scheme, animals were assigned to a random crossover dosing paradigm. Five minutes before presentation of food, animals received an intravenous injection of GLP-1 (5, 100, or 500 µg/animal). Statistical analysis of intergroup treatment variation was carried out using factorial analysis of variance (ANOVA) followed by Scheffe post hoc analysis.
Experiment 2: single dose of NN2211.
Normal adult male Wistar rats (n = 16) and MSG-treated rats (n = 16) were housed individually in metabolic cages with free access to a rat diet and water for at least 1 week before experimentation. Animals were kept in a 12:12 light:dark cycle, and the effect of NN2211 on nighttime food intake was assessed by injecting animals subcutaneously 23 h before lights out. Animals were left without food and water in the period from dosing to the onset of darkness. Three doses of NN2211 and vehicle were tested in a random crossover experiment (10, 50, and 200 µg/kg). Food and water intake and diuresis were monitored every 30 min for the first 2 h after onset of nighttime (lights out) and finally 12 h later at lights on (t720). All measurements were done in complete darkness with the assistance of night vision goggles (Bausch and Lomb, Rochester, NY). The minimum interval between experiments was 48 h. Statistical analysis of intergroup treatment variation was carried out using factorial ANOVA followed by Scheffe post hoc analysis.
Experiment 3: continuous administration of NN2211.
Beginning 1 week before the first dose was administered, normal adult male Wistar rats (n = 16) and MSG-treated rats (n = 16) were housed individually in metabolic cages with free access to a rat diet and water. Animals were kept on a 12:12 light:dark cycle, and the subchronic effect of two daily subcutaneous injections of NN2211 or vehicle on body weight, food and water intake, diuresis, and feces excretion was monitored. All parameters were measured between 9 and 11 A.M. while animals received their morning dose. On the basis of daily food intake, animals were stratified to different treatment groups ensuring comparable baseline values for food and water intake. Animals received two daily injections of NN2211 (100 or 200 µg/kg b.i.d.) at 8:00 A.M. and 7:00 P.M. throughout a 10-day period followed by a 5-day drug-free recovery period.
Animals were weighed every morning (between 9 and 11 A.M.) together with measurement of daily food and water intake as well as diuresis and feces excretion. At day 0, orbital blood samples were taken from all animals before the first dose was administered. Further orbital blood samples were taken at day 7 and 14 (i.e., during and after treatment with NN2211). At the final day of experimentation, animals were decapitated and trunk blood was collected as described. Different dose administrations were conducted in two separate experiments, each with its own vehicle-treated control groups. Data from these control groups were pooled.
Statistical analysis of effect of treatment on body weight, food and water intake, diuresis, and feces excretion was carried out using factorial ANOVA followed by Bonferroni correction for multiple comparison. Biochemical data from plasma samples were analyzed using factorial ANOVA followed by Fishers or Scheffes post hoc analysis.
Experiment 4: effect of subchronic NN2211 treatment on energy expenditure and body composition.
Twenty-four 12-week-old male rats were used in this study. The rats were housed individually in a temperature- (20°C) and light-controlled environment (12:12 light:dark cycle; lights on from 7:00 A.M.) with free access to food and water for at least 7 days before experimentation. The rats were stratified into three groups (G1, G2, and G3) according to weight 3 days before study start (n = 7 per group). The rats in G1 and G3 were treated with vehicle, and the rats in G2 were treated with NN2211 (200 µg/kg b.i.d.). The rats in G1 and G2 had free access to food and water during the 7-day treatment period, whereas the rats in G3 were "single" pair fed after the rats in G2. Body weight and food and water intake were recorded daily. By the end of the study (day 7), oxygen consumption and body composition were determined by indirect calorimetry and DEXA, respectively. Likewise, blood samples were collected for determination of hematocrit as well as for plasma levels of triacylglycerol (TG), glycerol, free fatty acids (FFAs), and total cholesterol.
DEXA.
Body composition was determined by DEXA (pDEXA Sabre, Stratec Medizintechnic; Norland Medical Systems, Phörzheim, Germany). The instrument settings used were as follows: a scan speed of 40 mm/s, a resolution of 1.0 x 1.0 mm, and automatic/manual histogram width estimation. The coefficient of variation as assessed by 10 repeated measurements (with repositioning of the rat between each measurement) was 3.48, 3.17, and 3.73% for bone mineral content, lean tissue mass, and fat tissue mass, respectively. By the end of the study (day 7), the rats were killed. The carcasses were stored in plastic bags at -20°C before determination of body composition; which was determined on defrosted carcasses.
Indirect calorimetry.
Oxygen consumption, CO2 production, EE, and the respiratory exchange ratio (RER) were determined by indirect calorimetry (Oxymax System; Columbus Instruments, Columbus, OH). The rats (n = 1 per chamber) were placed in airtight acrylic chambers (10.5 l). Oxygen and CO2 concentrations in the chamber in- and outlet gas were determined simultaneously every 20.25 min over a period of 4.4 h. Instrument settings used were as follows: a gas flow rate of 1.86 l/min, settle time of 90 s; measure time of 40 s, and system recalibration for each eight-chamber measuring cycle. By the end of the study (day 7), the nonfasted rats were subjected to indirect calorimetry (from 8:00 A.M. 1:00 P.M.). In contrast to the previous 6 days, the nonfasted rats did not receive NN2211 between 7:30 and 8:30 before indirect calorimetry. On the day of indirect calorimetry, the rats received treatment at 9:30after four pretreatment measurements. The rats had no access to food or water during their stay in the acrylic chamber. The indirect calorimetric measurements were performed over 3 days. As a "positive" instrument control, every day included a reference animal "treated" with the EE increasing compound 2,4-dinitrophenol (DNP, Sigma). On the basis of the measurements of O2 and CO2 in the chamber in- and outlet gas, estimates of O2 consumption, CO2 production, EE, and RER were calculated.
Blood sampling and biochemical assays
Blood sampling.
Orbital blood samples were obtained by puncture of the orbital venous plexus with glass capillary tubes. Samples were taken in standard heparinized EDTA (0.18 mol/l) glass tubes (Vacutainer) to which aprotinin (1,500 KIE/ml) and bacitracin (3%) were added. After sampling, tubes were kept on ice before being centrifuged (4°C at 5,000g for 10 min), and the resulting plasma was stored at -80°C before being analyzed. Trunk blood was obtained by decapitating animals and sampling into heparinized (500 IU/tube) glass tubes to which aprotinin (1,500 KIE/ml) and bacitracin (3%) were added. Glycerol and FFA concentrations were determined in EDTA (0.18 mol/l) plasma containing 1% NaF (wt/vol).
Plasma glucose.
Plasma glucose was measured on a standard COBAS analyzer (Toxicology Projects & Planning, Novo Nordisk, Copenhagen, Denmark).
Plasma leptin.
Plasma leptin was measured using a commercially available mouse leptin enzyme-linked immunosorbent assay kit (Crystal Chemical, Chicago, IL), showing >95% cross-reactivity to rat leptin.
Plasma biochemistry.
Orbital blood samples (days 0, 7, and 14) were taken from rats that were receiving 100 µg/kg b.i.d. NN2211 and a set of corresponding vehicle-treated animals. Plasma values of sodium, TG, cholesterol, creatinine, carbamide, and total protein were measured on a standard COBAS analyzer. FFA and glycerol were measured on a standard Hitachi Automatic analyzer.
Plasma potassium.
Orbital blood samples (days 0, 7, and 14) were taken from rats that were receiving 200 µg/kg b.i.d. NN2211 and a set of corresponding vehicle-treated animals. Blood was collected in heparinized glass tubes (Vacutainer), and the potassium content in resulting plasma was measured potentiometrically with an ion selective probe on a standard COBAS analyzer.
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RESULTS |
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Experiment 3
Effect of subchronic administration of NN2211 on food intake and body weight.
Two daily injections of NN2211 to adult male Wistar rats dose-dependently decreased body weight over the entire 10-day treatment period with significant differences obtained with the 200 µg/kg b.i.d. dosing regimen between treatment days 7 and 14 (Fig. 4). Loss in body weight was preceded by decreased food and water intake and increased diuresis (Fig. 5). Food intake was significantly lower during the initial 3 days of treatment for normal animals that were treated with 100 µg/kg b.i.d. (data not shown). Thereafter, the anorectic effect of this low dose was no longer statistically significant. In animals that were treated with 200 µg/kg b.i.d., food intake was significantly lower throughout the 10-day treatment period. After cessation of the treatment, food intake normalized within a few days and body weight gradually increased toward that of vehicle-treated animals (Fig. 5).
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Effect of subchronic administration of NN2211 on water intake, diuresis, and feces excretion.
In contrast to food intake, water intake was lower in NN2211-treated animals only at the initial days of treatment, because from day 4 onward, NN2211-treated groups displayed considerably higher water intake than corresponding vehicle-treated groups (Fig. 5). However, these effects were not statistically significant and had no impact on long-term body fluid homeostasis (see below).
In normal rats that were receiving 100 µg/kg b.i.d. NN2211, increased diuresis was accompanied by increased water intake from treatment day 2 onward to cessation of dosing (data not shown). In animals that were receiving 200 µg/kg b.i.d., however, fluid homeostasis was severely affected during the first 2 days of dosing, because a state of very low water intake coexisted with markedly increased diuresis (Fig. 5). For normal rats that were treated with 200 µg/kg b.i.d., apparent fluid balance with water consumption and diuresis at levels similar to vehicle-treated animals occurred from day 4 onward. Despite marked increasing effects on urinary water excretion, plasma sodium and potassium remained unaffected in animals that were treated with 200 µg/kg b.i.d. NN2211 (Table 1). Also, plasma variables reflecting renal function (creatinine, carbamide, and total protein) were unaffected by NN2211 treatment (Table 1). Feces excretion was followed in normal animals that were receiving 200 µg/kg b.i.d. and was observed to decrease during administration of NN2211 coincident with the decrease in food intake (Fig. 5).
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Experiment 4
Effect of subchronic administration of NN2211 on EE, body composition, food intake, and body weight.
As seen in experiment 3, 7 days of NN2211 treatment significantly lowered body weight (373.3 ± 14.7 vs. 417.3 ± 15.3 g), and the body weights in the pair-fed group were reduced similarly (378.5 ± 10.5). After 7 days of treatment, EE was considerably lower in both NN2211 and pair-fed animals (control 1,775 ± 39; pair fed 1,634 ± 49; NN2211 1,641 ± 27 kcal/h; n = 67, average of 3-h measurements). However, when EE was expressed as oxygen consumption per kilogram of body mass, no such differences were seen throughout the observation period (Fig. 6). Also, the RER was unaffected by 7 days of NN2211 treatment (Fig. 7). In contrast, pair-fed animals displayed a switch toward lipid metabolism, probably reflecting that these animals had been starved for a longer period of time before the onset of experiment.
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DISCUSSION |
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Like native GLP-1, peripheral administration of a single dose of NN2211 significantly inhibited nighttime food intake in rats. The effect was not readily recognized during the initial 120 min of the dark phase, probably reflecting protracted pharmacokinetic mobilization from subcutaneous injection depot as demonstrated previously in female rats (18). Pharmacokinetic characterization of this GLP-1 derivative in humans revealed plasma half-lives of active GLP-1 derivative of 14 h, which is considerably longer than the half-life of endogenous GLP-1 (1.2 h) (19). Preliminary studies of NN2211 half-life in the rat have shown somewhat shorter values (single subcutaneous bolus: t1/2 = 4 h), probably because of aforementioned high activity levels of DPP-IV (L.B.K., unpublished observations).
The site of the anorectic action elicited by peripheral administration of GLP-1/NN2211 is different from the site that elicits anorexia in response to intracerebroventricular administration of GLP-1, i.e., it does not involve MSG-sensitive parts of the hypothalamus (12). However, participation of both central and peripheral sites in GLP-1induced anorexia should be considered because a recent study showed that radiolabeled GLP-1 readily gains access to the central nervous system (18). The nucleus of the solitary tract is adjacent to the blood-brain barrierfree area postrema, and a number of studies have shown that peripheral administration of neuropeptides not only labels the area postrema but also diffuses into the adjacent regions (20). Such a mechanism is supported by our recent observations from rats that were implanted with GLP-1synthesizing tumors in which anorexia develops irrespective of subdiaphragmatic vagal transection (P.J.L., unpublished observations). Thus, it seems likely that the anorectic effect of peripheral GLP-1 is mediated via a peripherally accessible site located either in the brainstem or on vagal afferents. In rats, the inhibitory actions of GLP-1 on gastric motility is mediated via the vagus nerve (11), but also direct inhibition of gastrin secretion as well as stimulation of somatostatin release may affect gastric emptying (21,22). Obviously, delayed gastric emptying counteracts excessive meal-related glucose excursions with consequent beneficial glucose homeostatic effects. Decreased gastric motility may also be part of a premature inhibition of further ingestion as it constitutes a prandial satiety signal mediated via vagal stretch receptive nerve fibers.
A full analysis of NN2211 effects on the feeding pattern (meal size, intermeal interval, etc.) was not carried out, but preliminary data suggest that acute as well as subchronic peripheral administration of NN2211 reduces meal size similar to what is known for other gastrointestinal hormones, such as CCK (22,23 P.J.L., unpublished observations). It has been suggested that signals that modify food intake via diminishing meal size are both direct and indirect (23). Direct signals are elicited when nutrients gain contact to various segments of the gastrointestinal tract, whereas indirect signals arise from other sources than the gastrointestinal tract. Indirect signals modify the gain of direct signals, thereby influencing the efficacy of acute meal-terminating signals. Thus, GLP-1 represents a direct signal that arises from fat and carbohydrate contact with duodenal and jejunal lumen. The signal to ileal L-cells may also be hormonal in as much as gastric inhibitory peptide links the proximal small intestine with more distal segments. GLP-1 released from the distal ileum is foremost an incretin with actions on pancreatic insulin secretion (and inhibition of glucagon secretion), but it also has actions as an ileal brake, reducing gastric emptying and subsequently halting nutrient delivery to absorptive portions of the gastrointestinal tract (24). According to the proposed model of direct and indirect meal-terminating signals, it seems plausible that peripherally released GLP-1 interacts with leptin-sensitive sites in the brain stem (25). With the advent of a long-acting GLP-1 derivative, future studies of additive pharmacological effects of leptin and NN2211 will help to elucidate whether leptin exerts synergy with GLP-1 as is seen for another gastrointestinal tract hormone, CCK (26,27).
In the central nervous system, there is evidence that two independent GLP-1sensitive systems mediate the anorectic effects of centrally applied GLP-1. Firm evidence ascribes ascending GLP-1containing nerve fibers that originate in the caudal part of the nucleus of the solitary tract as mediators of visceral illness (28,29). However, the precise location(s) of the central GLP-1 receptors involved in this mechanism is unknown (28). Nonaversive reduction of food intake is elicited upon direct injection of GLP-1 into the hypothalamic paraventricular nucleus (13,30).
The regulatory role of both intestinal and central nervous system GLP-1 as either hormonal or neurotransmitter mediators of satiety has been questioned by observations from GLP-1 receptor null mutant mice (GLP-1R -/-) because these mice do not display an obese phenotype (31). However, initial statements about the lack of GLP-1R function in regulation of satiety are incorrect, because further evidence that GLP-1 has a role as an acute short-term mediator of satiety has actually been gained from studies on GLP-1R(-/-) mice. Careful analysis of the data presented by Scrocchi et al. (32) clearly demonstrates that GLP-1R(-/-) mice terminate the initial feeding period of the dark phase significantly later than wild-type animals, resulting in increased food intake during the initial 4 h of the dark phase. In GLP-1R(-/-) mice, other postprandial satiety factors probably take over the meal-terminating role of GLP-1 with resulting unaffected total caloric intake. So far, no loss of function mutations in the human GLP-1 receptor has been reported, but the therapeutically interesting question is whether long-term activation of peripherally accessible GLP-1 receptors constitutes a potential weight-reducing principle.
Using a novel long-acting GLP-1 derivative, NN2211, we extended single-dose experiments showing that subchronic administration of a GLP-1 agonist confers profound weight loss in both normal lean rats and in obese MSG-treated rats. Subchronic administration of NN2211 lowered plasma triglyceride levels in both normal and MSG-treated rats, probably reflecting enhanced peripheral postprandial insulin actions. However, NN2211-treated animals also underwent a mild state of catabolic degradation of adipose tissue stores inasmuch as leptin levels decreased in both normal and MSG-treated rats. Thus, the decreased levels of circulating triglyceride levels may have been the direct consequence of lowered food intake. Analysis of body composition by DEXA scanning was unable to reveal a significant loss of body fat in response to NN2211 or pair feeding. However, the interanimal variation, limited group sizes, and lack of instrument precision gave rise to relatively large standard deviations despite numerous repeated measurements. Thus, the lack of statistical significance of the observed drop in body adiposity of NN2211-treated animals may be due to a simple type 2 statistical error. First impressions of body weight curves and matched food intake suggest that GLP-1 agonists may enhance EE in addition to their anorectic effects. Measures of EE clearly showed that NN2211-treated and pair-fed animals had similar metabolic rates and body weights. However, the persistent weight loss and the apparent lack of rebound hyperphagia upon cessation of the NN2211 treatment are more enigmatic. Although long-term pharmacokinetic data are unavailable, it seems possible that the dosing regimen of NN2211 used in this study gives rise to accumulation of the compound in a subcutaneous compartment, thereby resulting in protracted slow release of the active GLP-1 analogue. Also, NN2211 had no impact on substrate utilization, ruling out that major shift toward fatty acid oxidation occurs during NN2211 treatment. It is worth keeping in mind that rodent and human substrate mobilization may vary considerably. Thus, future studies of EE in NN2211-treated humans are needed to elucidate fully the impact of this compound on human energy homeostasis.
A number of studies have investigated the potential anorectic effects of short-term intravenous GLP-1 infusion to human volunteers (<24 h). Both nonobese and obese humans respond to GLP-1 infusion by reducing their caloric intake without concomitant presence of gastrointestinal discomfort or increased equivalents of malaise (1,3). Subchronic administration of relatively high doses of the GLP-1 analogue exendin-4 to diabetic rodents decreases food intake and lowers body weight (33,34,35). Greig et al. (33) included a control group of normal-weight nondiabetic mice, in which both food intake and body weight remained unaffected by administration of 24 nmol/kg per day, suggesting that the anorectic effect may depend on increased plasma glucose levels or impaired insulin sensitivity. GLP-1induced anorexia is not mediated via pancreatic insulin secretion because normal rats displayed full sensitivity to NN2211 during euglycemia, when GLP-1 has no insulinotropic action. Extensive studies with NN2211 have shown that this derivative as native GLP-1 is incapable of causing serious hypoglycemia (36). Also, MSG-treated rats were fully sensitive to the anorectic action of NN2211 despite their well-known decreased peripheral insulin sensitivity (37,38). It is interesting to note that both circulating and postprandial levels of active GLP-1(7-36)amide are decreased in obese humans (39). In particular, obese individuals seem to have a selective attenuation of carbohydrate-induced postprandial GLP-1 secretion, and this defect may be related to the dyslipidemia experienced by most obese individuals because the degree of impaired GLP-1 release is tightly correlated to the circulating levels of nonesterified fatty acids (40). Therefore, acute regulation of feeding behavior in obese individuals may involve a weaker-than-normal postprandial GLP-1 satiety signal. In conjunction with carbohydrate ingestion, insulin-induced leptin secretion is markedly increased in comparison to levels seen during fat ingestion (41). Thus, lower-than-normal postprandial GLP-1 release in obese individuals may lead to an insufficient insulin release, causing both impaired glucose tolerance and lower leptin secretion.
In conclusion, we showed that peripheral administration of GLP-1 and a long-acting derivative hereof, NN2211, induce anorexia together with lowered water intake and increased diuresis. Furthermore, NN2211 confers lasting and reversible anorexia with accompanying weight loss and reduction of body adiposity. As seen for many other anorectic agents, NN2211 causes a moderate drop in total EE proportional to the induced weight loss. However, no change in substrate utilization was seen. Despite initial effects on water intake and diuresis, NN2211 administration has no debilitating effects on body water homeostasis.
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ACKNOWLEDGMENTS |
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The invaluable technical assistance with animal experiments by Frank Strauss, Aase Kirkeby, and Ken Heding is gratefully acknowledged. Analytical biochemistry was skillfully performed by Gitte-Mai Nelander and Conni Bech.
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FOOTNOTES |
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Received for publication 24 January 2001 and accepted in revised form 26 July 2001.
L.B.K and C.F. are employees of and hold stock in Novo Nordisk.
ANOVA, analysis of variance; DEXA, dual energy X-ray absorptiometry; DPP-IV, dipeptidyl peptidase-IV; EE, energy expenditure; FFA, free fatty acids; GLP-1, glucagon-like peptide-1; MSG, monosodium glutamate; RER, respiratory exchange ratio; TG, triacylglycerol.
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REFERENCES |
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