1 Institute of Neuroendocrinology, University of Lübeck, Lübeck, Germany
2 Department of Internal Medicine I, University of Lübeck, Lübeck, Germany
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ABSTRACT |
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Obesity is a health care problem of global scale, and its increasing prevalence strengthens the need for a thorough understanding of how food intake and body adiposity are regulated (1). Individual body fat content and body weight are of long-term constancy, pointing to a remarkably precise matching of caloric intake and energy expenditure (2). In recent years, research on the neuroendocrine networks maintaining energy homeostasis has made considerable progress in identifying the pivotal messengers that report the level of energy stored as body fat to the brain (3). Systemic insulin and leptin levels are proportional to body adiposity and decrease during fasting, enabling food intake to be triggered (4). Meals increase the concentration of insulin in the cerebrospinal fluid (CSF) and the hypothalamus (5). High concentrations of insulin receptors are found in brain regions important to food intake regulation, suggesting that insulin, independent of its ability to stimulate glucose uptake in peripheral cells, plays an essential role in modulating caloric intake (2). Furthermore, hypothalamic insulin signaling is necessary for the inhibition of hepatic glucose production (6).
Administration of insulin to the central nervous system (CNS) reduces food intake and body weight (7), whereas its antagonization has reverse effects (8). Accordingly, switching off neuronal insulin receptors increases body weight and susceptibility to diet-induced obesity (9). In animals, the catabolic effects of central insulin are well documented (10), and there are some reports on the disproportional relation between insulin secretion and weight gain in men (11,12); however, to our knowledge, there is no direct evidence for catabolic effects of brain insulin in humans.
Circulating insulin reaches the CNS via a saturable, active transport across the blood-brain barrier (13), with CSF insulin levels increasing during intravenous infusion in humans (14). However, inducing central nervous catabolic effects using long-term intravenous infusion is not practicable, as peripheral insulin is an anabolic factor in energy homeostasis. Intranasal administration has been demonstrated to increase the concentration of insulin in CSF without the insulin being absorbed into the blood stream (15,16). In previous experiments, we found a distinct increase in CSF insulin levels within 40 min after intranasal administration of 40 IU insulin, averaging 1.82 ± 0.76 µU/ml compared with baseline values before administration. This increase was also significant in comparison with a placebo condition (15). Furthermore, insulin was shown to distinctly alter brain functions after intranasal delivery (17). Thus, the nasal route provides an effective way of conveying insulin to the brain. In the present experiments, we examined the effects of 8 weeks of intranasal insulin administration (4 x 40 IU/day) on body weight, body composition, and plasma hormone levels in healthy male and female subjects. The single dose of 40 IU was chosen based on our previous studies confirming significant CSF accumulations for this dosage level.
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RESEARCH DESIGN AND METHODS |
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We randomly assigned subjects to either the insulin or placebo group (12 men and 8 women in each group). Both groups were comparable in age (24.6 ± 1.3 and 25.8 ± 1.2 years, respectively) and BMI (22.6 ± 0.3 and 22.7 ± 0.4 kg/m2, respectively) in the pretesting examination. During 2 weeks of baseline, all subjects received placebo. During the succeeding 8-week treatment phase, subjects were administered insulin or placebo intranasally four times a day: in the morning, around noon, and in the evening (each 30 min before mealtime) and before going to bed. Each dose consisted of 0.4 ml insulin (40 IU; Insulin Actrapid; Novo Nordisk, Mainz, Germany) or vehicle (HOE 31 dilution buffer for H-Insulin; Aventis Pharma, Bad Soden, Germany) administered within four 0.1-ml puffs (two per nostril), amounting to 1.6 ml (160 IU) of insulin or vehicle per day. Sprays were stored at
5°C and replaced every week. To ensure compliance, subjects kept a diary about their intake routine and were told that irregular intake would be detected via urine sampling.
Test sessions (scheduled between 0700 and 0900) took place at the beginning (session A) and end (session B) of the baseline phase and at the end of the treatment phase (session C). Subjects were kept unaware of the study aims by embedding examinations into the assessment of cognitive parameters, not described here in detail. Posttreatment interviews ensured all subjects had remained unaware of the treatment effects. In the beginning of a session, subjects were administered placebo (session A), insulin or placebo (B), and again placebo (C). Thus, session A was used to familiarize subjects with the intake routine, and sessions B and C allowed for the examination of acute and long-term treatment effects, respectively.
Immediately after substance administration, we weighed subjects and measured their body composition by standard bioelectrical impedance analysis (frequencies of 1, 5, 50, and 100 Hz; BIA 2000-M; Data Input, Frankfurt, Germany) indicating body fat, total body water, intracellular water, extracellular water, lean body mass, and body cell mass (Eurobody software; Data Input). Waist circumference was also measured, and subjects completed a questionnaire on their eating behavior (18). Subjects rated their hunger on a 10-point scale 60 min after insulin administration and at the end of the session. Thus we obtained difference values indicating the gradient of hunger feelings. Between ratings, resting energy expenditure was registered using indirect calorimetry (Deltatrac II, MBM-200 Metabolic Monitor; Datex-Engström Deutschland, Achim, Germany). Heart rate variability was measured simultaneously for an interval of 20 min (12-lead simultaneous electrocardiogram, sampling rate 1,000 Hz; ECG Lab Version 2.0; Meigaoyi, Beijing, China). Weekly, at 0800, we weighed subjects and sampled blood to determine leptin, insulin, and glucose concentrations. Levels of epinephrine and norepinephrine were taken from 12-h (20000800) nocturnal urine samples and measured by standard high-performance liquid chromatography.
To control for possible side effects, we monitored various other parameters in the weekly examinations, including plasma concentrations of ACTH, blood pressure, heart rate, and routine laboratory measurements (serum electrolytes; creatinine; HDL, LDL, and total cholesterol; and triglycerides). In a follow-up examination performed 45 months after cessation of treatment, we measured body weight, body composition, and blood parameters of the male subjects.
Blood hormone concentrations.
Blood samples were centrifuged immediately, and the plasma was stored at 20°C. Concentrations of leptin, insulin, and ACTH were assessed using standard radioimmunoassays (Human Leptin RIA KIT, Linco Research, St. Charles, MO; Pharmacia Insulin RIA100, Pharmacia & Upjohn, Uppsala, Sweden; Lumitest ACTH, Brahms Diagnostica, Hennigsdorf, Germany). Glucose was measured in fluoride plasma (Aeroset; Abbott, Wiesbaden, Germany).
Statistical analyses.
Comparisons between the effects of insulin and placebo were based on ANCOVA, with a group factor representing the treatment condition. In additional analyses, sex was included as a group factor, and treatment effects were evaluated separately for the male and female subgroups. Significant interaction effects were followed by pairwise one-sided contrasts. Respective values of session A (start of the baseline phase) served as covariates for parameters that could be subject to the acute influence of insulin (e.g., heart rate variability); values of session B (start of the treatment phase) were used as covariates in the assessment of long-term influence on body weight and related variables. All data are presented as means ± SE. P 0.05 was considered significant.
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RESULTS |
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Prolonged insulin administration induces weight loss and reduces adiposity in men.
In the men, insulin treatment reduced body fat content [F(1,21) = 4.65, P 0.05], body weight [F(1,21) = 4.50, P
0.05] (Fig. 1), and BMI [F(1,21) = 4.33, P
0.05]. Waist circumference was also decreased [F(1,21) = 3.98, P
0.05] (Table 1). Over the 8-week period, the insulin-treated men lost 1.28 ± 0.71 kg of their body weight (versus a slight gain of 0.45 ± 0.49 kg for the placebo group) and 1.38 ± 0.59 kg of body fat (versus a gain of 0.57 ± 0.61 kg for the placebo group). The BMI of insulin-treated men declined by 0.38 ± 0.21 kg/m2 (versus a gain of 0.13 ± 0.14 for the placebo group), and their waist circumference decreased by 1.63 ± 1.17 cm (versus a gain of 0.62 ± 0.88 for the placebo group). During the final examination after 8 weeks of treatment, hunger ratings were decreased [F(1,18) = 6.99, P
0.02]. Heart rate appeared to be enhanced, but this effect was not significant [F(1,19) = 3.15, P = 0.09].
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Insulin promotes weight gain via extracellular water storage in women.
In contrast to the effects in men, in the insulin-treated women, body weight increased as soon as 1 week after initiation of treatment [insulin, 61.85 ± 0.27 and placebo, 61.00 ± 0.27 kg; F(1,13) = 4.92, P 0.05] and remained constantly elevated thereafter (Fig. 2). The increase in body weight [F(1,13) = 7.49, P
0.02] and BMI [F(1,13) = 8.74, P
0.01] after 8 weeks appeared to be due to a rise in extracellular water [F(1,13) = 5.10, P
0.04] (Table 2, Fig. 2), in conjunction with an increased ratio of extracellular mass to body cell mass [F(1,13) = 4.49, P
0.05] and tendencies toward augmented total body water [F(1,13) = 3.65, P = 0.08] and lean body mass [F(1,13) = 3.28, P = 0.09]. The supplementary analysis of plasma sodium indicated a trend toward increased levels in the insulin-treated women over the 8-week period [139.41 ± 0.34 vs. 138.46 ± 0.34 mmol/l; F(1,13) = 3.57, P = 0.08], whereas corresponding values in men remained unaffected [139.87 ± 0.23 vs. 139.96 ± 0.23 mmol/l, F(1,21) = 0.08, P > 0.78; F(1,35) = 5.69, P
0.05 for treatment x sex]. There were no treatment effects on plasma hormone and glucose concentrations after 8 weeks of treatment (Table 2) or when average values during the treatment period were compared [leptin: 12.16 ± 0.57 vs. 11.96 ± 0.57 ng/ml, F(1,13) = 0.06, P > 0.81; insulin: 13.70 ± 1.82 vs. 11.54 ± 1.95 µU/ml, F(1,12) = 0.66, P > 0.43; glucose: 80.15 ± 1.46 vs. 83.90 ± 1.36 mg/dl, F(1,12) = 3.44, P > 0.09]. Also, average catecholamine levels were comparable [epinephrine: 6.50 ± 0.96 vs. 4.83 ± 0.96 µg/l, F(1,13) = 1.52, P > 0.24; norepinephrine: 20.00 ± 1.95 vs. 15.47 ± 1.95 µg/l, F(1,13) = 2.70, P > 0.13]. All other parameters, including those after acute insulin administration, remained unchanged in the female subjects.
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DISCUSSION |
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In animal studies, male rats decreased food intake after intracerebroventricular insulin infusion and had lost substantial weight after only 24 h of treatment, whereas age- and weight-matched female rats remained unaffected. Leptin infusion yielded a reverse pattern, with a greater impact being seen on the female rats (19). Our finding of different central nervous sensitivity to insulin also fits with this picture, as insulin treatment reduced waist circumference in men; visceral fat content is correlated with endogenous insulin secretion and is more prevalent in obese men than women, who carry more subcutaneous fat (21). The mechanism underlying the observed sex difference is unclear. No such difference has been found for downstream neuronal mediators of the catabolic effects of insulin and leptin (19,22). Thus, the observed sex difference may have its source in differentially regulated insulin and leptin signaling, an explanation that appears plausible given that in mouse brain, insulin signaling is essential for reproductive function (9).
In the women, insulin surprisingly increased extracellular water, as reflected by an increased ratio of extracellular mass to body cell mass, associated with rapidly increased total body weight and augmented BMI. A confounding effect of the menstrual cycle can be safely ruled out as all women were taking oral contraceptives and each woman was examined at identical times in the cycle. Body water distribution may have been altered by renal sodium reabsorption, which is known to be modulated by peripheral insulin sensitivity (23). A trend toward increased plasma sodium levels was indeed revealed in the insulin-treated women. Intranasal insulin may have mediated the effects on water storage by enhancing systemic insulin sensitivity, which depends on intact hypothalamic insulin signaling (24). It is also noteworthy that with reference to body weight, the women received a higher dosage of intranasal insulin than the men, which could have enhanced this type of effect.
The decline in body weight and adiposity after insulin administration in the male subjects may have stemmed mainly from reduced daily food intake. Insulins reducing influence on hunger has been inferred from previous studies where, under euglycemic conditions, rated hunger rose more slowly during infusion of higher versus lower dosages of insulin (25). Studies of long-term central nervous insulin administration in several species have indicated a rather slowly evolving attenuation of 24-h food ingestion, extending up to several weeks (7,26). These observations would fit the assumption that even a small decrease in hunger motivation, of which an individual may not necessarily be aware, can lead to reduced caloric intake. The effect of intranasal insulin on body weight in the men emerged after several weeks of treatment, a finding that supports the concept of a gradual impact of elevated brain insulin levels on the regulation of actual hunger and satiety. Correspondingly, rated hunger was not affected by acute insulin administration but decreased after 8 weeks of treatment. This observation does not exclude the possibility that effects on body weight could occur even sooner with higher CSF concentrations of the compound, although this might be difficult to achieve with intranasal insulin administration in humans. Central nervous insulin administration in animals is reported to not only inhibit food intake but also stimulate thermogenesis via enhanced sympathetic neuronal outflow to thermogenic tissue (5,27). The signs of increased sympathetic tone found in our study (i.e., acutely increased diastolic blood pressure and a trend toward elevated heart rate after long-term treatment) were too weak to justify corresponding conclusions. Likewise, catecholamine levels, resting energy expenditure, and heart rate variability were not elevated after insulin administration.
The central nervous pathways between hypothalamic nuclei and the caudal brainstem, where neural, endocrine, and duodenal nutrient and adiposity signals converge to terminate single meals, have been extensively reviewed (24,10). Insulin and leptin regulate body weight through joint downstream signaling via melanocortins, establishing a tonic catabolic output to avert excessive weight gain (28). In our group of men, weight loss was associated with decreased leptin concentrations. After the treatment period, the discontinued insulin in conjunction with diminished leptin signaling presumably permitted prevailing activation of anabolic pathways so that body weight and fat returned to pretreatment levels. Regaining weight might ultimately lead to counterregulation, as was expressed in the slight increase in insulin and leptin levels in the follow-up examination.
In conclusion, the present data strongly support the assumption that in humans insulin is a prominent adiposity signal in the brain and exerts catabolic effects on body weight and adiposity. Because insulin is pharmaceutically produced in large quantities, its intranasal administration may represent an economic but effective therapeutic means of reducing body weight in obese men. Besides impaired central nervous sensitivity to insulin and leptin (29), decreased blood-to-brain transport of both messengers possibly plays a crucial role in the pathogenesis of obesity (30). Reduced CSF levels of insulin in obese animals have been repeatedly reported (31,32), and data gathered in our laboratory point to a similarly degraded level of insulin in the CSF of obese men (33). Thus, enhancing central nervous insulin concentrations by intranasal administration appears to be a promising approach in the campaign against obesity.
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ACKNOWLEDGMENTS |
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We thank A. Hatke for her expert technical assistance and C. Otten and H. Ruf for their invaluable laboratory work.
Address correspondence and reprint requests to Manfred Hallschmid, PhD, Institute of Neuroendocrinology, University of Lübeck, Ratzeburger Allee 160, Haus 23a, 23538 Lübeck, Germany. E-mail: hallschmid{at}kfg.mu-luebeck.de
Received for publication June 16, 2004 and accepted in revised form August 6, 2004
CNS, central nervous system; CSF, cerebrospinal fluid
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REFERENCES |
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