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Department of Neuroendocrinology (C.B., J.B., M.H.) and Internal Medicine I (W.K.), University of Lübeck, 23538 Lübeck, Germany; and Interdisciplinary Obesity Center East-Switzerland (B.S.), Kantonsspital St. Gallen, CH-9400 Rorschach, Switzerland
Address all correspondence and requests for reprints to: Christian Benedict, Department of Neuroendocrinology, University of Lübeck, Ratzeburger Allee 160, Hs 23a, 23538 Lübeck, Germany. E-mail: benedict{at}kfg.uni-luebeck.de.
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Abstract |
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Objectives: Our objectives were to assess the effects of a single dose of intranasal insulin on food intake and memory function in men and women, and to determine any gender differences.
Methods: A total of 32 healthy, normal-weight subjects (14 men, 18 women) were intranasally administered 160 IU regular human insulin or vehicle before performing a hippocampus-dependent two-dimensional-object location task, a working memory task (digit span), and a hippocampus-independent mirror tracing task. Subsequently, food intake from an ad libitum breakfast buffet was measured.
Results: Insulin treatment decreased food intake in men but not in women (difference to placebo condition, men: –192.57 ± 78.48 kcal, P < 0.03; women: 18.54 ± 42.89 kcal, P > 0.67). In contrast, hippocampus-dependent memory and working memory were improved in women (P < 0.03, P < 0.05, respectively), whereas men did not benefit from acute insulin treatment (P > 0.17, P > 0.20). Performance on the hippocampus-independent mirror tracing task was not affected by insulin in women or men.
Conclusions: In accordance with animal data, results indicate that men are more sensitive than women to the acute anorexigenic effect of central nervous insulin signaling, whereas insulin’s beneficial effect on hippocampus-dependent memory functions is more pronounced in women. Our findings provide support for the notion of a fundamental gender difference in central nervous insulin signaling that pertains to the regulation of energy homeostasis and memory functions.
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Introduction |
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Although previous findings in animals and long-term observations in humans support the assumption that men are distinctly more sensitive to anorexigenic brain insulin signaling than women (7, 12), gender differences regarding the acute effect of central nervous insulin have not been systematically elucidated in humans. Here, we compared the effects of a single dose of intranasal insulin on food intake and memory functions in men and women. On the background of animal experiments indicating that brain insulin is particularly relevant for hippocampus-dependent spatial memory formation (18, 19), we also aimed at the functional differentiation of acute insulin effects on discernable memory systems, i.e. declarative spatial memory, working memory, and procedural memory.
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Subjects and Methods |
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Based on power calculations inferred from previous experiments on intranasal insulin administration, 32 healthy normal-weight subjects (18 women: body mass index 21.40 ± 0.48 kg/m2, age 22.44 ± 0.63 yr; 14 men: body mass index 22.94 ± 0.49 kg/m2, age 22.29 ± 0.62 yr) who were nonsmokers were included in the study. All women were taking oral (estrogen dominant, single-phase) contraceptives (Valette; Jenapharm, Jena, Germany) but were otherwise free of medication, as were the men. All relevant illness was excluded by clinical examination. Subjects fasted from 2200–0800 h before testing. They gave written informed consent to the study that conformed to the Declaration of Helsinki and was approved by the Ethics Committee on Research Involving Humans at the University of Lübeck.
Study design and procedure
Each subject participated in two conditions (insulin, placebo) spaced apart by 28 d, ensuring that the women participated on identical days in their menstrual cycle. Experimental days of the female subjects were scheduled not to take place during their respective menstruation phases. The order of conditions was balanced across subjects. Experimental sessions lasted from 0800–1100 h, starting with a 60-min baseline period (Fig. 1
). At 0900 h, subjects under the guidance of the experimenter intranasally administered 16 0.1-ml puffs (eight per nostril) of insulin and placebo, respectively, at 30-sec intervals, amounting to a total dose of 1.6 ml insulin (160 IU Insulin Actrapid; Novo Nordisk, Mainz, Germany) or vehicle (HOE 31 dilution buffer for H-Insulin; Aventis Pharma, Bad Soden, Germany). The nasal spray device applied here atomizes the insulin solution before inhalation so that it penetrates the nasal cavities more effectively.
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Cognitive tests
Digit span Twenty minutes and 75 min after substance administration, i.e. at 0920 and 1015 h, participants performed the Digit Span subtest of the Hamburg-Wechsler Adult Intelligence Scale-Revised 1991 (20) to assess the influence of intranasal insulin on verbal working memory. In this test, up to nine digits are read to the subject at a rate of one digit per second, e.g. five-eight-two. The subject is to repeat the digits chronologically (forward test) or in reverse order (backward test). Starting with three digits, their number gradually increases until the subject fails to repeat correctly the digits twice in a row. Correct responses after one presentation equal two points, and correct answers after two presentations equal one point. The total performance score is the sum of the forward and backward components (maximum score: 28 points).
Two-dimensional (2-D)-object location task Performance on this spatial learning task relies on temporal lobe structures, including the hippocampus (19). The task is a computerized version of the game "concentration," and it consists of 15 card pairs showing colored pictures of different animals and everyday objects. Card pairs are randomly distributed on a 15-in. screen at 30 possible locations displayed as gray squares ("the back of the cards") that are geometrically ordered in a checkerboard-like fashion (5 x 6 matrix). For the two experimental conditions, two different versions of the task are set up using different pictures and different locations. At learning (which in the present experiments took place at 0945 h), the first card of each card pair is presented alone for 1 sec followed by the presentation of both cards for 3 sec. After an interstimulus interval of 3 sec, the next card pair is presented in the same way. The whole set of card pairs is presented twice in different orders. Immediately after learning, cued recall is tested, i.e. the first card of each pair is presented, and the subject has to indicate the location of the second card with a computer mouse. The cued recall procedure is stopped after the first run.
Mirror tracing The hippocampus-independent mirror tracing task, which took place from 0950–1015 h, requires the subjects to trace a nongeometrical figure that they can only see reflected in a mirror four times in a run. Subjects are instructed to trace the line of the figure as fast and as accurately as possible. Before tracing the test figure, subjects train by tracing a simple star-shaped figure. The time needed for completion of the figure (speed) and the number of deviations from the prescribed 0.8-cm wide path (accuracy) are recorded and averaged over the four runs. For the two experimental conditions, two different figure versions were applied.
Blood hormone concentrations
Blood samples were centrifuged immediately, and the plasma was stored at –20 C. Serum insulin, C-peptide, cortisol, adiponectin, and leptin concentrations were assessed using ELISAs: insulin (Dako Cytomation, Cambridgeshire, UK), interassay coefficient of variation (CV) 7.5%, intraassay CV 6.7%; C-peptide (Dako Cytomation), interassay CV 5.2%, intraassay CV 4.7%; cortisol (Enzymun-Test Cortisol; Roche Diagnostics, Indianapolis, IN), interassay CV less than 3.9%, intraassay CV less than 2.0%; adiponectin (BioVendor GmbH, Heidelberg, Germany), interassay CV less than 7.3%, intraassay CV less than 6.4%; and leptin (Diagnostic Systems Laboratories, Sinsheim, Germany), interassay CV 4.4%, intraassay CV 3.8%. Glucose was measured in fluoride plasma (Aeroset; Abbott, Wiesbaden, Germany).
Statistical analysis
Comparisons between the effects of insulin and placebo were based on ANOVAs with the within-subject factor "Treatment" (as well as the factor "Time" where appropriate) and the between-subjects factor "Gender." Where appropriate, treatment effects were evaluated separately for the male and female subgroups. Significant interaction effects were specified by pairwise t tests. All data are presented as means ± SE. A P value less than 0.05 was considered significant.
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Results |
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Analysis of food intake revealed a significant interaction between the factors "Treatment" and "Gender" (P < 0.02), indicating differences in the response to intranasal insulin between men and women. In the men, intranasal insulin reduced average energy intake in comparison to placebo (insulin vs. placebo: 1158.81 ± 96.38 vs. 1351.38 ± 105.39 kcal; P < 0.03), whereas food intake remained unaffected in the women (insulin vs. placebo: 787.12 ± 41.25 vs. 768.58 ± 44.35 kcal; P > 0.67; Fig. 2A). ANOVA also revealed a main gender effect for food intake (P < 0.05). However, nonparametric analyses of male and female subgroups with comparable food intake (i.e. men with lowest and women with highest energy intake; each n = 6; P > 0.60, for main gender effect) again revealed that men but not women were sensitive to the anorexigenic impact of intranasal insulin (insulin vs. placebo: men, 889.60 ± 106.52 vs. 1087.61 ± 160.67 kcal, P < 0.05; women, 876.27 ± 55.50 vs. 963.30 ± 22.63 kcal, P > 0.25). In men as well as in women, the percentages of carbohydrates, fat, and protein in relation to total energy intake were not changed by treatment (P > 0.14, for all comparisons). Hunger ratings did not differ between conditions, neither at baseline (0800 h, insulin vs. placebo, 3.48 ± 0.43 vs. 3.87 ± 0.41 points; P > 0.33), 45 min after insulin administration (0945 h, insulin vs. placebo, 5.42 ± 0.46 vs. 5.84 ± 0.41 points; P > 0.28), nor after the end of the test meal (1050 h, insulin vs. placebo, 0.26 ± 0.10 vs. 0.23 ± 0.09 points; P > 0.75).
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Intranasal insulin overall did not alter performance on the 2-D-spatial location task (P > 0.49). However, ANOVA again indicated a significant "Treatment" x"Gender" interaction (P < 0.01). Women recalled significantly more card-pair locations after intranasal insulin compared with placebo (insulin vs. placebo: 51.9 ± 5.7 vs. 39.0 ± 2.9% correctly recalled card locations; P < 0.01), whereas men did not display any treatment effects (insulin vs. placebo: 44.6 ± 5.9 vs. 52.3 ± 3.1% correctly recalled card locations; P > 0.17; Fig. 2B
). A similar pattern was revealed for verbal working memory performance on the digit span test (Fig. 2C; P < 0.03 for treatment x gender) in which insulin treatment improved verbal working memory performance in women during the second run at 1015 h (insulin vs. placebo: 20.0 ± 1.11 vs. 18.64 ± 1.28 points, P < 0.05; first run: 19.0 ± 1.20 vs. 18.27 ± 0.87 points, P > 0.38). Working memory performance in men was unaffected by insulin (first run, 16.4 ± 1.26 vs. 15.5 ± 0.91 points, P > 0.35; second run: 15.7 ± 1.17 vs. 17.2 ± 1.34, P > 0.20). Performance on the procedural mirror tracing task did not differ between conditions (insulin vs. placebo: speed, 68.81 ± 2.28 vs. 70.84 ± 3.02 sec, P > 0.56; accuracy, 31.41 ± 1.94 vs. 30.70 ± 3.46 errors, P > 0.83), and there were also no gender-specific effects for this task (P > 0.20, for all comparisons).
Blood glucose and hormones
Blood parameters did not differ during baseline (P > 0.16 for all comparisons). In addition, there were no signs of a gender influence ("Treatment" x"Gender" x"Time"; P > 0.36, for all comparisons). ANOVA indicated a significant "Time" x"Treatment" interaction for plasma glucose and C-peptide levels (P < 0.004 and P < 0.05, respectively), indicating a decrease of both parameters after intranasal insulin administration (Table 1). However, introducing post-insulin glucose values as covariates in the analyses of food intake and memory performance did not change the respective treatment effects and the interactions between the factors "Treatment" and "Gender." Concentrations of circulating insulin, cortisol, leptin, and adiponectin were not affected by intranasal insulin (Table 1).
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Discussion |
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Plasma glucose concentrations decreased in response to intranasal insulin but clearly remained within the euglycemic range. The intranasal dose of 160 IU, which was applied here, by far exceeded those of previous studies in which blood glucose levels remained unchanged (11, 15). Thus, a small ratio of the compound may have entered the circulation via the nasal mucosa. Penetration of intranasal insulin into the circulation in turn was probably compensated by reduced secretion of endogenous insulin as suggested by the decrease in C-peptide levels, resulting in stable levels of circulating insulin. However, due to the fact that blood was sampled only twice, i.e. at baseline and 40 min after insulin administration, it cannot be excluded that significant changes in serum insulin levels may have elapsed our attention. Because brain insulin activates neuronal circuits that control endogenous glucose production (21), it might also be speculated that the decline in plasma glucose concentration was mediated by enhanced brain insulin signaling. Importantly, statistical analyses excluded any confounding influence of the slight reduction in plasma glucose concentration on the effects of insulin on food intake and memory performance. Unaltered levels of circulating cortisol, adiponectin, and leptin furthermore argue against a peripheral mediation. In addition, none of those parameters displayed any significant statistical interaction with the factors gender, treatment, and time.
Intranasal insulin decreased food intake in men only, indicating that men are more sensitive than women to the anorexigenic impact of the compound. This outcome demonstrates that elevating brain insulin levels results in an immediate decrease of caloric intake in men and strongly corroborates the notion that hypothalamic insulin signaling differs between genders. In rats, icv administration of insulin yielded strikingly similar results, reducing 24-h food intake and body weight of males but not females, whereas leptin administration induced an inverse pattern (7). Further examinations revealed that gonadal hormones determine the sensitivity to anorexigenic effects of central nervous leptin and insulin in both genders (22). We have previously shown that men, but not women, lose body weight and body fat during 8-wk intranasal treatment with 160 IU insulin/d (12). Interestingly, the acute decrease in energy intake of approximately 200 kcal observed here, if cumulated over 8 wk, would induce an estimated reduction in body fat comparable to the 1.4 kg the men of our previous study lost during long-term insulin treatment. Because in the present experiments hunger ratings were not affected, acute intranasal insulin administration most probably exerts its anorexigenic effects via meal-related signals that contribute to the termination of a meal, but not by affecting hunger motivation per se. Fittingly, in animal studies the effect of short-term satiety signals such as cholecystokinin has been enhanced by administration of insulin to the central nervous system (23). It is also possible that intranasal insulin suppresses food intake by inhibiting the expression of neuropeptide Y (24) as well as by stimulating the production of melanocortins (25).
In contrast to the anorexigenic effects on food intake in men, acute intranasal insulin administration improved declarative memory functions in the women. Previously, enhanced declarative memory performance in healthy men and women was observed only after 8-wk treatment, but not after acute intranasal insulin administration (17), albeit that cognitively impaired patients benefited also from acute treatment (14). Considering that the 160 IU dose of intranasal insulin used in the present experiments distinctly exceeded those of previous studies [i.e. 40 IU (15)], it might be assumed that in healthy humans, acute intranasal insulin improves memory functions only when administered at higher doses. Intravenous administration of insulin to healthy subjects yielded a more pronounced improvement of hippocampus-dependent memory at high rather than low doses (26), whereas cognitive facilitation in AD patients occurs at low rather than high doses of intranasal insulin (15). In addition, the spatial learning task used in the present experiments critically relies on the function of hippocampal regions (19) and might be more sensitive to the effects of insulin than the verbal memory task that was not affected by acute intranasal insulin in previous studies (17). Notably, our study is the first to demonstrate that intranasal insulin improves verbal working memory in humans that substantially relies on activation of the frontal cortex (27), a brain region displaying high densities of insulin receptors in rodents (28). Together, this pattern of effects suggests that intranasal insulin acutely enhances the function of the prefrontal-hippocampal loop that enables encoding and retrieval of declarative memory (29).
The improving effect of intranasal insulin on memory functions was restricted to the women, indicating a gender difference in the sensitivity to the neurocognitive effects of brain insulin signaling. The mechanisms underlying this difference cannot be derived from our data, although an interaction with central nervous estrogen signaling appears likely (22, 30). Because all the women of our study were taking oral contraceptives, they were by definition receiving pharmacological doses of an estrogen and a progestin. Thus, it cannot be excluded that gender effects reported here were mediated to some extent by this medication, and future studies should elucidate whether the menstrual cycle modulates the central nervous effects of intranasal insulin. In addition, neuroanatomical correlates of gender differences in the response to intranasal insulin are yet to be identified.
In sum, our data provide evidence that central nervous insulin plays a key role in the regulation of food intake behavior in humans and, in particular, in men. Furthermore, memory processing that involves hippocampal structures benefits from acute intranasal insulin treatment in women. Insulin transport to the brain is hampered in the obese state (31), and obesity represents a risk factor for dementia in women but not men (32). Convergent with these observations, our findings support the view that cognition depends more critically on insulin in women than in men. Furthermore, our results suggest that intranasal insulin might be an effective central nervous therapeutic compound in the treatment or prevention of disorders such as obesity and AD (15, 16, 33) that are assumed to result partly from a lack of brain insulin. A decisive factor to be considered in this context is gender.
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Acknowledgments |
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Footnotes |
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Author Contributions: All authors designed the study. C.B., J.B., and M.H. analyzed the data. C.B. and M.H. enrolled students. All authors contributed to writing the paper. C.B. and M.H. collected data or did experiments for the study. All authors participated in discussions concerning the results.
Disclosure Statement: The authors have nothing to disclose.
First Published Online January 29, 2008
Abbreviations: AD, Alzheimer’s disease; CV, coefficient of variation; 2-D, two-dimensional; icv, intracerebroventricular.
Received November 26, 2007.
Accepted January 22, 2008.
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