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Department of Endocrinology and Metabolism, Magdeburg University Medical School, Otto-von-Guericke University Magdeburg, 39120 Magdeburg, Germany
Address all correspondence and requests for reprints to: Dr. C. Schulz, Department of Endocrinology and Metabolism, Magdeburg University Medical School, 39120 Magdeburg, Germany. E-mail: carla. schulz{at}medizin.uni-magdeburg.de.
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Abstract |
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Introduction |
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Although in obesity serum leptin levels are elevated as a consequence of increased adipose tissue mass, cerebrospinal fluid (CSF) leptin levels do not appear to be elevated appropriately; the ratio between CSF and peripheral leptin is reduced in obese subjects (4). This is possibly caused by a saturation of the transport mechanism at high leptin levels (5) and a resistance of the leptin receptors at the BBB acquired in obesity, thus reducing the transport across the BBB.
A promising way to circumvent the BBB is via intranasal administration. Substances may traverse to the brain parenchyma or CSF via the olfactory membrane. A penetration into the brain through the nasal passage has been shown for a number of substances, including viruses (6) and peptides such as insulin and horseradish peroxidase (7). For example, central nervous effects were shown after intranasal application of insulin doses that did not affect serum levels (7).
The aim of our study, therefore, was to establish a reliable system for intranasal application of leptin in rats and to study the central nervous actions of this peptide in this novel experimental design.
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Materials and Methods |
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In a pilot study (our unpublished data), the intranasal administration of leptin in a dose of 0.03 mg/kg body weight showed no effect on body weight development; over a treatment/observation period of 15 d, leptin-treated male Wistar rats (n = 6) gained 53.8 ± 5.0 g, whereas control animals (n = 7) gained 52.4 ± 7.7 g. Based on these results, in the present study leptin was thus applied intranasally in two higher doses: 0.1 and 0.2 mg/kg body weight, dissolved in 25 µl physiological saline with sodium taurodihydrofusidate (STDHF) (1%) as adjuvant. In previous studies, STDHF has been shown to facilitate the intranasal uptake of substances (6) and to inhibit proteases in the nasal mucosa. The same volume of physiological saline with STDHF (1%) was used as control. Murine leptin was purchased from Sigma Chemie (Deisenhofen, Germany); STDHF was kindly provided as a gift by Leo Pharmaceuticals (Ballerup, Denmark).
At the beginning of the experiments, the mean body mass of the animals was 231.1 g; there were no significant differences of the initial weight in the different groups (see Results for details).
As described above, the animals were assigned to three experimental groups:
Group 0: 25 µl saline with 1% STDHF intranasally, n = 10;
Group 1: 0.1 mg/kg body weight leptin in 25 µl saline with 1% STDHF intranasally, n = 7;
Group 2: 0.2 mg/kg body weight leptin in 25 µl saline with 1% STDHF intranasally, n = 7.
The intranasal applications were performed daily at the beginning of the dark period between 1700 and 1900 h on a 28 consecutive days. After the application, animals were weighed on a Sartorius BP 2100 scale. Food and water intake were monitored subsequently.
At the end of the experiment, animals were decapitated 1 h after a final application of substances and trunk blood was collected on ice. Blood samples were centrifuged; serum was collected and frozen at –80 C until analysis of leptin and corticosterone.
The brains were removed, deep frozen on dry ice immediately, and stored at –80 C. In these brains, various neuronal structures (nuclei of the hypothalamus, amygdala) were examined by in situ hybridization for the expression of neuropeptide Y (NPY) and CRF mRNA.
The experimental protocols for animals and their care were in accordance with the German law and were approved by the committee on animal care. All experiments met the highest standards of humane animal care.
In situ hybridization
In situ hybridization was based on the method published by Erdtmann-Vourliotis et al. (8). The hybridization of one particular peptide mRNA was performed for all brain slices together in one experiment to exclude batch to batch variations in hybridization signals.
Preparation of brain sections
Frozen brains were cut with a cryostat into 14-µm slices at the level of the hypothalamus, mounted on microscopic slides and stored at –80 C. Brain slices were fixed in ice-cold paraformaldehyde in PBS (4%), washed in PBS, and dehydrated in graded alcohol.
Preparation of oligonucleotide probes
Single-stranded oligonucleotide probes were constructed complementary to mRNA sequences published in the NCBI Entrez Nucleotide database and chemically synthesized by NAPS (Goettingen, Germany).
CRF probe from molecule RNCRFR, length 45, starting position 686, 53% GC, melting temperature 100 C, 5'-TAACTGCTCTGCCCTGGCCATTTCCAAGACTTCCCTCAGAAGGTG-3'.
NPY probe from molecule NM_012614, length 48, starting position 266, 50% GC, melting temperature 100 C, 5'-GCCATATCTCTGTCTGGTGATGAGATTGATGTAGTGTCGCAGAGCGGA-3'.
More than five identical bases in a row and hairpins with a free energy lower than –4.7 kcal were avoided.
Probes were labeled with 35S-ATP using a terminal desoxyoligonucleotidtransferase. The labeled oligonucleotide was extracted by saturated phenol and chloroform/isoamyl alcohol and washed with chloroform/isoamyl alcohol. Subsequently the probes were precipitated by the addition of absolute ethanol at –80 C, washed, and after a final precipitation air-dried and resuspended in Tris-EDTA buffer.
In situ hybridization
For the in situ hybridization, 35S-labeled probes were diluted in hybridization buffer containing formamid and applied to the brain sections. Sections were covered and incubated over night at 42 C at 100% humidity.
Posthybridization washes
After hybridization microscopic slides were washed in sodium chloride/sodium citrate buffer to remove nonbound probes and dehydrated in graded alcohol.
Visualization and analysis
For visualization, Kodak (Stuttgart, Germany) Biomax MR film was exposed for 2 (NPY) or 3 wk (CRF) at –80 C and processed with Kodak GBX chemicals.
X-rays were digitized with a microscope and charge-coupled device camera (Olympus SZX9, Olympus DP10, Hamburg, Germany) and analyzed with Scion Image (Scion Corp., Frederick, MD). Using this software, the signal derived from the specific binding of the labeled probes to neuroanatomical structures was quantified in relation to the background (surrounding neuronal tissue with only nonspecific labeling).
RIAs
Serum samples were analyzed for leptin and corticosterone.
Leptin was determined with the Linco rat leptin RIA (IBL-Hamburg, Hamburg, Germany). This assay possesses a 100% cross-reactivity with mouse leptin, enabling the determination of the endogenous rat leptin and the murine leptin that was applied intranasally as well. Double determinations from 25 µl serum were made. The intraassay variation and detection limits were 6.2% (n = 5) and 0.4 ng/ml, respectively.
Corticosterone was determined in duplicates (10 µl samples) by the ICN rat corticosterone RIA (ICN Biomedicals GmbH, Eschwege, Germany). The intraassay variation was 5.9% (n = 5), and the detection limit was 19.3 ng/ml.
Statistical analysis
All values are given as mean ± SD. Statistical analysis was accomplished by using SPSS, version 10.0 for windows. ANOVA for repeated measures was performed to analyze for differences between body weight, food, and water intake over the whole observation period. At every single time point, Bonferroni’s t test was carried out to reveal differences between groups. In situ hybridization signals were also compared with Bonferroni’s t test. Values of P < 0.01 were considered highly significant, P < 0.05 significant, respectively.
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Results |
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, differences between control and leptin groups were first detected on d 7 of the treatment. The two leptin groups did not differ from each other. In a pilot study (our unpublished results), we had previously studied the intranasal administration of leptin in a dose of 0.03 mg/kg body weight. This treatment was without any effect on body weight development; over a treatment/observation period of 15 d, leptin-treated male Wistar rats (n = 6) gained 53.8 ± 5.0 g, whereas control animals (n = 7) gained 52.4 ± 7.7 g.
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). This effect was more pronounced in the 0.1 mg/kg leptin-treated group. In day by day comparisons, the differences between control and 0.1 mg/kg leptin groups were significant (P < 0.05) or highly significant (P < 0.01) from d 4 (food intake) or d 2 (water). For food intake, this is indicated by asterisks in Fig. 2.
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Serum corticosterone was 341.6 ± 137.8 ng/ml in control animals (n = 9), 364.2 ± 82.9 ng/ml in the 0.1 mg/kg leptin (n = 4) and 384.0 ± 144.0 ng/ml in the 0.2 mg/kg leptin group (n = 6).
Both hormones did not differ between the treatment groups.
mRNA expression
Expression of CRF and NPY mRNA in the hypothalamus and the amygdala was assessed with in situ hybridization. In the PVN, CRF mRNA in the higher leptin group showed a tendency toward up-regulation; however, it was not significant when compared with control animals (Fig. 3). In contrast, NPY mRNA expression was diminished in leptin-treated groups; however, this effect was not statistically significant (Fig. 4). As shown in Fig. 5, in the central nucleus of the amygdala (CeN), CRF mRNA expression was elevated significantly in the 0.1 mg/kg group when compared with controls.
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Discussion |
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Thus far, no studies measuring the rate of leptin-uptake in intranasal application have been published. The rate of transport through the nasal passage is dependent on the size, structure, and chemical properties of a substance; furthermore, a possible degradation by the enzymes in the nasal mucosa has to be taken into account. The bioavailabilities of drugs in nasal administration are almost linearly linked to their molecular weight and it has been shown that dextran of a molecular weight comparable to leptin reaches the CSF directly, not via an uptake into peripheral circulation (9). The usage of absorption enhancers has been shown to be highly effective for drug delivery (6), e.g. enabling peptides of higher molecular weight to penetrate into the brain (10). Leptin has a molecular mass of 16 kDa compared with 5.8 kDa for insulin, therefore the rate of STDHF-enhanced absorption can be roughly estimated to be one third of insulin, indicating that approximately 6% of the leptin administered would have been taken up—equaling 6 and 12 µg/kg body weight at the given dosages of 0.1 and 0.2 µg/kg. Given the hydrophobicity of leptin, the absorption rate might even be underestimated.
A wealth of data exists in rodents for various doses of leptin applied peripherally or centrally. For rats, icv doses of 2.5 µg/kg·d and above have been shown to affect metabolic parameters (11), whereas via peripheral application, doses of 0.16 mg/kg·d were shown to be either ineffective (12) or effective by others (13); profound effects of a peripheral application were found at doses of 0.5 mg/kg·d and above (14). Our findings on metabolic parameters indicate that a leptin dose in the low (if at all) effective range when given through the ip or sc route, apparently led to central concentrations in the bioactive range when given intranasally. This is strongly suggestive of a pronounced uptake directly into the CNS because, with the applied doses of 0.1 and 0.2 mg/kg body weight, no bioactive plasma concentrations were achieved. Our data from serum leptin determinations confirm a direct access for peptides to the CSF as described by others (7): serum levels did not differ significantly between groups, indicating that there was no significant uptake from the nasal cavity into circulation. A leakage from the brain to the periphery can also be excluded, because the transport of leptin across the BBB is unidirectional into the CNS (1).
Surprisingly, the effects of the lower leptin dose of 0.1 mg/kg body weight on food intake were more pronounced than in the higher dose. It can be speculated that the application of the higher leptin dose might have led to a central leptin resistance (15). The lower leptin dose of 0.1 mg/kg was also more effective in reducing water intake than the higher dose; however, this is likely secondary to food intake.
In contrast to these observations, the development of body weight was identical in the two leptin-treated groups and significantly lower than in controls. Together with the observation of a higher food intake of the 0.2 mg/kg group compared with the lower leptin group, the 0.2 mg/kg group must have had a higher energy expenditure to counterbalance the higher food intake compared with the 0.1 mg/kg group, resulting in an identical net effect on body weight. This can be explained by the finding that the effects of leptin on energy expenditure are less susceptible to a down-regulation process than the effects on appetite (16). This may be attributed to the distinct hypothalamic sites involved in leptin actions on food intake and energy expenditure respectively (17). Rozhavskaya-Arena et al. (18) speculate about the involvement of different domains of the leptin molecule in appetite or thermoregulation, respectively.
A sufficient central nervous availability of leptin after intranasal application inducing metabolic effects is supported by our in situ hybridization data. In accordance with findings from other studies employing both peripheral and central leptin administration, we demonstrated effects of intranasal leptin on CRF expression. The expression of NPY, another pivotal neuropeptide in central nervous appetite regulation, was not affected significantly, but showed a trend toward down-regulation.
It is well established that CRF and/or CRF-like peptides play a crucial role in central nervous appetite regulation (19, 20). Furthermore, we could demonstrate that endogenous CRF-like peptides are involved in the regulation of body weight (21).
It was demonstrated that leptin exerts its effects on food intake and energy expenditure at least in part via CRF receptor-mediated pathways (22). Conversely, the effects of changes on the nutritional status on CRF neurons require leptin (23).
The anatomical substrate for the effects of leptin on CRF is the localization of leptin receptors on CRF neurons in various nuclei of the hypothalamus, including arcuate nucleus (ARC) and PVN (24). Leptin icv elevates CRF release from a number of hypothalamic nuclei including the PVN in vivo and in vitro (25, 26).
In our study, we observed a trend toward up-regulation of CRF mRNA in the PVN; however, this was not significant. The effects were weak when compared with other studies employing icv administrations of leptin in acute treatment schemes (27, 28). The smaller effects in our study may be addressed to the prolonged elevated leptin levels in our chronic application design. Similar observations were made after long-term application of leptin in ob/ob mice (29). Furthermore, the increase of CRF mRNA after an acute application of leptin was lost in a chronic treatment scheme (30). These controversial results from long- and short-term studies imply that the marked effects of single leptin administrations on the CRF system are diminished, maybe even lost, after prolonged administration.
NPY is regarded as one of the central molecules in appetite regulation. The expression of the long form of the leptin receptor in this nucleus identifies NPY neurons that are activated by fasting (31); the fasting induced up-regulation of NPY expression can be blunted by the administration of leptin (28). Similarly, it has been demonstrated that single or repetitive injections of leptin decrease NPY mRNA in the ARC of rodents (29).
As shown in Fig. 4
, we observed a trend toward down-regulation of NPY mRNA in the ARC after intranasal application of leptin. Given the profound effects on metabolic parameters demonstrated in this study, a stronger down-regulation of NPY mRNA might have been expected. Because NPY is up-regulated in fasting and down-regulated in feeding conditions, the already low expression levels in our ad libitum-fed animals may not have allowed a further down-regulation of NPY mRNA. In addition, many of the studies on the action of leptin on central NPY expression were performed in ob/ob mice (e.g. Ref. 29), which are particularly sensitive to exogenous leptin. Furthermore, our methodology of comparing the gray values of x-rays underestimates the binding of labeled probes to the tissue because there is no linear relationship between the gray value and the hybridization signal in the given range; thus, the elevation of the hybridization signal is not adequately reflected in the gray values and vice versa.
In this study, we also demonstrated an increase in the expression of CRF mRNA in the CeN (Fig. 5). The amygdala is part of the limbic system and involved in processing of emotional stimuli. Its role in the acquisition of motivational value in conditioning is well examined (32).
The role of the amygdala in terms of body weight homeostasis is less well studied. Lesion experiments revealed an inhibitory effect of the CeN on food intake, whereas the basolateral nucleus of the amygdala exerts opposite effects (33). From in situ and RT-PCR studies, it is known that the long form of the leptin receptor is expressed in the amygdala in CRF neurons, implicating a possible direct action of leptin in this structure, which has been demonstrated in vitro (34). However, to our knowledge thus far, no in vivo data on the effects of leptin on CRF release, synthesis, or content in the amygdala exist.
A few studies focus on the CRF/amygdala system in the processes of feeding or fasting: it has been shown that ingestion elevates CRF tissue content in (35) and release from the CeN (36). These findings and our observation of CRF mRNA changes in the amygdala indicate a possible role of this structure in the regulation of body weight. A possible neuroanatomic substrate are neuronal projections from the amygdala to the ARC. Behavioral experiments demonstrating an involvement of the amygdala in taste aversion processes support the findings from physiological and anatomical studies (37).
It can thus be assumed that the amygdala is involved in affective and/or emotional aspects of food intake; CRF may be one of the participating neurotransmitters/neurohormones.
The intranasal application thus represents a useful tool to administer leptin in a noninvasive way with a rapid permeation into the CNS. This is of particular interest in the condition of leptin resistance, as found in human and experimental animal obesity.
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Acknowledgments |
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Footnotes |
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Abbreviations: ARC, Arcuate nucleus; BBB, blood brain barrier; CeN, central nucleus of the amygdala; CSF, cerebrospinal fluid; CNS, central nervous system; CRF, corticotropin-releasing factor; icv, intracerebroventricular; NPY, neuropeptide Y; PVN, paraventricular nucleus; STDHF, physiological saline with sodium taurodihydrofusidate.
Received October 23, 2003.
Accepted for publication March 1, 2004.
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