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Endocrinology Vol. 143, No. 6 2333-2340
Copyright © 2002 by The Endocrine Society

CRH-ACTH-POMC-ADRENAL

Inactivation of the GR in the Nervous System Affects Energy Accumulation

Christoph Kellendonk*1,2, Sandra Eiden*1, Oliver Kretz, Günther Schütz, Ingrid Schmidt, François Tronche3 and Eckhart Simon

German Cancer Research Center (C.K., O.K., G.S., F.T.), Im Neuenheimer Feld 280, D-69121 Heidelberg, Germany; and Max-Planck-Institut fuer Physiologische und Klinische Forschung (S.E., I.S., E.S.), W.G. Kerckhoff-Institut, Parkstraße 1, D-61231 Bad Nauheim, Germany

Address all correspondence and requests for reprints to: Günther Schütz, German Cancer Research Center, Im Neuen-heimer Feld 280, D-69121 Heidelberg, Germany. E-mail: . g.schuetz{at}dkfz.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The homeostatic regulation of body weight protects the organism from the negative consequences of starvation and obesity. Glucocorticoids (GCs) modulate this regulation, although the underlying mechanisms remain unclear. To address the role of central GRs in the regulation of energy balance, we studied mice in which GRs have selectively been inactivated in the nervous system. Mutant mice display marked growth retardation. During suckling age this is associated with normal fat deposition causing a 60% temporary increase of percent body fat, compared with control littermates. After weaning, fat and protein depositions are reduced so that adults are both smaller and leaner than their controls. Decreased food intake and, after weaning, reduced metabolic efficiency account for these developmental disturbances. Plasma levels of leptin and insulin, two important energy balance regulators, are elevated in young mutants but normal in adults. Leptin/body fat ratio is higher at all ages, suggesting disturbed control of circulating leptin as a consequence of chronically elevated GC levels in mutant animals. Adult mutants display increased hypothalamic CRH and NPY levels, but peptide levels of melanin concentrating hormone and Orexin A and B are unchanged. The increased levels of plasma GCs and hypothalamic CRH may act as catabolic signals most likely leading to persistently reduced energy accumulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCOCORTICOIDS (GCs) are steroid hormones, which are secreted from the adrenal gland into the circulation under the control of the hypothalamic-pituitary-adrenal axis (HPA-axis). They regulate many physiological processes by activating two transcription factors, the GR and the MR (1). It is long known that GCs modulate the regulation of body weight and fat storage.

GCs affect fat deposition. Chronic hypercortisolism as observed in patients with Cushing’s syndrome is associated with altered fat distribution and visceral obesity (2). In addition, in several obesity models, the contribution of GCs to excess fat deposition has been documented. For instance, mice homozygous for a mutated leptin gene (ob/ob) develop obesity that is dependent on GCs (3). Similarly, adrenalectomy ameliorates excessive fat deposition in adult rats homozygous for a genetic defect in the leptin receptor gene (fa/fa) (4, 5). The fact that adrenalectomy does not reduce fat deposition in suckling-age fa/fa rats suggests developmental differences in the impact of GC deprivation (6).

GCs affect food intake. They increase appetite in humans (7) and restore decreases in food intake in rats following adrenalectomy (8). In contrast, long-term treatment of adult mice with high levels of GCs inhibits food intake and evokes body weight loss (9). These discrepancies could have several origins. They could reflect differential actions of GCs on peripheral vs. central organs, depending on their concentrations. In addition, they may reflect differential actions of activated GRs and MRs. Indeed, both receptors are expressed in the brain, but whereas high GC levels activate both, low levels mainly activate the high-affinity MR (10, 11, 12).

Food intake and energy balance are centrally controlled by hypothalamic neuropeptides. The expression of some of these peptides is regulated by GCs (13, 14, 15, 16, 17). Through their central actions, GCs modulate the somatic and autonomic nervous system outflow that controls energy expenditure and the release of hormones such as insulin (13, 17). To dissociate central and peripheral functions of GRs, we analyzed animals in which the GR gene is selectively inactivated in the nervous system. Using the Cre/loxP recombination system, we deleted the third exon of the GR gene in neural precursor cells (18). This leads to the absence of GRs in neurons and glia from an early prenatal stage. Mutant mice (GRNesCre mice) showed an activated HPA-axis leading to elevated basal GC levels in combination with increased CRH levels in the paraventricular nucleus (PVN). In this study, we addressed the effect of central GR absence on the development of body growth and energy balance.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
The GR gene was inactivated in the mouse as previously described (18). GRNesCre mice express Cre recombinase under the control of the rat nestin promoter and enhancer and are homozygous for the GRLoxP allele. Control GRLoxP/LoxP littermates do not express Cre. Mice were housed with chow (type 1314, Altromin, Lage, Germany) and water ad libitum in a 12:12 h light:dark cycle at 22 C except for measurements under thermoneutral conditions. For measurements during the first 5 wk of life, we used animals derived from 11 litters, killed at different developmental stages, with weaning on the postnatal d 21. Experiments in adults were carried out in three different experimental groups of female mice. The animal experiments described were conducted in strict compliance with European Convention and institutional regulations.

Plasma measurements
GH, IGF-1, and T4 plasma concentrations were determined following the instructions of commercial RIA kits (rat GH RIA kit, Amersham Pharmacia Biotech, Freiburg, Germany), rat IGF-1 RIA kit, DRG, and T4 RIA kit, ICN, Eshwege, Germany) in samples collected shortly after lights-on, by tail phlebotomy or decapitation. For developmental studies on plasma insulin and leptin levels, animals were decapitated 2 h before dark phase. For insulin measurements we used a Serono diagnostic RIA kit with rat insulin (Linco Research Inc., St. Charles, MO) as standard; for leptin we used a mouse RIA kit (Linco Research Inc.). Measurements were independently duplicated and variability was decreased by correcting the data for interassay variability and buffer dilution using internal correction factors.

Determination of energy balance
The oxygen consumption of 7 weanlings and 20 adult mice was continuously recorded over several days in an open flow system as described (19). Energy expenditure was determined using a conversion factor of 20.4 kJ/liter O2 corresponding to a respiratory quotient of 0.85. Body composition—water, fat mass, and fat-free dry mass (FFDM)—was determined as described (19). The energy equivalents used were 38 kJ/g for fat mass and 20 kJ/g for FFDM. In weanling mice the intake of chow (type 1314, Altromin) was recorded daily at the end of the light phase. Energy content of feces was determined by bomb calorimetry. Energy expenditure and body energy content were used as the most reliable parameters to calculate metabolizable energy intake. Energy expenditure and intake were first considered in absolute terms for energy balance calculations. Additionally, we expressed energy turnover in relation to the two-thirds power of body mass, following the frequently used convention to account for potential thermoregulatory modifications of energy dissipation.

Immunohistochemistry
Immunohistochemistry was performed on paraffin-embedded sections using polyclonal antibodies against NPY (dilution 1:400, UCB, Braine-C’Alleud, Belgium) (20) human CRH (dilution 1:200, UCB) (21, 18), melanin concentrating hormone (MCH, a gift from Joan Vaughan and Wylie Vale) (22), and Orexin A and B (dilutions 1:200, Santa Cruz Biotechnology, Santa Cruz, CA) as primary antibodies. Antigen-antibody complexes were visualized by horseradish-peroxidase-conjugated goat antirabbit immunoglobulins (dilution 1:50, DAKO Corp., Hamburg, Germany) using 3,3'-diaminobenzidine (Sigma, St. Louis, MO) as the chromogene.

Quantification was performed by microdensitometric measurements using an image processing system IBAS (release 2.0, Kontron Instruments Ltd., Eching, Germany). The system consists of a black-and-white television camera attached to a light microscope, an image processor, and a host computer. The following values were measured: 1) mean gray reference value (mean gray value of a tissue-free area [calibration of the variation of the brightness]); 2) mean gray value of a background area (mean gray value of an area of unspecifically stained tissue [standard area in the lateral hypothalamus]); 3) SD of gray values within the background area; and 4) mean gray value of a specifically stained area (MGSSA). Specifically stained structures were defined by a gray-threshold discrimination. Within the PVN and the arcuate nucleus, respectively, all pixels with gray values larger than the mean gray value of a background area + 3 x SD of the mean gray values within the standard area (see previous text) were defined as specifically stained. MGSSA is the mean gray value of this discriminated area. ODs were calculated by ODSSA = -log (MGSSA). The method is described in detail elsewhere (23).

Statistical analysis
Data for metabolic balance, leptin, and insulin concentrations were evaluated by ANOVA with genotype as factor. If more than one litter or experimental group of the same age were investigated, data were evaluated by additionally considering a combined litter-sex factor (24) in the juveniles or the experimental group (only female adults) by two-way-ANOVA. Relationships between plasma leptin levels and body fat content were also evaluated by regression analysis to consider interlitter differences in growth and total fat mass (25). Least square means (±SE) provided by two-way ANOVA or arithmetic means (±SE) provided by one-way ANOVA were used to describe averages.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To address the function of central GRs in growth, the body masses of GRNesCre mice and control mice were compared. Mutant mice show progressively reduced growth rates irrespective of gender (Fig. 1AGo). The body mass of mutant mice differs significantly (P < 0.05) from that of control littermates from postnatal d 2 onward. By 1 yr of age, their body mass is only 50% of that of controls (19.1 ± 2.3 g vs. 36.7 ± 3.4 g, n = 12/12). In adult mutants, this reduction is associated with reduced body length (10.6 ± 0.8 cm for controls vs. 8.9 ± 0.4 cm for mutants, n = 12/12, P < 0.001). This is in line with elevated GC levels in mutant mice (control vs. mutant morning values: 1.12 + -0.56 µg/dl vs. 14.4 + -11.02 µg/dl, P < 0.05; evening values 8.67 + -6.91 µg/dl vs. 62.42 + -23.58 µg/dl, P < 0.05), which may act catabolically (26) and inhibit bone formation (27). In addition to a 50% smaller FFDM (Fig. 1CGo), adult mutants also have a 75% smaller total fat mass (Fig. 1BGo). Thus, adult GRNesCre mice display reduced body fat content as well as altered fat distribution (18). Translated into terms of body energy content, these differences result in strongly reduced body energy content of mutant animals (Fig. 1EGo).



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  		Figure 1. A, Body mass growth of individual control (gray) and mutant mice (black). From the start of weighing on postnatal d 2, least square means differ significantly between genotypes (P < 0.05, ANOVA with genotype and litter-sex as the factors). B through E, Developmental changes in body fat mass (B), FFDM (C), body fat content (D), and whole-body energy content (E) of control (gray lines) and mutant (black lines) mice from suckling age to adulthood. Least square means (±SE), N > 15 for sucklings and adults and N > 8 for 35-d-old. *, P < 0.05; ***, P < 0.001 for differences between genotypes. F, G, Box plots for GH and IGF-I plasma levels in adult control (gray) and mutant (black) mice, n = (con/mut): GH = 10/10, P = 0.36, IGF-1 = 7/7, P < 0.001 in two-sided t test.

 
Following the metabolic development of GRNesCre mice from suckling age to adulthood shows that deposition of fat mass is normal during suckling age (Fig. 1BGo). However, FFDM growth lags persistently behind that of the controls from the beginning (Fig. 1CGo). Consequently, at weaning (d 21) the percentage of body fat content (Fig. 1DGo) is increased by 60%. After weaning, however, body fat mass increases little further, and, as the result, percent body fat content of adult GRNesCre mice is reduced in comparison with adult control mice (Fig. 1DGo). Thus, body energy content of GRNesCre mice lags progressively behind that of the control animals (Fig. 1EGo). The reduced body mass of adult GRNesCre animals is associated with a decrease in plasma levels of IGF-1 (Fig. 1GGo), whereas GH levels (Fig. 1FGo) do not seem to be affected by the absence of GR in the brain. The observed combination of reduced body mass with reduced IGF-1 but normal GH levels in adult GRNesCre mice corresponds to the observation of retarded growth in mice with a mutation causing deficient IGF-1 at normal GH signaling (28).

To understand the reasons for the postweaning change in body composition, we continuously measured energy expenditure from weaning to d 35 and calculated metabolizable energy intake and efficiency of energy deposition (Fig. 2Go). Food intake and energy expenditure are both significantly lower (Fig. 2, A and CGo), but the energy contents of the feces (not shown) do not differ between GRNesCre and control littermates (16.8 ± 0.1 vs. 17.0 ± 0.1 kJ/g). In total, during the first 2 wk after weaning, the metabolizable energy intake in mutant mice is reduced by 30%, and the deposited energy by 50% (350 ± 16 kJ vs. 530 ± 18 kJ, P < 0.001, 30 ± 7 kJ vs. 58 ± 8 kJ, P < 0.05, respectively). This corresponds to a reduction in net efficiency of metabolizable energy utilization by 25% in mutant, compared with control mice (8.3% ± 1.5% vs. 11.0% ± 1.5%). A lower energy deposition in mutant animals could be owing in part to an increased requirement for thermogenesis at the laboratory temperature of 22 C, caused by their smaller body size. Indeed, when normalized using the commonly accepted two-thirds power of body mass relation, both food intake and energy dissipation were similar for both genotypes (Fig. 2, B and DGo).



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  		Figure 2. Means (±SE) of daily food intake and energy expenditure (EE) of control (gray) and mutant (black) littermates between postnatal d 21 and 35. Absolute values (A, B) and values per body mass (BM) to the two-thirds power (C, D) to normalize for differences in body surface/mass ratio. N (con/mut) = 3/4. **, P < 0.01; ***, P < 0.001 for genotype differences in one-way ANOVA.

 
We repeated the measurements of energy expenditure at thermoneutral conditions in adult animals (36 C for mutant and 34 C for control mice, Fig. 3AGo). Expressing energy expenditure in relation to the two-thirds power of body mass (Fig. 3BGo) indicated a slightly but significantly higher body surface-related metabolic rate in adult GRNesCre mice, compared with their controls at 22 C ambient temperature. At thermoneutrality, however, this difference disappeared. The most likely explanation is that in addition to their smaller size, a lower thermal insulation of adult GRNesCre mice becomes effective at moderately cold temperatures (22 C). Indeed, we observed a clearly reduced fur density of the adult GRNesCre mice, compared with the controls.



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  		Figure 3. Means (±SE) of energy expenditure (A, B) and plasma T4 (C) of adult control (gray) and mutant (black) mice under cold (22 C) and thermoneutral conditions (TN = 34 C for controls and 36 C for the smaller mutants). Absolute values (A) and energy expenditure per body mass (BM) to the two-thirds power (B). N (con/mut) = 3/3 at 22 C and 10/10 at TN. For T4 data N (con/mut) = 26/26 at 22 C and 10/5 at TN. *, P < 0.05; ***, P < 0.001 for genotype differences in ANOVA with experimental group (or assay number) as the second factor if more than one experimental group (assay) was run. The difference between T4 values at TN and 22 C was also significant (P < 0.01) in the mutants.

 
Because T4 is essential for metabolic cold defense and because the pituitary-thyroid axis is modulated by GCs (29), we measured T4 levels at 22 C and at thermoneutrality. In both adult mutants and controls, the T4 levels are higher under moderately cold conditions than at thermoneutrality, indicating a normal response of the thyroid gland to cold stimulation (Fig. 3CGo). Generally, however, the T4 levels of adult GRNesCre mice were lower than in the control animals.

Two key hormones involved in the regulation of energy homeostasis are insulin and leptin (16, 30). Changes in the concentration and sensitivity of both hormones could be an underlying cause for the reduced fat deposition in GRNesCre mice. Both plasma insulin and leptin levels are higher in mutants than in control littermates at 10, 21, and 35 d of age (Fig. 4, A and BGo), although for insulin the difference is significant only at d 21. However, in adult animals the concentrations of both no longer differ between GRNesCre and control mice. Because plasma leptin levels are normally very closely correlated with body fat, we performed a comparative regression analysis of the relationships between these two variables as functions of age and genotype. As demonstrated in Fig. 4CGo, the slopes of these regressions decrease with age, but for each age group, the slopes determined for the GRNesCre mice are significantly steeper than those for the controls. Similar results were obtained when plasma leptin concentrations were correlated with percent body fat content rather than with total fat mass (not shown). The data indicate a consistently altered control of plasma leptin levels in the mutants.



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  		Figure 4. Plasma insulin (A) and leptin (B) levels of control (gray) and mutant (black) mice from suckling age to adulthood. Least square means (±SE). *, P < 0.05; **, P < 0.01; ***, P < 0.001 in ANOVA with genotype and litter-sex or experimental group as the factors. C, Plasma leptin as a function of body fat in 10-d-old (dotted lines), 21-d-old (dashed-dotted lines), 35-d-old (dashed lines), and adult (solid lines) control (gray) and mutant (black) mice. N (con/mut): d 10 = 16/15, d 21 = 18/20, d 35 = 11/8, adult = 12/12. In each age group, regression lines (0.60 <= r <= 0.95) for control and mutant mice differ significantly (P < 0.05 for differences in slopes).

 
Reduced energy deposition and dysregulated plasma leptin levels may result from a disturbed central regulation at the level of the hypothalamus. Because a direct role of GCs in the central regulation of body fat content has been postulated (31, 32), we determined whether the absence of GRs in the brain affects the expression levels of several hypothalamic neuropeptides known to control energy balance. Using immunohistochemistry we observed an up-regulation of NPY levels (Fig. 5Go). NPY immunoreactivity is enhanced in mutant vs. control mice in the ARC, where the peptide is produced (4-fold, n = 5/5, OD: 4.0 ± 0.6 vs. 1.1 ± 0.3, P < 0.01, two-sided t test) and in the PVN, where it is released (2-fold, n = 5/5, 2.5 ± 0.6 vs. 1.2 ± 0.3, P < 0.01). As already described we also measured an up-regulation of CRH in the PVN (Ref. 18 and Fig. 5Go). No differences between mutant and control littermates were observed in neuropeptide concentrations of MCH and Orexin A and B (Fig. 5Go).



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  		Figure 5. Immunoreactivity (IR) for hypothalamic peptides in adult control (left) and mutant (right) mice. Shown are immunohistochemical stainings with antibodies against these peptides. From top to bottom: NPY IR in the arcuate nucleus; NPY IR in the PVN; CRF IR in the PVN; MCH IR in the lateral hypothalamus; Orexin A and B IR in the lateral hypothalamus.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To study the function of central GRs in body weight regulation, we analyzed mice in which the GR gene has been selectively inactivated in the nervous system. Postnatal development of mutant mice deviates from control mice in a nonmonotonal fashion. Before weaning, growth retardation is associated with increased body fat content and thereafter with reduced body fat content. The immediate consequences of central GR deficiency, enhanced paraventricular CRH levels, and peripheral hypercorticosteronism are known to exert primary direct effects on energy balance. However, neuroendocrine deviations also develop secondarily, in part as the result of compensatory mechanisms. Primary and secondary deviations mutually interfere with one another and, together with environmental influences, affect the energy balance from early developmental phases until adulthood. Therefore, a continuous and multiparametric analysis is required from suckling age to adulthood (33).

Onset of abnormal energy balance regulation in suckling-age mutants
A decrease in food intake may cause the reduced accumulation of lean body mass in suckling-age GRNesCre mice, whereas reduced thermoregulatory energy expenditure may be responsible for the preweaning maintenance of normal body fat deposition. These conclusions are indirectly supported by studies performed with mutated (fa/fa) and normal suckling-age rats. Pups with defective leptin receptors (fa/fa) display reduced energy expenditure but normal food intake resulting in increased body fat content but normal growth (34, 35). Conversely, cold exposure (34), norepinephrine (36), or leptin treatment (25, 37) that increase energy expenditure but do not affect food intake selectively decrease body fat content but never compromise growth of lean body mass in wild-type rat pups (33). Only in response to under- and overnutrition at uncompromised metabolic cold defense, parallel decreases and increases of lean body mass and fat content, respectively, occurred (38, 39). The observation that in the suckling-age GRNesCre mice of this study lean body mass grew less, but fat deposition remained normal thus suggests that both food intake and thermoregulatory energy expenditure were decreased.

A lowered adrenal responsiveness, typical in suckling wild-type mice, may be excluded as an explanation for the temporarily increased body fat content of GRNesCre mice. Generally, hypercorticosteronism as opposed to a lack of glucocorticoid action is associated with enhanced body fat content that develops independently of changes in food intake (40). We measured strongly increased CRH and GC levels in adult GRNesCre mice. Rather, prenatal hypercorticosteronism is a likely assumption because the central GR gene is already inactivated during embryogenesis because of the activity in neuronal precursor cells of the nestin promoter driving Cre recombinase. The consequence would be an early disruption of the paraventricular negative feedback mechanism, similar to what has been observed in ubiquitous GR knockout mice. GR knockout mice show strongly increased paraventricular CRH and blood corticosterone levels during embryogenesis and at birth (41, 42), and CRH was identified as the major target for the hypothalamic feedback control (21).

Postweaning changes in energy balance of mutants
After weaning, both lean body mass growth and fat accumulation are reduced in mutant animals. The weaning process is characterized not only by transition from milk as a high fat diet to chow as a carbohydrate-rich diet but also by enzymatic and hormonal changes that include the HPA-axis (6, 43). A change in the relation between energy expenditure and food intake is also observed in other models of deviant metabolic regulation, such as the fa/fa Zucker rat or the monosodium-glutamate-treated rat (44, 45, 46, 47). For the GRNesCre mice, low rates of food intake and, consequently, reduced growth of lean body mass persist unaffected by the weaning process, emphasizing their primary character. In contrast, fat deposition is reduced only after weaning, in line with the permanently enhanced thermoregulatory energy expenditure that is required under standard laboratory conditions. The ability of mutants to activate T4 release in the cold indicates that their ability to cope metabolically with the thermoregulatory requirements of the laboratory environment is not impaired. Their T4 levels are, however, generally lower than in the controls, probably because of the suppressive action of elevated GC levels on the pituitary-thyroid axis (29).

The role of CRH in determining metabolic efficiency
One primary and very important consequence of central GR deficiency is the strong up-regulation of CRH in the parvocellular neurons of the PVN. CRH acts catabolically by inhibiting food intake and stimulating energy expenditure. In ob/ob mice, blockade of CRH production by elevated plasma GC is proposed to be a major factor responsible for the observed hyperphagia and reduced brown adipose tissue thermogenesis leading to obesity. Adrenalectomy normalizes energy balance in ob/ob mice and GC resubstitution abolishes this effect most likely by suppression of CRH expression (3). Thus, following the rationale that central GR deficiency generates a condition that is with respect to CRH analogous to that of adrenalectomy, the reduced efficiency of energy deposition in postweaning mutants is not surprising. The absence of changes in MR expression in the brain of adult GRNesCre mice excludes adaptation of the MR system as a compensatory process for the loss of GR receptor function (Kretz, O., unpublished data). This is in line with data obtained from mice with general GR deficiency, which remain fully glucocorticoid resistant (48) and in which the CRH system remains the major target for the hypothalamic glucocorticoid feedback control (21).

Hyperleptinemia and hyperinsulinemia: effects and causes
In addition to hypothalamic CRH levels, absolute plasma concentrations for two major metabolic hormones, leptin and insulin, are up-regulated in young GRNesCre mice, but this difference disappears in adults. The hyperleptinemia observed throughout the suckling period should prevent the prevalence of fat deposition over lean body mass growth (25, 37, 49); hence, the high body fat of GRNesCre weanlings suggests leptin resistance. Hyperleptinemia, relative to body fat content, persisted in GRNesCre mice from suckling age until adulthood. It was neither affected by the weaning process nor by the biphasic development of body fat content with initial obesity and subsequent progressive leanness. In mutant mice, chronic elevation of plasma GC as a primary factor and hyperinsulinemia as a secondary factor most likely enhance leptin production (16, 17, 31).

When considering the cause-and-effect relation, hyperinsulinemia as a potential cause of leptin resistance has to be taken into account during suckling age, considering recent observations in overnourished and leptin-treated rat pups (39). With the normalization of the absolute leptin levels after weaning, hyperinsulinemia is also diminished in GRNesCre mice despite persisting hypercorticosteronism. One explanation for this could be a ß-cell cytotropic effect of GCs that would imply the development of diabetes when the functional reserve of the pancreas becomes limiting (50).

Possible mechanisms underlying increased NPY levels and their lack of effect
GCs directly affect the central regulation of body weight by regulating the expression of hypothalamic neuropeptides. Therefore, we measured the expression levels of several neuropeptides known to be involved in the hypothalamic regulation of body weight. In addition to the already discussed up-regulation of paraventricular CRH, we observed increased NPY levels in the ARC nucleus in which NPY is synthetized and in the PVN in which it is released. CRH and NPY differentially affect food intake and energy expenditure. Whereas CRH has anorexic and thermogenic properties, NPY has the opposite effects (13, 17). In view of this antagonism, knowing the sequence of their actions is important. Adrenalectomy was shown to prevent anabolic effects of NPY infusions (51). This could be owing to NPY requiring central GC signaling for its function (52) or to simultaneously increased CRH levels. Functional impairment of hypothalamic CRH neurons was shown to enhance NPY-induced food intake (53). Evidence that the CRH level in the PVN is inversely related to the orexigenic action of NPY was provided in rats by showing that central administration of the CRH antagonist {alpha}-helical CRH 9–41 potentiated the orexigenic effect of NPY injected into the same locus (54). Conversely, NPY deficiency induced by targeted genetic deletion did not impair the antiorexigenic action of CRH (55). Thus, the lower the level of the CRH signal, the stronger is the action of NPY, but the action of CRH is independent of the NPY signal. This may explain why the high NPY levels found in GRNesCre mice do not effectively counteract the suppression of food intake and the general catabolic conditions.

The question remains why the NPY signal is enhanced in these animals. Its increased expression in the ARC is probably not a direct consequence of GR inactivation because GCs increase the expression of NPY mRNA levels in wild-type animals (56, 57). Orexin may be excluded as a stimulator of NPY because the Orexin signal is not enhanced. Most likely, inputs from the periphery stimulate NPY expression as a meaningful response to massively reduced food intake and energy storage that characterize postweaning GRNesCre mice. Changed plasma levels of metabolites and hormones other than leptin might reflect the reduced energy reserves and increase NPY expression in the highly redundant control system designed to prevent starvation (58).

In conclusion, inactivation of the GR gene in the nervous system affects energy accumulation in two ways. First, it leads to a reduction in the growth of lean body mass from suckling age on. Second, it is associated with an increased body fat content until weaning but a reduced accumulation of fat after weaning. The missing negative feedback control of CRH and the resulting hypercorticosteronism are the main primary alterations. They do not only affect growth directly but also modulate other feedback regulations in ways raising difficulties for the analysis of cause-and-effect relationships. The present study on GRNesCre mice highlights the problems encountered in analyzing the pathophysiological development of chronic metabolic disturbances especially for a genetic lesion that becomes effective already at early stages of development. Altered hormone levels because of the genetic lesion at critical stages of development (59) may have permanent effects on the organism (33), which may differ from the consequences of the genetic lesion in the adult animal. The HPA-axis is a good example for this because GC levels around birth have an influence on the HPA-axis activity in the adult animal (60).


   Acknowledgments
 
We are grateful to Edgar Weigand, Giessen, for competently carrying out the bomb calorimetric measurements. We thank Sally Till for critically reading the manuscript.


   Footnotes
 
This work was supported by DFG (Schm 680/4-1).

1 *C.K. and S.E. contributed equally to the study. Back

2 Present address for C.K.: Center for Neurobiology and Behavior, Columbia University, 722 West 168th Street, New York, New York 10032. Back

3 Present address for F.T.: Molecular Genetics and Neurophysiology, Collège de France, CNRSFRE2401, 11 Place M. Berthelot, 75231 Paris Cedex 5, France. Back

Abbreviations: FFDM, Fat-free dry mass; GC, glucocorticoid; HPA-axis, hypothalamic-pituitary-adrenal axis; MCH, melanin concentrating hormone; MGSSA, mean gray value of a specifically stained area; PVN, paraventricular nucleus.

Received October 10, 2001.

Accepted for publication February 21, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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