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Developmental Endocrinology Branch, National Institute of Child Health and Human Development (S.R.B., E.L.W., D.J.T., S.J.R., G.P.C.); Medicinal Chemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases (D.B.L., K.C.R.); and Department of Pathology, National Cancer Institute (N.M., M.T.), National Institutes of Health, Bethesda, Maryland 20892; and the Department of Internal Medicine III, University of Leipzig (S.R.B.), Leipzig 04103; and the Institute of Pharmacology and Toxicology, Technical University of Aachen (M.J., H.G.J.), Aachen, Germany
Address all correspondence and requests for reprints to: Stefan R. Bornstein, M.D., Clinical Center, National Institute of Child Health and Human Development, National Institutes of Health, Building 10, Room 10N262, Bethesda, Maryland 20892.
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Abstract |
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We conclude that chronic administration of antalarmin does not affect body weight, carbohydrate metabolism, or leptin expression, whereas it reduces adrenocortical function mildly, without anatomical, clinical, or biochemical evidence of causing adrenal atrophy. These results are promising for future uses of such an antagonist in the clinic.
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Introduction |
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The nonpeptide CRH antagonist 154,526 and a close analog, antalarmin, have a high affinity for the CRH type 1 receptor and in acute rat studies block CRH-stimulated ACTH release and carrageenan-induced inflammation and produce anxiolysis (17, 18, 19, 20). The existence of redundant hypophysiotropic factors for ACTH release, such as arginine vasopressin (AVP) and norepinephrine, and the finding that a CRH gene knockout mouse does not have complete deficiency of HPA function (21), raises the possibility that chronic administration of a CRH antagonist may be met with escape and normalization of ACTH and cortisol release, due to increased release of alternate ACTH secretagogues from the hypothalamus. In addition, the known relations between the HPA axis and the regulation of food intake and energy expenditure generate questions as to the potential effects of a chronically administered CRH antagonist on the body weight and metabolic status of an individual. Thus, data on the chronic effects of a CRH antagonist are quite critical in predicting the clinical utility of such an agent. We designed an 11-day study to analyze the chronic effects of antalarmin on pituitary-adrenal function and maintenance of body weight and metabolic status.
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Materials and Methods |
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Animal procedure
Adult male Sprague-Dawley rats were purchased from Taconic Farms
(Germantown, NY) and were housed three per cage under controlled
environmental conditions. Rats were exposed to a standard 14-h light,
10-h dark cycle, and feed and water were supplied ad
libitum. Animals were given ip injections of either vehicle alone
or 20 mg/kg antalarmin twice daily at approximately 10- to 12-h
intervals for 11 days. They were weighed once per day in the morning.
On the 11th day they were decapitated, and trunk blood was collected
into EDTA-containing tubes on ice for hormone determinations. The
decapitation procedure was performed sequentially in a separate room,
and each animal was killed within 3 min of being removed from its home
cage. Abdominal fat and adrenal tissues were rapidly dissected and
either frozen on dry ice and stored at -70 C or stored in fixative as
described below.
A dose of 20 mg/kg was chosen for the chronic studies, as this quantity was previously shown to block the biological effects of CRH on ACTH secretion and carrageenan-induced sc inflammation (18).
General histology and morphometric analysis
Adrenal tissue was fixed in 4% formalin solution, paraffin
embedded and sectioned, then mounted on slides and deparaffinized.
For histology, the slides were stained with hematoxylin-eosin. Sections were cut from the largest diameter of the gland, and the average width of the cortex was determined in each gland (n = 4), with an average of at least six different sections from each adrenal.
Electron microscopy
For the ultrastructural investigations, small pieces of adrenal
tissue were fixed in 4% paraformaldehyde-1% glutaraldehyde in 0.1
mol/liter phosphate buffer (pH 7.3) for 3 h, postfixed for 90 min
in 2% OsO4 in 0.1 mol/liter cacodylate (pH 7.3),
dehydrated in ethanol, and embedded in epoxy resin. Ultrathin sections
(70 nm) were stained with uranyl acetate and lead citrate and examined
at 80 kV under a Phillips electron microscope 301 (Phillips
Electronics, Mahway, NJ).
In situ end labeling
Detection of apoptosis on tissue sections was performed with the
terminal deoxynucleotidyl transferase (TdT)-mediated deoxy-UTP nick
end-labeling (TUNEL) assay. This technique enables identification of
individual cells undergoing apoptosis in tissue sections with
heterogeneous cell populations.
The In Situ Cell Death Detection kit, POD (Boehringer Mannheim, Indianapolis, IN), was used according to the instructions of the manufacturer. Briefly, after deparaffinization and rehydration, the tissue sections were treated with 20 µg/ml proteinase K (Life Technologies, Gaithersburg, MD) at 37 C for 30 min. Endogenous peroxidase activity was quenched in methanol containing 0.5% H2O2 for 30 min. The slides were then washed twice with PBS and incubated with the TUNEL reaction mixture (TdT with fluorescein-labeled nucleotides in TdT buffer) at 37 C for 1 h. Subsequently, they were washed three times with PBS and incubated with Converter-POD (POD-conjugated antifluorescein antibody) at room temperature for 30 min. Diaminobenzidine was used to visualize the apoptotic nuclei. Heavily stained, dark brown nuclei were considered apoptotic.
Quantification of apoptotic rate
The number of positively stained nuclei was analyzed in
representative quadrangular areas of identical size within the zona
fasciculata of the adrenal cortex. In each section, positive steroid
cells per total number of cells were obtained by counting 100 cells;
areas of necrosis were excluded. The results for each group are given
as the percent mean ± SEM of four different areas in
four animals in each group.
RNA isolation and Northern blot
Fat was dissected and immediately frozen in liquid nitrogen. The
samples were homogenized with a Polytron homogenizer (Brinkmann
Instruments, Westbury, NY) in 4 mol/liter guanidine thiocyanate
supplemented with 7% mercaptoethanol. Lysates were layered on a cesium
chloride cushion (5.88 mol/liter) and centrifuged at 28,000 rpm (rotor
SW40) at 20 C for 29 h. Pelleted RNA was dissolved on 300 µl
sodium acetate-Tris buffer and neutralized by the addition of 50 µl 2
mol/liter potassium acetate (pH 5.5). Samples of total RNA were
separated by electrophoresis on 1% agarose gels containing
formaldehyde and transferred onto nylon membranes (Hybond
N+, Amersham-Buchler, Braunschweig, Germany). Before
transfer, gels were stained with ethidium bromide to ascertain that
equal amounts of total RNA had been separated. Probes were generated
with the Klenow fragment of DNA polymerase I and
[
-32P]deoxy-CTP from the partial ob
complementary DNA, as previously described (22), by random
oligonucleotide priming. The nylon membranes were hybridized at 42 C,
and blots were washed once with 0.8 x SCC (standard saline
citrate)-1% SDS for 10 min at 55 C. Autoradiographs were analyzed with
the LKB laser densitometer and the software GelScan 2.0 from Pharmacia
(Piscataway, NJ).
Hormone measurements
For all measurements, blood was taken after decapitation of the
animals into a chilled vial with EDTA and immediately centrifuged at
1600 x g at 4 C for 15 min. Plasma was stored at -70
C until analysis. Leptin was measured by a commercially available RIA
(Linco Research, St. Charles, MO). Controls were used in the low and
high sections of the standard curve. Samples were used in duplicate,
and standards were used in triplicate. The intra- and interassay
coefficients of variation were both below 5%. ACTH and corticosterone
were measured by RIAs (Brahms Diagnostica, Berlin, Germany). The intra-
and interassay variations were less than 8%.
Statistical analysis
Data analyses were performed on an IBM-compatible computer using
SigmaPlot (Jandel Scientific, Chicago, IL) and Statistica (StatSoft,
Tulsa, OK). Differences in plasma ACTH, corticosterone, glucose, and
leptin levels were assessed by unpaired Student’s t test.
Data are expressed as the mean ± SEM.
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Results |
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). Mean morning blood glucose levels
were not significantly different in the two groups (Fig. 1b). Mean
plasma leptin values were 3.4 ± 1 pmol/liter in
antalarmin-treated animals vs. 3.2 ± 0.7 pmol/liter in
controls (Fig. 1c). Northern blot analysis demonstrated variable
ob mRNA expression, with no significant differences between
antalarmin- and vehicle-treated animals (Fig. 1d).
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, a and b).
There was a mild decrease in the adrenocortical widths of
antagonist-treated animals (0.95 ± 0.09 mm) compared with those
of vehicle-treated animals (1.38 ± 0.10; P <
0.005; Fig. 3).
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). In the antalarmin-treated animals,
adrenocortical cells demonstrated an increased number of liposomes and
fewer mitochondria with tubulovesicular inner membranes (Fig. 4B). In
vehicle-treated animals, subcapsular glomerulosa cells demonstrated
characteristic oval or elongated tubulovesicular mitochondria,
perinuclear Golgi apparatus, and filopodia (Fig. 5A). In the antalarmin-treated animals,
mitochondria were more rounded with vesicular inner membranes,
demonstrating a fasciculata-type appearance (Fig. 5B).
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). Cell
shrinkage and condensation of nuclei were observed, whereas
mitochondrial structures were preserved during the early phase in
antalarmin-treated animals (Fig. 7A).
Cells exhibited a blackened nucleus, cell blebbing, and formation of
apoptotic bodies containing cell membranes during later stages (Fig. 7B).
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Discussion |
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Alteration of organelles in adrenocortical cells reflects the functional status of the steroid-producing cells (28). Particularly, the SER and mitochondria reveal an astonishing capacity to adapt to the varying demands for steroidogenesis (29). While hypophysectomy leads to a decrease in SER and inner mitochondrial membranes, reflecting the hypofunctional status of the cells, administration of CRH or stress induces an increase in SER, volume of mitochondria, and volume of the area covered by mitochondrial membranes in the zona fasciculata (29, 30). Consistently, in the animals treated with the CRH antagonist, there was a reduction in SER and inner mitochondrial membranes, providing evidence for chronic understimulation of adrenocortical steroidogenic activity.
Programed cell death or apoptosis preserves adrenocortical tissue homeostasis, zonation, and remodeling (31). Hypophysectomy-induced decreases in ACTH secretion lead to adrenal atrophy by increasing apoptosis of the adrenal cortex, whereas administration of ACTH restores the normal apoptotic rate (32, 33, 34). This is in accordance with our findings suggesting that an increased apoptotic rate in the outer zona fasciculata resulted in a reduction of adrenocortical width in the animals treated with the CRH type 1 receptor antagonist.
Although hypophysectomy does not affect the structure of the zona glomerulosa (34), CRH antagonist-treated animals had less well differentiated glomerulosa cells with a fasciculata-type appearance, as previously described in adrenal autotransplants (35). The expected inhibition of the central autonomic system by the CRH antagonist may be of relevance. Both the sympathoadrenal system and local medullary CRH participate in the regulation of adrenocortical steroidogenesis (35, 36, 37, 38, 39, 40). Therefore, both central and local inhibition of adrenal CRH might contribute to the greater reduction of plasma corticosterone levels than what would be expected from the concurrent decrease in ACTH levels. This is further underscored by our previous finding that CRH reduced adrenocortical atrophy in the absence of pituitary ACTH (40). Indeed, apoptotic cell death is more widespread when the adrenal cortex is disconnected from its innervation and interaction with the adrenal medulla (41), whereas neurally dependent compensatory adrenal growth after unilateral adrenalectomy has been demonstrated (42).
Finally, there was a conspicuous reduction in the vascularization of the inner zona fasciculata and reticularis in animals treated with antalarmin compared with that in controls. It is of interest to note that CRH acts as a very potent vasodilator of the adrenal gland (43). Hence, the antagonist may block the action of CRH released from the adrenal medulla in response to splanchnic nerve stimulation, preventing dilation of the adrenal vasculature (44, 45, 46). Therefore, the dual actions of CRH on the adrenal cortex, via inhibition of ACTH secretion and through the sympatho-adrenal system, may explain why chronic treatment with the CRH antagonist does not fully reproduce the effects of hypophysectomy or treatment with glucocorticoids.
There is a well established marked relation between the nutritional status of mammals and the activity of the HPA axis (47, 48, 49). The striking findings that adrenalectomy can restore the sensitivity to centrally given insulin in obese Zucker (fa/fa) rats that have a defect in the leptin receptor and that there is a marked effect of adrenalectomy on the leptin dose-response curve for the reduction of food intake underscore the important interaction between the HPA axis and the leptin system (4). CRH might, therefore, inhibit feeding and reduce body weight by decreasing the activity of leptin (4). However, chronic antalarmin administration induced no significant changes in body weight, leptin secretion, or leptin mRNA expression, whereas plasma corticosterone levels and adrenal size were reduced. Several explanations may account for these findings. First, the endocrine and metabolic effects may be mediated by different types of CRH receptors. Although CRH stimulates the pituitary-adrenal axis via type 1 CRH receptors (for review, see Ref.50), it has been postulated that the anorectic effect of CRH may be mediated via type 2 CRH receptors. Indeed, urocortin, a CRH-related neuropeptide that appears to be the natural CRH type 2 receptor ligand, had appetite-suppressing effects at doses that did not activate the HPA axis (51). As antalarmin is a selective type 1 CRH receptor antagonist, our data support the concept that the anorectic effect of CRH might be exerted primarily via type 2 CRH receptors.
Chronic administration or excess endogenous glucocorticoids lead to weight gain (49, 52, 53, 54). High doses of glucocorticoids acutely increase leptin concentrations in vitro and in vivo, whereas adrenalectomy can partially normalize or prevent weight gain and obesity (55, 56). While antagonizing the effect of CRH, antalarmin may concurrently suppress its central anorectic effect. However, the simultaneous reduction of adrenal glucocorticoid production may counteract such suppression and its sequelae of weight gain and associated metabolic changes. Glucocorticoids are necessary for sustaining normal glucose concentrations by stimulating gluconeogenesis and causing insulin resistance. The animals were kept in an ad libitum feeding state, which would in-crease the threshold for glucocorticoid deficiency-induced hypoglycemia.
Finally, CRH is only one of a large family of catabolic systems that
induce anorexia, increase thermogenesis, and promote weight loss (4).
Thus, serotonin, urocortin, and the melanocortin MSH (50, 51, 57, 58) or other as yet unknown factors may come into play when CRH is
inhibited. Although leptin is a stress-related peptide and acts on the
HPA axis at central and peripheral levels (7, 9, 10, 11, 12, 59), CRH does
not change the leptin system. In accordance with our data,
administration of ovine CRH to patients with Cushing’s syndrome before
and 10 days after curative surgery resulted in no changes in plasma
leptin levels when measured over a 2-h period after iv CRH
administration, whereas the appropriately elevated baseline plasma
leptin levels in these obese patients before and after surgery remained
unchanged (60).
In conclusion, chronic treatment with antalarmin was well tolerated and had a mild, but clear, suppressive effect on the activity of the pituitary-adrenal axis. ACTH and corticosterone levels were lower, and adrenal size was reduced due to increased apoptosis in the outer zona fasciculata. Antalarmin did not induce significant body weight or metabolic changes, a highly desired property for a clinically useful CRH antagonist.
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Footnotes |
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Received October 7, 1997.
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