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CRH-ACTH-POMC-ADRENAL |
Department of Biochemistry and Molecular Biology (M.S.S., I.L.) and Department of Pathology (A.W.Z.), Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112
Address all correspondence and requests for reprints to: Iris Lindberg, Ph.D., Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1901 Perdido Street, New Orleans, Louisiana 70112. E-mail: . ilindb{at}lsuhsc.edu
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Abstract |
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Introduction |
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7B2 null mice exhibit a very complex pathology that in many respects resembles Cushing’s disease; this gene deletion represents a lethal phenotype in 129/Sv mice (7). 7B2 nulls exhibit severe pathologies of liver, pancreas, and pituitary and die in the prepubertal or pubertal ages. The Cushing’s-like disease developed by 7B2 null mice takes the form of pituitary-dependent hyperadrenocorticosteronism, severe hypoglycemia, hyperproinsulinemia, adrenal hypertrophy, pituitary hypotrophy, and altered islet cell morphology (7). Unlike classical Cushing’s disease, which results from excess ACTH secretion from pituitary adenomas, 7B2 null mice develop this disease from intermediate lobe ACTH hypersecretion. These mice resemble mice deficient in the dopamine receptor 2, which also develop a specific form of Cushing’s-like disease; both kinds of null mice suffer from adrenal hypersecretion of corticosterone because of excess circulating ACTH (8, 9).
The diagnosis of Cushing’s disease seems to be insufficient to account for the sudden death of 7B2 nulls within a specific period of life, the prepubertal to pubertal phase (4–6 wk old). Because they are devoid of PC2 activity, 7B2 null mice exhibit deficiencies in the processing of proglucagon and to a lesser extent proinsulin (7). This lack of PC2-mediated peptide processing is not a symptom of typical Cushing’s disease and most likely accounts for many of the differences of this null model with Cushing’s. However, certain clinical symptoms of 7B2 null mice are consistent with those of Cushing’s, such as abnormal fat disposition, especially on the back and around the neck. Other clinical symptoms that are consistent with severe Cushing’s include changes in skin; compared with wild-type (WT) mice, 7B2 null mice exhibit marked thinning of the skin and dermal atrophy with hyperkeratosis. Livers of 7B2 nulls were previously found to be markedly abnormal, with severe fat vacuolization. However, all of these clinical signs are not sufficient to explain the sudden expiration of 7B2 null mice, in which rapid deterioration and death occur within a 6-h time frame. In this report we have further investigated the pathological changes occurring the 7B2 null, with emphasis on the actual cause of death.
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Materials and Methods |
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When used, anesthesia using Avertin produced better results in terms of recovery and was better tolerated than ketamine-xylazine anesthesia. Animals were anesthetized ip with Avertin (administered at a 2.5% solution in 0.9% NaCl; 0.015–0.020 ml/g mouse body weight) before venopuncture from the retroorbital plexus or heart. Avertin in the dose mentioned above produced short-term anesthesia without serious side effects (such as convulsions and respiratory failure) and yielded rapid recovery.
Necropsy
All animals in these experiments were subjected to necropsy, performed either immediately after death or within a short period after death (2–3 h), maintaining carcasses at 4–8 C. Samples for biochemical investigations were collected and frozen at -20 C, and samples for histopathologic evaluation were placed in tubes containing zinc formaldehyde. Representative sections of organs and grossly identifiable lesions were processed, blocked in formalin, and then stained with hematoxylin and eosin for microscopic analysis.
Biochemical and clinical chemistry analysis
Blood glucose.
Every second day blood glucose was measured using a standard glucometer (Lifescan One Touch, Milipitas, CA). Blood was obtained either by lateral tail venopuncture or cardiac or retroorbital plexus venopuncture. Plasma glucose was also measured using a kit for glucose (Sigma, St. Louis, MO) for comparison with data obtained with a Lifescan glucometer. We did not detect significant differences in glucose levels between the two methods.
Analysis of tissue free glucose and glycogen
Free tissue glucose was measured by a quantitative, enzymatic method for glucose determination (10). After homogenization of previously weighed tissue in 1 N acetic acid and centrifugation at 10,000 rpm, the supernatants were deproteinized using 0.3 N barium hydroxide and zinc sulfate. After centrifugation the clear supernatant was used for quantitative enzymatic glucose determination using a kit (Sigma). The reaction is based on the coupled enzymatic reactions of glucose oxidase and peroxidase followed by reaction with the chromatogenic oxygen acceptor o-dianisidine. The intensity of the brown color was measured at 475 nm and is proportional to the original glucose concentration. Plasma for glucose analysis was prepared immediately after venopuncture (subanesthesia using Avertin) from the retroorbital plexus for 7B2 nulls (in light of their very bad health) and via cardiac puncture for 7B2 WT animals.
For glycogen analysis (11), fresh wet liver and muscle tissues were measured and minced in a Petri dish with a small amount of PBS solution and placed in an tube (Eppendorf, Brinkmann Instruments, Westbury, NY). Homogenization with sonication was performed in 500 µl ice-cold 1 N acetic acid, and samples were then boiled in a water bath for 45 min for digestion of glycogen. After boiling, samples were placed on ice and centrifuged at 10,000 rpm 4 C/10 min. Supernatants were collected in separate tubes. Samples were deproteinized and used for the analysis of enzymatic determination of glucose residues by the protocol described above for free-tissue glucose. Tissue glucose content was calculated as gram of glucose residues per gram of wet tissue after acid digestion.
Metyrapone and glucagon treatment
Metyrapone (2-methyl-1, 2-di-3-pyridyl-1 propane, obtained from Aldrich Chemical Co., Inc., Milwaukee, WI) was dissolved in 0.5% methylcellulose (Sigma) and force fed to mice at 1 mg/10 g body weight twice per day (12). Groups treated with metyrapone alone as a control received 0.9% saline solution intramuscularly. Groups treated with metyrapone and glucagon (50 µl of 0.05 mg/ml, Elli Lilly and Co., Indianapolis, IN) received drugs im. Animals were fed twice per day at the same time.
RIA of corticosterone and ACTH
Plasma was prepared from blood obtained through venopuncture. Plasma was collected in tubes (Eppendorf) and stored at -70 C until the assay procedure. The ImmuneChem 125I Corticosterone RIA (ICN Biomedicals, Inc., Costa Mesa, CA), specifically designed for use in laboratory mice and rats, was used to measure plasma corticosterone. For measuring plasma ACTH, we used the ACTH RIA kit (human ACTH 1–39 assay kit, Nichols, San Juan Capistrano, CA). This assay is specific for intact ACTH 1–39.
Lactate and magnesium levels in plasma and tissues
The level of plasma lactate and magnesium in plasma and tissues was determined using Sigma kits. Lactate was measured using an enzymatic method and the absorbance of the colored product was measured at 540 nm. Magnesium was measured using a quantitative, colorimetric method, and the absorbance of the product, a pink magnesium-calmagite complex, was measured at 520 nm.
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Results |
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Gross pathology of 7B2 null mice at necropsy
7B2 nulls exhibited a typical Cushingoid phenotype, with small muscle mass, and typical alterations of the skin with hyperemia and dermal atrophy. The fur was significantly thinner, with regional alopecia and a specific buffalo hump in the dorsal cervical region.
Features of the gross pathology of the 7B2 null mouse are shown in Fig. 1
. Significant hepatomegaly was observed (Fig. 1B; compare with control in Fig. 1A), and the liver was yellow, indicating macroscopic signs of fatty change. The total mass of wet liver in 7B2 null mice was 1.1 ± 0.08 g, n = 8, compared with WT mice, which had a weight of 0.41 ± 0.01 g (n = 7; P < 0.001, compared with controls). The hepatomegaly occasionally produced strong tension on the diaphragm. Perforations of the gallbladder (Fig. 1C), hemothorax (Fig. 1C), and/or intraabdominal hematomas were often present. The spleens were decreased in size and retroperitoneal fat deposits were increased. The total mass of spleen in 7B2 null mice was 14.1 ± 0.4 mg wet weight, compared with the mass detected in 7B2 WT mice, 68.1 ± 2.6 mg (P < 0.001). Polyphagia in 7B2 nulls was recognizable by the frequent presence of a greatly distended stomach, always full of food. Significant lymphoid atrophy was present. Cardiomegaly (141 ± 13 mg wet weight in 7B2 nulls, compared with 52 ± 4 mg in WT mice; P < 0.001) was present with pigmentation on the ventral side of the heart, and significant myocardial calcification was detectable on palpation. Cryptorchidism was often detected in males. We observed hypotrophic alterations in the anterior pituitaries in all null mice. In contrast, the adrenal was hypertrophic, sometimes presenting an intensive red color.
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Histopathologic (microscopic) findings in 7B2 null mice at necropsy
The most significant organ microscopic findings were observed in the liver. All mice had microvesicular and macrovesicular steatosis (Fig. 2A) in a perivenular (acinar zone 2 and 3) or panacinar pattern. Hepatic necrosis was also present and ranged from focal to widespread in a midzonal, periportal, or combined pattern (Fig. 2B). For comparison, normal liver histology (WT mouse) is shown in Fig. 2C. In one instance, massive necrosis was observed. Pulmonary edema and congestion were present and most likely represented an agonal event. The gross observations of myocardial calcifications were confirmed by the presence of dystrophic calcification in the myocardium by microscopy.
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129/Sv 7B2 null mice are severely hypoglycemic at the time of death
Blood glucose, measured in blood obtained by lateral tail venopuncture, was less than 20 mg/dl several hours before death (Fig. 3A). The normal level of blood glucose in 7B2 nulls is approximately 75 mg/dl (7). One to 2 d before death, the level of glucose significantly decreased; at this time the animals also appeared to become hypothermic.
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). 7B2 nulls exhibited a high level of free tissue glucose in the liver. Because circulating glucose is low, this free glucose is apparently unable to be released into the peripheral circulation. However, 7B2 null brains had a higher level of free tissue glucose, compared with WT animals. Muscle tissues (musculus gastrocnemius and musculus soleus) of 7B2 nulls showed a significantly decreased level of free tissue glucose, compared with the levels detected in 7B2 WT controls.
7B2 null mice exhibited levels of liver glycogen five times higher than those found in the livers of WT mice (Fig. 3C). Significant differences between the sexes of 7B2 null mice in the concentration of liver glycogen were detected (P < 0.05; data not shown). 7B2 males had less liver glycogen than 7B2 females of both genotypes, both in WT and null (P < 0.05).
Blood glucose is elevated in the 7B2 null after treatment with metyrapone
Treatment of 7B2 WT animals with the steroid synthesis inhibitor metyrapone (at a concentration of 1 mg/10 g of body weight twice per day) resulted in a significant increase in the level of blood glucose (Fig. 4A) (P < 0.001 vs. control group treated with vehicle). Metyrapone treatment was carried out for 21 d; no significant change was detected between 5 d and 21 d of treatment.
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) exhibited a significant increase in blood glucose. During the first 3 d of treatment with metyrapone, animals in the metyrapone group and the group with the combination of metyrapone and glucagon were tested for reactions to glucagon administered intramuscularly. Both groups showed significant changes in level of blood glucose after im administrations of glucagon, compared with the untreated group and the group of mice treated with methylcellulose. After the third day, one group was continued on treatment with metyrapone alone, and another group was continued on a combination of metyrapone with glucagon. Only the group treated with the combination of metyrapone and glucagon exhibited a significant increase in blood glucose, compared with untreated animals and animals treated with the metyrapone vehicle, methylcellulose. The level of glucose was maintained at about 120 mg/dl until the end of treatment. Changes in behavior such as increased activity and increased aggressiveness during handling were noticeable during the first 10 d of treatment, particularly in the group treated with the combination of metyrapone and glucagon. However, this change was transient, and treated animals in both groups died in the same critical period, between 4 and 5 wk old.
Hypercorticosteronism can be rescued by metyrapone treatment in 7B2 null mice
Figure 5A shows results of the corticosterone RIA in 7B2 null mice, supporting previous results (7) that indicate that 7B2 nulls exhibit much higher levels of corticosterone, compared with WT controls. Treatment of 7B2 WT mice with metyrapone produced a significant decrease of plasma level of corticosterone vs. the group of 7B2 WT animals treated with vehicle. A significant decrease in plasma corticosterone was detected, from 150 ng/ml plasma corticosterone in the control group to less than 50 ng/ml of plasma corticosterone in a group treated with metyrapone (Fig. 5B). 7B2 nulls treated with metyrapone alone or metyrapone in combination with glucagon exhibited a significant decrease (P < 0.0001) of circulating corticosterone (less than 100 ng/ml), compared with animals in the group treated with methylcellulose (circulating corticosterone < 500 ng/ml) (Fig. 5C).
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). The plasma ACTH of 7B2 nulls treated with metyrapone was not significantly different vs. both control groups (Fig. 5E).
7B2 nulls exhibit severe lactic acidosis and hypomagnesia
The level of lactate in the plasma of 7B2 null mice was five to six times higher than in WT mice (Fig. 6A). No significant differences were observed between the sexes, but significant differences were noted between genotypes (P < 0.0001).
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). However, the levels of magnesium in the livers of 7B2 null mice were significantly increased 2-fold, compared with 7B2 WT mice. A similarly increased level of magnesium was also detected in the brains of 7B2 null mice. Muscle tissue showed no significant differences between WT and null genotypes. |
Discussion |
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Interpretation of necropsy findings
During necropsy of 19 7B2 null animals, all animals exhibited certain typical pathological alterations present in Cushing’s disease as well as several pathologies not typical of that disease. Severe fatty and necrotic changes in the livers as well as hepatomegaly and intensive bleeding in the thoracic and abdominal cavities were always observed.
The patterns of steatosis were not specific but resembled those seen in acute fatty liver of pregnancy (acinar zones 2 and 3), Reye’s syndrome (panacinar), and Cushing’s syndrome (13) in humans. The hepatic findings in these mice also indicate acute liver failure and the observations of apparent bleeding diathesis most likely result from a decrease in liver-dependent coagulation factor synthesis, a common, and one of the first, clinical manifestations of liver failure. Another possible contributing factor to the observed hemorrhage could be vascular fragility, a typical manifestation of Cushing’s disease. These processes, in concert, could explain the systemic hemorrhage seen in the 7B2 null mice at necropsy. Intraabdominal bleeding has been frequently noted in 7B2 null newborns (7 and unpublished results).
Glucose and fat metabolism are altered in 7B2 nulls
Severe hypoglycemia is a common metabolic abnormality seen in human and veterinary medicine. A life-threatening condition, hypoglycemia is most often easily diagnosed and rapidly treated with a satisfactory outcome. If not recognized and promptly treated, hypoglycemia may cause irreversible central nervous system injury and, rarely, results in death (14). During their first 3 wk, 7B2 nulls exhibit moderate hypoglycemia with daytime blood glucose levels of 75 mg/dl. This phenomenon is likely to be caused by the lack of circulating glucagon. Approximately 1–2 d before death, blood glucose rapidly decreases to 20–25 mg/dl. This severe hypoglycemic condition can occasionally be more even drastic immediately before death at 16 mg/dl (in four cases). Superimposed on a preexisting chronic hypoglycemic condition complicated by acute liver failure with associated bleeding diathesis and metabolic derangement, severe hypoglycemia most likely represents the precipitating cause of death in 7B2 nulls.
Liver glycogen measurements support the hypothesis that glucose is immobilized in 7B2 nulls. Indeed, the increase in liver glycogen suggests that 7B2 nulls exhibit a unique form of glycogen storage-like disease. Glycogen storage disease type 1a, caused by a deficiency in glucose phosphatase, is characterized by severe hypoglycemia, liver enlargement, growth retardation, hyperlipidemia, and hyperuricemia (15). The 7B2 null mice have a relatively similar clinical picture. It is interesting to note that male double nulls for PC2 and 7B2 do not exhibit a lethal form of Cushing’s (Laurent, V., and I. Lindberg, unpublished data) and also do not exhibit high levels of liver glycogen (0.072 g/g vs. 0.120 g/g for 7B2 nulls). Thus, high levels of liver glycogen are indicative of a metabolic abnormality associated with the disease process.
In the 7B2 null, glucose is apparently not able to diffuse from the tissues into the circulation. Abnormal distribution of free tissue glucose was especially prominent in the liver of 7B2 nulls, in which the free glucose level was four times higher than the level detected in 7B2 WT animals. The liver and brain represent two areas of aberration in glucose metabolism in the 7B2 null. In accordance with our observations of severe hypoglycemia, the liver and brains of 7B2 nulls contain a high concentration of free tissue glucose and glucose as glycogen. There are two explanations for this, a defect in the cleavage of glycogen molecules into molecules of glucose or a defect in the release of glucose from the liver into the circulation. 7B2 nulls do not produce substantial levels of mature glucagon, the hormone responsible for release of glucose from the liver into the circulation (7). However, the PC2 null, which also lacks glucagon, exhibits no comparable alterations in glucose metabolism (16). Correct levels of circulating glucagon, balanced with proper levels of insulin, are required to maintain normal blood glucose levels (16). The glycogen storage-like disease present in 7B2 null mice could represent a consequence of the effects of chronically elevated corticosterone on glucose metabolism combined with an increase in circulating proinsulin forms and the absence of mature glucagon in 7B2 nulls.
Pituitaries of PC2 null mice contain high levels of intact ACTH 1–39, but the PC2 null does not exhibit high levels of corticosterone, most likely because of the lack of highly increased circulating ACTH in this null model; the increased ACTH in the pituitary is apparently not subject to release in the PC2 null (16A ). Excepting slight hypoglycemia, PC2 nulls do not exhibit Cushingoid pathologies similar to the 7B2 nulls and like 7B2 nulls, PC2 nulls also lack active glucagon and exhibit increased proinsulin forms. A distinguishing feature between these two null models is the fact that PC2 nulls exhibit normal expression of the 7B2 protein, distributed in organs and in circulation; the fact that the PC2 nulls show no signs of illness points to additional important roles for the 7B2 protein unrelated to PC2-mediated effects.
Corticosterone normally plays an important counterregulatory role during hypoglycemia and augments glucose production, decreases glucose utilization, and accelerates lipolysis (17). Generally glucocorticoids counteract the effects of insulin and stimulate gluconeogenesis (18). Thus, chronically increased circulating corticosterone, in combination with effects on glucagon, would be expected to contribute to many of the pathological alterations in sugar and fat metabolism seen in the 7B2 null.
A major contributing cause of death in 7B2 nulls is likely to be the process of fat replacement in the liver. Hepatic steatosis, observed in our pathohistological analysis, is the end result of abnormal triglyceride synthesis and secretion. Our results are in agreement with other studies of the causes of sudden death resulting from hepatic steatosis with hypoglycemia, hypomagnesemia, and high serum levels of FFA (19).
Treatment of WT mice with metyrapone, a potent inhibitor of corticosterone synthesis, produced a significant increase in blood glucose. However, treatment of 7B2 nulls with metyrapone produced the opposite result. These differences between WT and null mice confirm our hypothesis as to significantly different patterns of glucose utilization in the 7B2 null. Glucagon treatment of 3-wk-old animals was very successful and increased blood glucose to above 100 mg/dl in many animals. However, animals from the metyrapone-treated group and in the group treated with the combination of metyrapone and glucagon died in the same time period, with the same clinical signs as untreated 7B2 nulls.
Hypomagnesemia
A high level of magnesium in the liver can indicate serious alterations in normal liver function. Hypomagnesemia in the plasma and heart tissue of 7B2 null mice suggests that a contributing cause of immediate death in 7B2 nulls might be hypomagnesemia. This pathological alteration is present in chronic liver and renal disease, heart failure, and coma and generates a life-threatening condition. The reason that 7B2 nulls exhibit lower magnesium levels is unclear but might be secondary to alterations in blood chemistry.
Lactic acidosis
Dying 7B2 nulls are in severe lactic acidosis, with levels of plasma lactate in dying 7B2 null mice about 6-fold higher than levels measured in 7B2 WT mice. Excessive concentrations of circulating lactate can produce severe tissue oxygen deprivation, tissue hypoxia, weakness, stupor, fatigue, and circulatory failure with coma (20, 21). Chronic lactic acidosis might be a factor in the process of organ necrosis (confirmed in our histopathologic observations), particularly in the liver. Blood lactate reflects both the production and metabolism of lactate. Normally, the liver is able to remove more lactate than is produced by the body, but the liver in 7B2 nulls is apparently not able to metabolize lactate. This observation supports our conclusion that the liver in 7B2 nulls is highly dysfunctional. Lactic acidosis is a highly critical condition, and, with hypoglycemia, potentially also represents a major cause of immediate death in 7B2 nulls.
In conclusion, 7B2 null mice exhibit a unique form of Cushing’s disease with many atypical symptoms, such as hypoglycemia. Cushing’s and non-Cushing’s symptoms in the 7B2 null join with other metabolic abnormalities to generate a multisystem disorder. The severe hypoglycemia caused by the lack of glucagon, increased proinsulin forms, and chronically elevated corticosterone, coupled with alterations in blood coagulation, all combine to result in multiple organ pathologies including liver failure and precipitate further abnormalities in blood coagulation and blood chemistry, such as lactic acidosis. A hypoglycemic crisis coupled with respiratory distress and intensive internal thrombosis then results in abrupt death.
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Acknowledgments |
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Footnotes |
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Abbreviations: PC2, Prohormone convertase 2; WT, wild-type.
Received September 17, 2001.
Accepted for publication January 22, 2002.
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