Expression of the POMC gene in the adrenal gland and ...



Expression of the POMC gene in the adrenal gland and adrenocortical tumors causing hypercortisolism in dogs

Written by J. Burghard

December 2009

Supervisors:

S. Galac

H.S. Kooistra

J.A. Mol

Department: Clinical Sciences of Companion animals, Faculty of Veterinary medicine, Utrecht University

Abstract

Introduction: Hypercortisolism the most common endocrine disease in dogs. Chronic elevated plasma levels of cortisol result in clinical signs such as polyuria and polydipsia, polyphagia and alopecia. One of the causes for cortisol expression is an adrenocortical tumor (AT). So far, it was believed that AT secrete cortisol independently from the pituitary and ACTH. However, human research has shown that there is expression of adrenocorticotropic hormone (ACTH) and its precursor POMC in the tumors. In this research, the expression of POMC in ATs in dogs was studied in 26 carcinomas and 12 adenomas. For comparison also 15 normal adrenal cortexes served as control.

Materials and methods: RT-PCR was performed on all the adrenal tissue samples. PCR products have been sequenced. After this, a qPCR was performed. Finally immunohistochemistry was done.

Results: In the PCR, POMC expression was found in different samples, in both ATs and normal adrenal tissue. qPCR gave no reliable results. Results of the immunohistochemistry seemed to match with the results of the PCR, except for the normal tissue slides.

Contents

1. Abstract 2

2. Introduction

- Hypothalamic-Pituitary-Adrenal axis 4

- Hypercortisolism 5

3. Materials and methods

- Primers 9

- cDNA 9

- RT-PCR 10

- qPCR 10

- Immunohistochemistry 11

4. Results

- RT-PCR 12

- qPCR 12

- Immunohistochemistry 13

5. Discussion 14

6. Acknowledgements 15

7. References 16

8. Appendices

1: Sample names 19

2: Protocol Immunohistochemistry 21

3: Results RT-PCR 22

2. Introduction

Hypothalamic-Pituitary-Adrenal axis

Adrenal cortisol secretion is regulated by the hypothalamus-pituitary-adrenal axis. In the hypothalamus corticotrophin-releasing hormone (CRH) is produced and secreted in the portal vessels of the pituitary gland. CRH-secretion is increased by stress and hypoglycaemia, plasma cortisol provides the negative feedback [1].

In the pituitary gland, the CRH stimulates corticotrophs, which account for 15% of the pituitary cells. They produce POMC, a precursor molecule which is cleaved in to multiple products (fig 1). One of them is adrenocorticotropic hormone (ACTH), a 39-amino acid peptide [2,3]. ACTH in the dog is secreted in pulses, ranging from six to twelve per 24 h period. ACTH is secreted into the blood and transported to the adrenal glands by the circulation [4].

The adrenal gland consists of a cortex and medulla. The cortex consists of 3 different zones: The outer zona glomerulosa that produces mineralocorticoids, the middle zona fasciculata which produces glucocorticoids (cortisol and corticosterone) and the inner zona reticularis which produces androgens and, to a minor degree, cortisol [2]. Negative feedback of pituitary corticotrophin-secretion is regulated by plasma cortisol [1].

Cortisol acts on the metabolism of carbohydrates, proteins and lipids. By doing so the plasma glucose levels are increased. Gluconeogenesis is increased by stimulation of the enzymes of the gluconeogenic pathway. For gluconeogenesis amino acids are needed. Cortisol provides these by stimulating the protein catabolism. Another action is reducing uptake of glucose by muscle and fat tissue. All of the above can lead to hyperglycemia, which can lead to insuline resistance. In order to still have a energy supply for the muscles, lipogenesis is decreased and lipolysis is increased, leading to a higher level of glycerol and free fatty acids.

Cortisol also suppresses inflammatory and immunological responses, leading to destruction of tissue and fibroplasia. At high levels, cortisol can reduce the resistance to, for instance, bacteria and viruses.

Hypercortisolism/Cushing’s syndrome

Introduction

Cushing’s syndrome or hypercortisolism is one of the most common endocrinological disorders in dogs. It is characterised by a chronic excess of plasma cortisol levels.

Estimated incidence is 1 to 2 cases/1000 dogs/ year [5]. The median age in which Cushing’s syndrome occurs is 10 years. Some breeds seem to have a predisposition. In, among others, Poodles, Dachshunds, Beagles, Labrador retrievers and German Shepherds have a higher incidence of hypercortisolism then other breeds. Typical clinical signs in dogs are polyuria, polydipsia, polyphagia, panting, abdominal enlargement, endocrine alopecia, mild muscle weakness and lethargy [6, 7, 8].

There are three forms of this disease: a pituitary or ACTH dependent form, an ACTH independent form and an iatrogenic form. The first form, the pituitary dependent form, also called Cushing’s disease or the ACTH-dependent form. It is caused by a tumor in the pituitary that leads to an overproduction and excretion of ACTH. This tumor is usually located in the anterior lobe [6, 7, 9]. Because ACTH stimulates cortisol production in the adrenal gland, this results in hypercortisolism. In patients with hypercortisolism, this is the most common form. It occurs in about 80-85% of the patients [4, 6, 7].

In ACTH-independent hypercortisolism, elevated cortisol secretion is a result of an adrenocortical tumor. This tumor produces cortisol independent from ACTH and suppresses ACTH secretion by negative feedback. The tumor can be either an adenoma or carcinoma and is usually unilateral. About 15-20% of the patients with Cushing’s syndrome have an adrenocortical tumor. Adenomas and carcinomas occur at the same incidence [7]. An andrenocortical tumour is considered carcinoma when the tumor size exceeds 2 cm in diameter and when there is histological evidence of invasion of neoplastic cells into blood vessels, peripheral fibrosis, capsular invasion, a trabecular growth pattern, hemorrhage, necrosis, and single cell necrosis. Typical histological characteristics for adenomas are hematopoiesis, fibrin thrombi, and cytoplasmic vacuolization [11]. They can metastasize to the lungs and liver. [5, 6, 7, 10].

The third form, the iatrogenic form is caused by long-term or excessive administration of glucocorticoid drugs to the dog, usually by a veterinarian.

Ectopic ACTH secretion has been described in dogs, but is rare [12].

Diagnosis

For diagnosing one of the three forms above, there are several endocrinological tests available. There are different screening tests, which can only indicate if hypercortisolism is present or not.

The first and least invasive is measuring the urinary corticoid creatinine ratio (UCCR). For this test the owner collects morning urine samples on two consecutive days, the UCCR in these samples is averaged. This way the UCCR gives an indication of cortisol secretion over a period of time and is adjusted for fluctuations in plasma levels caused by the pulsatile release.

Secondly, the sensitivity of the pituitary-adrenocortical system to suppression is tested with the low-dose dexamethasone suppression test. Dexamethasone is a glucocorticoid, which normally acts as a suppressor of the ACTH secretion from the pituitary. In dogs with hypercortisolism, the pituitary is very resistant to the glucocorticoid feedback. Therefore in these dogs, after intravenously administrating dexamethason, there should be no decline in the plasma cortisol levels, while there should be a decline in healthy dogs. This test can have a false positive result due to stress. This can be avoided by combining UCCR measuring and administrating the dexamethason orally, which can be done by the owner at home [4, 6-9, 13,].

The next step to determine the actual cause of hypercortisolism is a high dose dexamethasone suppression test. In dogs with ACTH-dependent hypercortisolism, higher doses of dexamethasone usually overcome the resistance of the pituitary to glucocorticoid feedback. In dogs with ACTH-independent hypercortisolism the pituitary gland is chronically suppressed by the cortisol form the adrenal tumour, therefore, no matter how high the dose; it will not suppress plasma cortisol levels. Therefore a decrease of >50% of plasma cortisol confirms ACTH-dependent hypercortisolism.

Often the test for diagnosing hypercortisolism is combined into one test with differentiating between the forms by using UCCR measurement together with oral dexamethason administration.

When the high-dose dexamethason suppression test gives a decrease of ................
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