Fourth Assignment: Review of a paper on Hyperadrenocortisolism in Dogs

Paper:

J.N. Midence, K.J. Drobatz, and R.S. Hess. (2015)
Cortisol Concentrations in Well-Regulated Dogs with Hyperadrenocorticism Treated with Trilostane.
Journal of Veterinary Internal Medicine. 29:1529-1533
*Click the name of the article to be transported there!*

                                              (Taken from: http://usercontent2.hubimg.com/4667577_f520.jpg)

Summary:

This paper is a veterinary research paper on the affects of the drug known as Trilostane. Trilostane is a drug which affects the activity of the hormone ACTH. When ACTH binds to receptors in the adrenal cortex, it stimulates the production and secretion of steroid hormones in a number of ways. One being, the stimulation of gene expression for the enzymes involved in the conversion of cholesterol to steroids such as Cortisol. Trilostane acts on a specific enzyme responsible for the conversion of pregnenolone into progesterone. The name of this enzyme is 3β-Hydroxysteroid dehydrogenase. Trilostane binds to this enzyme and inhibits its activity. Since the creation of progesterone from pregnenolone is a critical step in the synthesis of Cortisol, the binding of Trilostane directly inhibits the creation of the steroid hormone Cortisol.

Figure 1: A pathway diagram illustrating the production of Cortisol and the activity of Trilostane. Original image taken from http://ucsdlabmed.wdfiles.com/local--files/chapter-10/Slide1.jpg

The purpose of the experiment was to determine the effectiveness of Trilostane as a treatment option for hyperadrenocorticism in dogs. Hyperadrenocorticism is a condition in which the dogs pituitary secretes too much ACTH, which causes the excessive secretion of steroid hormones such as Cortisol. Cortisol causes the blood glucose levels to ride, and thus, dogs suffering from hyperadrenocorticism will have an unhealthy level of blood glucose. Symptoms include polyuria, polydipsia, polyphagia, and excessive panting. The scientists predicted that Trilostane treatment would suppress cortisol expression and reduce symptoms associated witht eh condition. In order to test Trilostane as a Cortisol suppressant, the reseachers used approximately 13 different dogs as subjects for the experiment. In order to be considered for the experiment, the dogs must be healthy and their hyperadrenocorticism symptoms were well-regulated. Meaning they had normal urination, drinking, eating, and activity level. Their blood serum was also required to be about < 2.0 μg/dL before the tests started. An approximately even number of both male and female dogs were used with a median age of 9.9 years.

For the procedure, dogs were treated with Trilostane for 152 days before enrollment into the experiment. Researchers continued to give each dog a daily dose of Trilostane averaging 7.3mg/kg of weight. They than took measurements of blood Cortisol and ACTH levels at four different time frames during the days of the experiment and injected synthetic ACTH to stimulate Cortisol production at various points:

Pre1: A measurement taken 3-6 hours after the Trilostane injection, but BEFORE ACTH stimulation.

Post1: A measurement taken 3-6 hours after the Trilostane injection, AFTER the first ACTH stimulant injection.

Pre2: A measurement taken 9-12 hours after the Trilostane injection, BEFORE the second ACTH injection.

Post2: A measurement taken 9-12 hours after the Trilostane injection, AFTER the second ACTH stimulant injection.

This experimental format was used in order to determine the effects of Trilostane over time and under repeated exposure to stimulation of Cortisol production.

Results:

Figure 2: The results summary graph for the experiment, showing measured cortisol levels taken at different time intervals, both before and after ACTH stimulation.

Pre1 Cortisol measurements were lower on average than Pre2 Cortisol levels. Also, Post1 levels of Cortisol were much lower than in Post2 Cortisol measurements. Pre1 and Post1 Cortisol measurements were at similar levels on average. While Post2 levels were much higher than Pre2 levels of Cortisol.

The acceptable range of Cortisol levels for normal functioning in dogs with hyperadrenocorticism is around 4.0 μg/dL. Pre1, Post1, and Pre2 Cortisol measurements were all within range of normality for all dogs measured. Only some of the dogs measure at a Post1 time interval were measured to have Cortisol levels within normal range.

None of the 13 dogs used in the final clinical experiment showed symptoms of hypoadrenocorticism brought on by the treatment of Trilostane.

Findings:

- These results are indicative that Trilostane is effective in normalizing Cortisol levels in dogs hyperadrenocorticism.
- Trilostane can have strong effects in ~75% of tested dogs for a period of up to 12 hours after administered dose.
- Trilostane has high resistance to high ACTH stimulation of the adrenal cortex.
- Trilostane has strong effects in both male and female dogs.
- Trilostane may safely be used in most young dogs without causing symptoms of hypoadrenocorticism.

Critique:

The results obtained definitely align well with the predictions of the research team. After 152 days of treatment with Trilostane, most of the dogs had shown normalized levels of Cortisol in the blood and were no longer showing symptoms of hyperadrenocortisolism. As it turns out, Trilostane is now used as a patented drug called Vyritol for the treatment of Cushing's Syndrome in dogs and cats!
The article was fairly interesting and clearly written. However, I definitely have some criticisms. There was little to no background information provided on the endocrine axis affected by their drug in the article. Knowledge of both the drug mechanism of action, and the hormones involved are critical in understanding how the drug can potentially treat hyperadrenocortisolism. As well, there is only one figure provided in the article, which is the summary graph I have pasted above. The graph is useful for comparison purposes of cortisol levels at different time intervals. However, I believe a clearly organized table with specific cortisol measurement values would be useful in allowing the reader to see the results in the experiment. They currently have these numbers listed in the results section which is somewhat disorganized and hard to read. As well, there is no information provided on dog breeds used in the experiment which may affect the results and effects of the drug.

Future Experiments:

I originally chose this article because I am very interested in both medicine and disease. I think the endocrine axis involved in hyperadrenocorticism is extremely interesting in how it affects the body and how small imbalances can cause problems.
To expand on the research present in this article, I think my first experiment would be to see how Trilostane affects puppies and young dogs. The median age for dogs in this experiment was 9.9. As well, 13 dogs is a small sample size. I would like to see how this medication affects a wider age range and population of dogs. Adjusting dosage within different dogs would be interesting to find the optimal dosasge for treatment. I think I would carry out this experiment by selecting approximately 50 dogs to experiment on over a two year period. 5 dogs from each year of age from 1-10. I would carry out dosage and procedure as in the previous experiment and adjust as necessary with each age group in order to find optimum dosage and identify any side-effects.
Secondly, I would definitely be interested to study a similar disease, hypoadrenocorticism. Dog's with this disease have a lower than normal level of cortisol in the blood as opposed to a higher level. I would like to test different types of synthetic ACTH or glucocorticoid as possible vectors for replacement therapy. The proceure would also be similar to what the researchers in the above article have done, but instead of testing a drug, we would administer synthetic hormone. We would measure levels of this hormone in the blood at various time intervals and determine how often and how effective dosage of the synthetic hormone is in the dogs.

Third Assignment: The Functions of Adrenocorticotropic Hormone!

General Functions and Localizations:     

Adrenocorticotropic hormone has a plethora of functions throughout the mammalian anatomy. Chief of which, is its role in metabolic activities when the body is under stress and during the early morning. ACTH is the tropic hormone involved with the release of Cortisol from the adrenal cortex (Wang and Majzoub, 2011). As stated in a previous assignment, ACTH is a derivative of the precurser hormone Proopiomelanocortin, and thus is classified as a melanocortin. Melanocortin hormones bind to receptors known as melanocortin receptors. ACTH and its derivatives currently affect three out of five of the different melanocortin receptor derivatives that are known in mammals (Wang and Majzoub 2011). Depending on which receptor is bound, the hormone will have a different effect on the target cell. I will discuss the function, effects, and localizations of each receptor once bound by ACTH or its derivatives in this post.

The first receptor I will be discussing is receptor MC1R. Receptor MC1R is primarily localized to melanocytes, keratinocytes, and dermal fibroblasts in mammals (Wang and Majzoub, 2011). These cells are mostly found in the epidermal and dermal layers of the skin. MC1R receptor recruitment is controlled by calcium concentrations and exposure to UV light. When calcium is high, and UV light contacts the skin, it stimulates the recruitment of more MC1R receptors to the cell membrane which increases the cells sensitivity to the receptor ligand. MC1R is primarily acted on by α-MSH, which is a derivative of ACTH. When α-MSH binds to MC1R receptor, it activates a cAMP secondary messenger pathway (Wang and Majzoub, 2011). High levels of cAMP stimulate phosphorylation of tyrosinase which is the main enzyme involved in the movement of itracellular pigment. When tyrosinase is active, it causes conglomeration of vessicles containing eumelanin (A black-brown pigment), and diffuses vessicles containing pheomelanin (red-yellow pigment). This mobilization causes darkening of the cells and thus leads to darkening of the skin. The darkening process is much more active in non-human animals, due to a higher concentration of α-MSH in the blood (Thody, 1999). The darkening of the skin is often essential for protection from high intensity UV rays. The human MC1R receptor is highly polymorphic, and is often associated with the traits of fair/light skin and blond hair (Wang and Majzoub, 2011). In addition to its role in pigmentation, α-MSH is involved in the human inflammatory response. MC1R receptors can be found on macrophage. When α-MSH levels are high, they bind to these recptors on the surface of the macrophage, causeing stifled antibody release and lowering inflammation (Wang, and Majzoub, 2011). Systemic injection of α-MSH has been found to treat arthritis, cancer inflammation, and even inflamed regions of the brain (Schaible et al. 2013). α-MSH is also involved in an antibody regulating axis that is involved with food intake in the intestines (Coquerel et al. 2011).
 

 

Figure. 1: Flow chart demonstrating the relationship between ACTH and
α-MSH, as well as some of their functions. Taken from https://classconnection.s3.amazonaws.com/779/flashcards/1382779/jpg/pomc1334541252805.jpg

 

The next receptor I will be discussing is the main receptor, on which ACTH acts directly. This receptor is known as MC2R, and is found mainly in the human adrenal cortex. As stated in a previous post, ACTH release is controlled by the release of CRH from the hypothalamus. CRH release is regulated by glucose and cortisol levels in the blood, as well as by circadian rhythms (Your bodies 24-hour clock). In the early morning, glucose levels are low, but your body will need glucose in order to wake and be alert. The hypothalamus releases CRH which in turn releases ACTH. ACTH travels through the blood to the adrenals where it binds to MC2R receptors. The binding of the MC2R receptor causes a cascade of effects activated by a secondary messenger pathway (Wang and Majzoub, 2011). Binding causes the cell to increase uptake of lipoproteins into lipid droplets in the cell and begins the hydrolysis of cholesterol within the lipid droplets. Cortisol is a steroid hormone, therefore, it is a cholesterol derivative. The binding of ACTH also causes an increase of cholesterol transport by the steroidogenic acute regulatory protein (StaR) into the mitochondria. Once inside the mitochondria, the cholesterol is converted to pregnenolone which is the precurser to all steroid hormones. The pregnenolone is evetually converted to cortisol and is released into the blood stream where it is bound to a transport protein and sent to other cells (Wang and Majzoub, 2011). Cortisol is responsible for increasing gluconeogenesis in the liver, and inhibiting glucose uptake by adipose tissue and muscle, increasing the concentration of glucose in the blood. Cortisol levels peak in the early morning when the body is waking, and slowly decrease during the day. Thus, ACTH is directly responsible for regulating cortisol levels, and thus regulating blood glucose levels (Ben-Shlomo et al. 2011).

The final receptor that ACTH and α-MSH have an effect on is the MC5R receptor. Studies on this receptor are still limited and so not much is known yet. However, current studies have shown that this receptor is highly concentrated in the aldosterone producing cells of the adrenal cortex, in the zona glomerulosa. It has been proven that high levels of ACTH and α-MSH have caused high levels of aldosterone excretion from these cells (Wang and Majzoub, 2011). Aldosterone is responsible for increasing blood volume and sodium concentration when the blood is at a low blood pressure.

Pathologies:

There are many pathologies associated with ACTH and cortisol levels. One of the most prominently known is Cushing's Syndrome. Cushing's Syndrome, or hypercortisolism, is caused by high levels of ACTH and/or cortisol in the blood (Ben-Shlomo et al. 2010). The raised levels of these hormones are usually caused by one of two things. The first, is cortisol like drugs such as prednisone. These drugs often euther mimic the effects of cortisol, or cause increased cortisol release. The second, is a tumor known as a pituitary adenoma. These tumors occur in the anterior pituitary gland where ACTH is produced. The growth of the tumor causes increased secretion of ACTH, which in turn causes the increased secretion of cortisol (Ben-Schlomo et al. 2010). High levels of cortisol cause increased blood glucose anf thus, cause many problems in the mammalian anatomy. Symptoms of Cushing Syndrome include high blood pressure, abdominal obesity, stretch marks, rounded face, fat lumps on the shoulders, weak muscles, weak bones, acne, and in women, an irregular menstrual cycle (Ben-Shlomo et al 2010).

 

 

 

Another common condition related to ACTH/Cortisol release is known as Addison's Disease.  Addison's Disease, or hypocortisolism, is caused by insufficient cortisol release by the adrenal cortex (Lovas and Husebye, 2005). The adrenals do not release enough cortisol typically because of damage caused by the immune system of the affected. Immune cells break down the cells of the adrenal cortex and thus stymy the production and release of cortisol. Addison's Disease can also be caused by infection in the adrenal cortex (Lovas and Huesebye, 2005). Symptoms typically include fatigue, muscle weakness, lightheadedness, joint pain, sweating, and especially hyperpigmentation. Addison's disease often causes high levels of ACTH in the blood, which cause MC1R receptors to be activated and thus causing high melanin levels in the skin (Lovas and Husebye, 2005).

References:

1) Oulu Wang & Joseph A. Majzoub. (2011). Adrenocorticotropin. In The Pituitary (Third ed., pp. 47-81). San Diego, California: Blackwell Science
2) Anthony J. Thody. (1999). a-MSH and the Regulation of the Melanocyte Function. Annals of the New York Academy of Sciences. Vol 885. pp.217-229
3) Eva-Verena Schaible, Arne Steinsträßer, Antje Jahn-Eimermacher, Clara Luh, Anne Sebastiani, Frida Kornes, Dana Pieter, Michael K. Schäfer, Kristin Engelhard, and Serge C. Thal. (2013). Single Administration of Tripeptide α-MSH(11–13) Attenuates Brain Damage by Reduced Inflammation and Apoptosis after Experimental Traumatic Brain Injury in Mice. PLoS one. 8(8). pp. 1-10
4) Quentin Coquerela, Maria Hamze Sinnoa, Nabile Boukhettalaa, Moïse Coëffiera, Mutsumi Terashia, Christine, Bole-Feysota, Denis Breuilléb, Pierre Déchelottea, Sergueï O. Fetissov. (2011). Intestinal inflammation influences α-MSH reactive autoantibodies: Relevance to food intake and body weight. Psychoneuralendocrinology. 37(1). pp. 94-106
5) Anat Ben-Shlomo, Ning-Ai Liu, and Shlomo Melmed. (2010). Pathogenesis of Corticotropic Tumors. Cushing's Syndrome pp. 1-40). New York City. Humana Press. 
6) Dr Kristian Løvås and  Prof Eystein S Husebye. (2005). Addison's Disease. The Lancet. 365(9476). pp. 2058-2061
7) https://classconnection.s3.amazonaws.com/779/flashcards/1382779/jpg/pomc1334541252805.jpg
8) https://blog.dlvrit.com/wp-content/uploads/Cortisol-Peak-Hours.png
9) https://s-media-cache-ak0.pinimg.com/236x/5b/82/4f/5b824f69dc82065ed723015c12410979.jpg

Second Assignment: The Structure of ACTH, and its precurser Proopiomelanocortin!

Adrenocorticotropic hormone (ACTH) is a 39 amino acid long peptide hormone that is actually the product of post-translational processing. It's precursor is a prohormone known as POMC or Proopiomelanocortin. Proopiomelanocortin is 241 amino acids long after the dignal peptide has been removed, and can be cleaved into a variety of different hormone messengers (Tanaka, 2003). ACTH is cleaved from POMC in the rough endoplasmic reticulum and the golgi apparatus by enzymes known as prohormone convertases. PC1 cleaves the ACTH from an already cleaved ABI product during post-translational processing (Tanaka, 2003). Since ACTH is cleaved from another peptide, it is not directly coded for by a gene. Therefore, in this post we will be looking at the amino acid sequence of POMC as coded for by the DNA of several species of frogs and toads.

 

Figure 1:  This image illustrates the action of PC1 cleaving ACTH from the  prohormone POMC. Taken from (Tanaka, 2003). Along with the 2D structure of ACTH. Taken from http://pubchem.ncbi.nlm.nih.gov/image/imagefly.cgi?cid=16132265&width=300&height=300.

 ACTH is only 39 amino acids long, and as such, has a secondary structure of a single alpha helix (Brunton et al. 2013). Amino acids 1-10 are responsible for binding and signalling the MC2R receptor in the Zonae Fasciculata of the adrenal medulla (Gao & Wong 1998). This area contains many serine acids which help to create hydrogen bonds with the receptor. Once bound, the receptor changes conformation and activates a G-protein linked secondary messenger pathway eventually leading to the production and release of glucocorticoids. 

Figure 2: This figure shows the alignment for the Marshfrog (Accession number M62770.1), African Clawed Frog ( Accession number: NM_001087369.1) and the oriental fire bellied toad (Accession number AY692246.1), as well as the approximate position of ACTH in the POMC prohormone.

KEY: " * " (Asterisk) indicates the position of a single fully conserved residue, " : " (Colon) indicates conservation of groups with strongly similar properties. " . " (Period) indicates conservation of groups with weakly similar properties.


As we can see from the above alignment, the section of POMC corresponding to ACTH is highly conserved, showing that the sequence is functionally very important. We can see that specifically, the serine residues at the beginning of the peptide are conserved, due to their function in binding of the MC2R receptor.

 Figure 3: This figure shows the Percent Identity Matrix for the POMC alignments of the Marsh Frog, African-clawed Frog, and the Oriental fire-bellied toad.

  From the Percent Identity Matrix, we can see that ~ 70-75% of amino acid sequence is conserved between each genus of toad and frog. All of these species are fairly analagous, and even though they are extremely geographically seperated, their hormonal structure is still similar.

References:
 
1) Shigeyasu Tanaka. (2003). Comparative Aspects of Intracellular Proteolytic processing of Peptide Hormone Precursers: Studies of Proopiomelanocortin Processing. Zoological Science. 20. P. 1183-1198
2) http://pubchem.ncbi.nlm.nih.gov/image/imagefly.cgi?cid=16132265&width=300&height=300
3) Brunton, Lawrence L., Lazo, John S., & Keith L. Parker. (2013). Goodman and Gilman's The Pharmological Basis for Therpeutics. 12th Ed. McGraw and Hill. New York. 
4)Gao, Xinfeng., & Tuck C. Wong. (1998). Studied of the Binding and Structure of Adrenocorticotropin Peptides in Membrane Mimics by NMR Spectroscopy and Pulsed-Field Gradient Diffusion. BioPhysical Journal. 74(11). P. 1871-1888

First Assignment: An introduction to Adrenocorticotropic hormone!

Adrenocorticotropic hormone (ACTH) is a peptide hormone. It is created from a 266 amino acid precursor known as proopiomelanocortin (POMC). ACTH is cleaved from POMC into a 39 amino acid long protein (Schlomo Melmed, 2011). This hormone is created in the adenohypophysus, or anterior pituitary, by corticotroph cells. ACTH can be detected in mammals such as dogs as early as the eighth week of the fetal stage, as Rathke's diverticulum (the predecessor of the anterior pituitary) extends to merge with the diencephalon (Schlomo Melmed, 2011).

As the term "Adrenocortico" suggests, ACTH is highly involved in mediation of function for the cortex of the adrenal glands. ACTH is released from the anterior pituitary into the blood where it stimulates the release of glucocorticoids such as the steroid hormone cortisol. Glucocorticoids are highly involved in digestion, the stress response, and physiological development (Bundgaard & Rehfeld, 2010). Thus, regulation for the release of ACTH is extremely important. ACTH works on a 24 hour circadian rhythm and peaks in the morning when it is time for the body to become awake and alert. The release of ACTH is stimulated by the release of corticotropin releasing hormone (CRH) from the hypothalamus. When ACTH is released into the blood, it binds to receptors of secretory cells in the adrenal cortex and triggers a G protein linked secondary messenger pathway. Glucocorticoids such as cortisol are then released into the blood stream where they cause gluconeogenesis from the liver. Glycogen is broken down and released as glucose into the blood stream where it provides energy for the body. Other functions of the glucocorticoids include moderating the metabolism of proteins, fat and carbohydrates, as well as controlling leukocyte production and inflammation during the immune response (Bundgaard and Rehfeld, 2010). The release of ACTH is mediated by the concentration of glucose and cortisol in the bloodstream. The hypothalamus registers when cortisol levels are peaking and thus releases less CRH, which in turn causes the stymied release of ACTH from the adenohypophysis.

The importance of glucocorticoids can be seen by the symptoms of patients who suffer from Cushing Syndrome, also known as hyperadrenocorticism. This condition is caused by an excess release of CRH in the hypothalamus or cortisol from the adrenal glands into the blood stream. The disease is often caused by a hormone secreting tumor in the glucocorticoid pathway (Constantine A. Stratikis, 2012). Since cortisol is highly involved in the metabolic production of fats and proteins, cushings syndrome causes rapid weight gain.

ACTH plays an extremely integral role in the metabolic pathway of the body. We still do not know many of its functions. Research has proven that synthetic ACTH can even be used to treat mild cases of epilepsy (Croiset & De Weid, 1992). There is so much still to be discovered about metabolic hormones such as ACTH, which is why I chose it as my favorite hormone!

Figure 1: The CRH, ACTH, and Cortisol secretory pathway showing how cortisol levels and stresses affect secretion. Taken from http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/adrenal/feedback.gif

References:

1) Melmed, S. (2011). Adrenocorticotropin. In The Pituitary (Third ed., pp. 47-50). San Diego, California: Blackwell Science.
2) Croiset, G., & De Wied, D. (1992). ACTH: A structure-activity study on pilocarpine-induced epilepsy. European Journal of Pharmacology, 229(2-3), 211-216.
3) Rehfeld, J., & Bundgaard, J. (2010). ACTH: Cellular Peptide Hormone Synthesis and Secretory Pathways. In Cellular Peptide Hormone Synthesis and Secretory Pathways (pp. 70-88). New York, New York: Springer.
4) Stratakis, C. (2012). Cushing Syndrome in Pediatrics. Endocrinology and Metabolism Clinics of North America, 41(4), 793-803.
5) http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/adrenal/feedback.gif