Kimberly Dalton and Dr. Allan Judd, Physiology and Developmental Biology
The amount of cortisol within the human body must remain at just the right level to adequately mediate the physiological stress response. With elevated cortisol levels, the immune system is suppressed, and the organism becomes vulnerable to infection. With reduced cortisol levels, the immune system is hyperactive, and the organism becomes vulnerable to autoimmune disorders. Too much or too little cortisol is lethal: clearly, cortisol levels have a great effect on human pathophysiology. The project I did provided one step in the mission to characterize the mechanism behind cortisol release. Once this is done, we will have a more thorough understanding of stress-related pathological disorders, so the door will be open to discover effective treatments for these disorders.
The traditional model of cortisol regulation by adrenocorticotropic hormone (ACTH) only holds true under basal cortisol levels and during acute stress. But during chronic inflammatory stress, various cytokines, interleukin-6 (IL-6) in particular, mediate the response (Bornstein et. al 2008). In other tissues, the IL-6 mediated effects are carried out through activation of AMPK (Carey et. al 2006). Dr. Judd of BYU is currently investigating the role of AMPK in mediating the IL-6 response in bovine adrenals. Not long ago, Dr. Judd showed that AMPK upregulates the expression of steroidogenic acute regulatory (StAR) protein, the protein involved in the rate-limiting step of steroidogenesis, which is the transport of cholesterol across the mitochondrial membrane (Dayton et. al 2010). In the project that I did this semester, I studied the effect of AMPK on the enzyme P450scc by treating adrenal tissue with various compounds and measuring the expression of both mRNA and protein through RT-PCR and Western blots, respectively. Other students in Dr. Judd’s lab acquired the adrenal zona fasciculata tissue samples from bovine adrenal glands collected from the Deseret slaughterhouse in Spanish Fork. Once they had dissected out the zona fasciculata tissue of the adrenals (which contain the cells that secret cortisol), they divided the tissue into samples that received different treatments. The treated samples from the experiments explained below incubated with the indicated treatments, and the experiment was terminated by flash freezing the tissue in liquid nitrogen. Protein and mRNA were extracted from the tissue and we ran them through Western blots and RT-PCR, respectively. With the Western blots, I showed the differences in P450scc protein levels between the four samples, and with the RT-PCR, I showed the differences in P450scc mRNA levels between the four samples. Both methods measured the same thing, the expression of the P450scc gene, but both are necessary to take into account posttranscriptional regulation of gene expression.
Experiment 1: time course for AICAR
Zona fasciculata tissue was exposed to the same dose of AICAR for varying lengths of time. This experiment found the optimal time of AICAR treatment for best AICAR-induced results.
Experiment 2: dose response for AICAR
Zona fasciculata tissue was exposed to varying doses of AICAR at the optimal length of time. This experiment found the optimal dose of AICAR treatment for best AICAR-induced results.
Experiment 3: AICAR and compound C
The first sample received no treatment and acted as a negative control. The next sample was treated with AICAR, a known AMPK activator. This sample showed that AMPK is involved in P450scc expression because the expression level in this sample differed from the expression level in the control sample. The next sample was treated with compound C, a known AMPK inhibitor. The final sample was treated with compound C and AICAR together. Compound C was used to assure that the AICAR-induced differences in expression level were due to AICAR-induced AMPK activation and not a different method. This experiment showed that AMPK is involved in P450scc expression. Compound C decreased the AICAR-induced increase in P450scc expression, which showed that AICAR does indeed increase P450scc expression via activation of AMPK.
Experiment 4: IL-6 and compound C
Adrenal tissue samples were treated with IL-6 in the presence and absence of compound C. the effect of IL-6 on P450scc expression was decreased with compound C, which shows that IL-6 increases P450scc expression via activation of AMPK.
Since AMPK was shown to upregulate P450scc expression in experiment 3, and since the effect of IL-6 on P450scc is decreased with compound C, I conclude from the data that IL-6 induces an upregulation of P450scc expression through a pathway that involves the activation of AMPK.
Scholarly Sources
- Bornstein SR, Engeland WC, Ehrhart-Bornstein M, Herman JP. Dissociation of ACTH and Glucocorticoids. Trends in Endocrinology and Metabolism 2008; 19(5):175-180.
- Andrew L. Carey, Gregory R. Steinberg, S. Lance Macaulay, Walter G. Thomas, Anna G. Holmes, Georg Ramm, Oja Prelovsek, Cordula Hohnen-Behrens, Matthew J. Watt, David E. James, Bruce E. Kemp, Bente K. Pedersen, and Mark A. Febbraio. Interleukin-6 Increases Insulin-Stimulated Glucose Disposal in Humans and Glucose Uptake and Fatty Acid Oxidation In Vitro via AMP-Activated Protein Kinase. Diabetes, Vol. 55, October 2006; 2688-2697.
- AW Dayton, RM Argyle, TB Walker, BD Burrows, JC Smart, KA Dalton, TL Ogzewalla, AM Judd. AMP-Activated Protein Kinase Increases the Expression of StAR in Adrenocortical Tissues. ABS P3-616 92nd Annual Meeting of the Endocrine Society. San Diego, CA. June 19-22, 2010.