Kaitlyn Ehmann

Investigating the Role of Genetics in the Successful Outcome of Bariatric Surgery

The global obesity epidemic is accelerating at an alarming rate carrying significant health and economic consequences. Given that obesity is not a single disease, but a collection of disorders that share obesity as a common symptom, a uniform approach cannot be applied for the prevention, diagnosis, or treatment of obesity. Cortisol imbalances, such as hypercortisolism, have been linked to obesity due to its effects on the body that can contribute to weight gain. Cortisol has far-reaching effects across numerous organ systems, thanks to the presence of its receptors in almost every tissue in the body. High cortisol levels are associated with increased eating, the production of visceral fat, and muscle resistance to insulin — all leading to weight gain. Bariatric surgery is the most effective and long-lasting treatment option targeting weight reduction; however, a significant portion of patients experience weight regain following surgery. Interestingly, many post-bariatric surgery patients with normal cortisol levels still experience weight regain leading us to hypothesize that single nucleotide polymorphisms associated with cortisol hypersensitivity are a factor leading to weight regain post-bariatric surgery. Several SNPs that have been studied show an association with heightened cortisol sensitivity. Our current research is investigating the relationship between the allele frequency of certain SNPs (rs41423247, rs1360780, and rs12086634), and weight regain post-bariatric surgery. Through a collaboration with Ellis Hospital Bariatric Care Center, we collected patient DNA and clinical information to track patient weights following bariatric surgery. Our findings indicate no significant correlation between weight regain and SNPs rs414 and rs136. However, a correlation exists between SNP, rs120 and weight regain following bariatric surgery. The ability to predict the risk of weight regain following bariatric surgery is a valuable tool that can be used in the post-operative management of patients, potentially improving their chances of long-term success.

Arundhati Gore

Co-immunoprecipitation of the human follitropin hormone receptor (hFSHR) with caveolin

Follitropin or follicle stimulating hormone (FSH), a glycoprotein hormone secreted by the pituitary gland, is essential for male and female reproductive competency. Improper functioning of FSH and its cognate receptor, hFSHR, can have severe effects such as impaired puberty, precocious puberty, primary ovarian failure, azoospermia and others. The hFSHR receptor is a G Protein-Coupled Receptor (GPCR) found in specialized lipid rafts and caveolae. The function of other GPCRs has been shown to be influenced by interactions with scaffolding proteins like caveolin-1, which stimulates the localization and subsequent de-localization of signaling molecules and proteins within the membrane lipid raft. This begs the question as to whether caveolin-1 is involved in the FSH signaling pathway. To address this, my project focuses on determining whether hFSHR and caveolin-1 have direct physical interactions.
To determine this interaction, co-immunoprecipitation was used. Human embryonic kidney cells stably expressing hFSHR and transiently transfected with myc-tagged caveolin-1 were extracted with different buffers and detergents to optimize extract hFSHR while maintaining any protein-protein interactions that occur in the cellular environment. An antibody specific for hFSHR was allowed to interact with the cell extracts and then isolated. Western blotting was then used to detect the presence of myc-tagged caveolin-1 in the isolates. Preliminary research suggests that hFSHR and caveolin-1 co-immunoprecipitate but only weakly. Future research to optimize the interaction will utilize chemical cross-linking to bind proteins in the local environment of hFSHR followed by immunoprecipitation and immunoblotting.
It is crucial to develop improved techniques to study hFSHR interactions with other proteins in its environment to better understand the function of hFSHR in vivo and potentially develop better treatments for infertility associated with lack of FSH function.

Morgan Malinow

Mutations in Caveolin Binding Motif Alter Human Follicle Stimulating Hormone Receptor Signaling

Understanding the function of follicle-stimulating hormone (FSH), is a key to making strides in reproductive healthcare. FSH targets Sertoli cells in the testes which are responsible for promoting spermatogenesis, and studies of signal transduction from the human follicle-stimulating hormone receptor (hFSHR) are essential for understanding issues related to fertility and contraception. hFSHR is found in microdomains of the cell membrane called lipid rafts. Lipid rafts contain high levels of cholesterol and sphingolipids and frequently include the protein caveolin. hFSHR contains a caveolin binding motif (CBM) sequence which includes four aromatic amino acids (all phenylalanine in the case of hFSHR). To assess the importance of the CBM in hFSHR function, mutants were created by changing phenylalanine residues to leucine. CMB’s mutated sites at F479/481 (AB), F481/486 (BC), and F486/489 (CD) were tested to determine the effect of the mutations on activation of the p44/42 MAP kinase pathway. Previous studies from our lab using mutants of 3 (ABC and BCD) or 4 (ABCD) phenylalanines showed an increase in signaling compared to the wild type cells. However, mutating just the AB or CD sites resulted in decreased signaling compared to the wild type receptor. Future studies are aimed at understanding the difference in signaling of double mutants versus the more extensively mutated (triple or quadruple mutants) caveolin binding motifs and the effect of the double mutants on other signaling pathways such as the production of cAMP. These studies will allow for a greater understanding of the role of caveolin in hFSHR function and provide insights into the role of lipid rafts in regulation of reproduction.

Grace Nicol

Investigating the Role of Beta-Arrestin in Ovarian Hyperstimulation Syndrome

Approximately 13.4% of women between the ages of 15 and 49 in the United States are infertile. Infertility treatments include fertility drugs that can stimulate the ovaries to release an egg; yet, these treatments are not free of negative side effects. Ovarian hyperstimulation syndrome (OHSS) is one side effect thought to be caused by hyperactivity of the human follicle-stimulating hormone receptor (hFSHR). OHSS leads to painful growth of the ovaries, blood clots, nausea, and an increased risk of miscarriage. Normal hFSHR functioning is regulated through the deactivation of the receptor through internalization after receptor signaling, a process mediated by the protein beta-arrestin. We hypothesized that reducing beta-arrestin would result in hyperactivity of the hFSHR, which would mimic the condition that leads to OHSS. Hyperactivity of the hFSHR would be shown through reduced extracellular signal-regulated kinase (ERK) phosphorylation and increased cyclic adenosine monophosphate (cAMP) compared to wild-type (WT).
We used small interfering RNA (siRNA) to decrease the levels of beta-arrestin 2 in human embryonic kidney (HEK) 293 cells expressing hFSHR prior to FSH stimulation. Using western blot analysis, we found that ERK phosphorylation was reduced following FSH stimulation in HEK 293 cells transfected with β-arrestin 2 siRNA. This suggested that internalization of the receptor was reduced in the KO cells compared to WT cells. However, when evaluating cAMP signaling there was a reduction compared to WT following a five-hour FSH treatment, which does not correlate with our initial hypothesis.
Understanding normal internalization of the hFSHR will provide insights into the pathology of OHSS due to the potential contribution of hFSHR hyperactivity. Studies like this will further research into interventions to improve in vitro fertilization outcomes by preventing OHSS.

Davis Richard

Investigation on the impact of sphingolipid disrupting agents on follicle-stimulating hormone receptor function

With over 121 million unintended pregnancies occurring each year worldwide, the study of sexual reproduction hormones is necessary to provide better contraceptive measures. A hormone responsible for sexual reproduction in males and females is follicle-stimulating hormone (FSH). When FSH binds to its receptor (FSHR), signaling cascades are activated, resulting in spermatogenesis in males and oogenesis in females. FSHR resides in microdomains of the cell membrane called lipid rafts, characterized by their high concentrations of cholesterol and sphingolipids. Lipid rafts have been shown to play a role in controlling and regulating the signaling of proteins that reside in them, such as FSHR. Recent research suggests that sphingosine-1-phosphate mediates FSH-induced cell viability but not steroidogenesis. Due to this, we hypothesized that disruption of the sphingolipids would lead to alteration in FSHR signaling, connecting the role of sphingosine-1-phosphate to lipid raft structure. HEK293 cells stably expressing FSHR were treated with myriocin (Myr) or Fumonisin B1 (FB1) to disrupt sphingolipid synthesis. Cells were put through a 30-minute time course of FSH treatment and FSHR signaling was evaluated through western blots monitoring the activation of the p44/42 MAPK pathway. Compared to the control, it was seen that FB1 decreased activation of the p44/42 MAPK pathway due to a reduction in band intensity, while Myr increased activation due to an increase in band intensity. Cyclic adenosine monophosphate (cAMP) levels were observed through the use of a luciferase assay, where it was seen that both FB1 and Myr led to a decrease in cAMP levels compared to the control. Collectively, these results demonstrate that FSHR signaling is dependent on sphingolipids, most likely through their role in lipid raft structure and function. Results from this research provide a better understanding of sexual reproductive pathways, hopefully assisting in the creation of more effective and readily available contraceptives.

Hannah Shames

Investigating the Role of Caveolin in Human FSH Receptor Activity

It is estimated that approximately 10-15% of couples in the United States struggle with infertility, meaning that they have trouble getting or staying pregnant. Within these cases approximately one third are the result of male fertility problems with another third being the result of female fertility problems with the final third being a combination of male and female fertility problems. Treatment for infertility can consist of a combination of medications, surgical interventions and assisted reproductive technologies such as in vitro fertilization (IVF). IVF normally includes the use of human follicle stimulating hormone (hFSH), a gonadotropic hormone that is produced in the anterior pituitary gland that is involved in fertility in both males and females. Further understanding of hFSH, its receptor and its signaling pathway could provide alternate treatments for infertility. The hFSH receptor (hFSHR) is a g protein-coupled receptor (GPCR). Previous research in our lab has shown that the hFSHR localizes in lipid rafts which are specialized sections of the plasma membrane enriched with sphingolipids and cholesterol, making them more rigid compared with other sections of the plasma membrane. The structural protein caveolin is also thought to play a role in the localization and removal of GPCRs from lipid rafts which has implications for the regulation of hFSHR activity. In this study, human embryonic kidney cells cells expressing hFSHR were transfected with a dominant negative (S80E) caveolin mutant to investigate the role of caveolin in the hFSHR activity. We found that disrupting the function of caveolin resulted in decreased signaling via the p44/42 and CREB pathways. Further understanding of hFSH and its receptor’s activity can help provide alternative infertility treatments that are more effective for both male and female infertility.

Allison Smith

Investigating the Association of Cortisol Hypersensitivity Single Nucleotide Polymorphisms with Metabolic Syndrome

Global obesity rates have nearly tripled since 1975. Obesity puts individuals at an increased risk of type II diabetes, fatty liver disease, obstructive sleep apnea, and many more conditions. Although the WHO defines obesity as a BMI greater than or equal to 30, this unfortunately is only one small piece of the puzzle. There are many subtypes of obesity including imbalances in steroid hormones such as cortisol. Cortisol, commonly known as the body’s stress hormone, possesses the ability to affect various functions throughout the body that extend beyond its role in the stress response. Mediated by the HPA axis, when dysregulation occurs, disorders within the body can arise such as Cushing’s Syndrome (CS), caused by the overproduction of cortisol. A disease that is quite similar to CS is Metabolic Syndrome (MSX). While the presentation of these two diseases may be difficult to differentiate between, people with MSX have normal cortisol levels. This paradox suggests that some other mechanism could be at play that is causing this hypercortisolemia phenotype in the presence of normal cortisol levels for MSX patients. One possible mechanism is that people with MSX might have a hypersensitivity to cortisol. Past research has indicated there have been SNPs, or single nucleotide polymorphisms, found in the genes associated with hypersensitivity to cortisol as well as many genes with known SNPs that play a pivotal role in the regulation and action of cortisol in the body. We hypothesized that the presence of these SNPs leads to a hypersensitivity to cortisol and is associated with the development of MSX. In collaboration with the Ellis Bariatric Care Center, we are collecting buccal DNA swabs from patients with obesity and determining SNP allele frequencies by qPCR. Specific SNPs are showing correlation with individual characteristics of MSX such as elevated triglyceride levels. Identifying genetic markers that are associated with MSX should give physicians the ability to intervene early on to hopefully prevent its further development.