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NEWELL-FUGATE COMPARATIVE ENDOCRINOLOGY LAB

Focused on the Sexually Dimorphic Steroid-Driven Control of Cellular and Organismal Metabolism

Investigating how androgens and androgen metabolites control adipose tissue and liver metabolic function

Androgen imbalance causes metabolic dysfunction in males and females 

The Newell-Fugate Comparative Endocrinology Lab research focuses on revealing the sexually dimorphic, androgen-driven mechanisms that regulate immunometabolism in the adipose tissue and liver, how cell-specific steroidogenic processes modulate these mechanisms, and how these processes are influenced by energy balance (nutrition and exercise). Our laboratory is particularly interested in how androgens, via their direct and indirect effects on the mitochondria, impact cellular insulin signaling and nutrient utilization.  

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Recent Newell-Fugate Lab Publications
*click on the abstract to be taken to the publisher's website

Single-nucleus transcriptomics of epicardial adipose tissue from females reveals exercise control of innate and adaptive immune cells

Ahmad,I et. al. 2024
Cell Communication and Signaling

The aim of  this study was to assess the ability of exercise to promote an anti-inflammatory microenvironment in epicardial adipose tissue of females. Yucatan pigs were assigned to sedentary (Sed) or exercise (Ex) treatments and coronary arteries were occluded (O) with an ameroid to mimic CAD or remained non-occluded (N). EAT was collected for bulk and single nucleus transcriptomic sequencing (snRNA-seq). Exercise upregulated G-protein coupled receptor, S100 family, and FAK pathways and downregulated the coagulation pathway. Exercise increased the interaction between immune, endothelial, and mesenchymal cells in the insulin-like growth factor pathway and between endothelial and other cell types in the platelet endothelial cell adhesion molecule 1 pathway. Sub-clustering revealed nine cell types in EAT with fibroblast and macrophage populations predominant in O-Ex EAT and T cell population predominant in N-Ex EAT. Coronary occlusion impacted the largest number of genes in T and endothelial cells. Genes related to fatty acid metabolism were the most highly upregulated in non-immune cells from O-Ex EAT. Sub-clustering of endothelial cells revealed that N-Ex EAT separated from other treatments. In conclusion, aerobic exercise increased interaction amongst immune and mesenchymal and endothelial cells in female EAT. Exercise was minimally effective at reversing alterations in gene expression in endothelial and mesenchymal cells in EAT surrounding occluded arteries. These findings lay the foundation for future work focused on the impact of exercise on cell types in EAT.

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Androgenic steroids induce pathologic scarring in a preclinical porcine model via dysfunctional extracellular matrix deposition 

Reich, E. et. al. 2024
The FASEB Journal

Hypertrophic scarring is a major source of morbidity.  Based on increased frequency of hypertrophic scarring in patients on testosterone, we hypothesized that androgenic steroids induce abnormal scarring and developed a preclinical porcine model to explore these effects. Mini-swine underwent castration, received no testosterone (noT) or biweekly testosterone therapy (+T), and underwent excisional wounding. To create a delayed wound healing model, a subset of wounds were re-excised at 2 weeks. Scars from postoperative day 42 (POD42) and delayed wounds (POD28) were harvested 6 weeks after initial wounding for analysis via histology, bulk RNA-seq, and mechanical testing. Histologic analysis of scars from +T animals showed increased mean fibrosis area (p = .007) and thickness (p < .001) compared to noT. XX+T and XY+T scars had greater tensile burst strength (p = .024 and p = .013, respectively) compared to noT swine. Color deconvolution analysis revealed greater deposition of type I and type III collagen as well as increased collagen type I:III ratio in +T scars. Dermatopathologist histology scoring showed that +T exposure was associated with worse overall scarring (p < .05). Gene ontology analysis found that testosterone exposure was associated with upregulation of cellular metabolism and immune response gene sets, while testosterone upregulated pathways related to keratinization and laminin formation on pathway analysis. In conclusion, we developed a preclinical porcine model to study the effects of the sex hormone testosterone on scarring. Testosterone induces increased scar tissue deposition and appears to increase physical strength of scars via supraphysiologic deposition of collagen and other ECM factors. The increased burst strength seen in both XX and XY animals suggests that hormone administration has a strong influence on scar mechanical properties independent of chromosomal sex. Anti-androgen topical therapies may be a promising future area of research.

Fabrication of silastic testosterone enanthate implants to achieve virilizing levels of serum testosterone in swine

Mahabir, N. and Newell-Fugate, A.E. 2024
Methods X

The aim of this study was to create a continuous drug delivery system that would yield serum testosterone levels in sexually mature, female pigs consistent with serum testosterone levels of human transgender men. Testosterone enanthate was mixed with medical grade silicone at a ratio of 20 % by weight, placed in silastic tubing to cure at room temperature for 24 hours, then removed from the tubing mold and stored at 4 ° C until surgical implantation. Testosterone enanthate oxidizes at high temperatures which is why the implants had to be stored cold. Each implant was 9 cm long and contained 0.56 g of testosterone enanthate. A minimum of one implant (0.56 g testosterone enanthate) and a maximum of four implants (2.24 g testosterone enanthate) were placed in the cervical subcutaneous fat of each pig. After implantation, serum testosterone was assessed over 40 days. Silastic testosterone enanthate implants increase serum testosterone and maintain it in a relatively constant state in a dose-dependent manner for ∼ 21 - 25 days post-implantation.

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Biomarkers of reproductive health in wildlife and techniques for their assessment

Ghosal, R. et. al. 2023
Theriogenology Wild

Historically, reproductive health in wildlife species has been evaluated primarily via immunoassay detection of fecal and urinary steroid hormone metabolites. This combination of sample type, biomarker category, and assay has been preferred for decades due to the ease of assessing reproductive health through the evaluation of stable compounds in easily collected biological samples using a cost-effective method. Increasingly, beginning with high performance liquid chromatography (HPLC) and more recently with convergence chromatography and ultra HPLC coupled with mass spectrometry (MS), wildlife studies are incorporating more sensitive and specific high-throughput technologies for the assessment of not only steroid hormone metabolites but proteins as well. Of note, a comprehensive health evaluation requires the measurement of biological readouts that modulate reproduction such as: glucocorticoids, leptin, insulin, thyroid hormones, melatonin, the microbiome, and markers of inflammation. Emerging modulatory biomarkers of reproductive health include acute phase proteins, microRNAs, and reactive oxygen species. Several of these biomarkers require application of newer technologies such as LC-MS/MS and sequencing, which demonstrates the need for the field of wildlife reproductive biology to diversify from its reliance on immunoassays. Importantly, endocrine disrupting chemicals adversely affect many aspects of reproductive function and evaluation of these compounds requires high throughput technology such as LC-MS/MS. The application of sequencing, particularly Next Generation Sequencing of bulk RNA (RNA-Seq) and single cell RNA-Seq, is uncommon in studies of wildlife reproductive health. However, as the cost of these methods decreases and consortiums of wildlife researchers band together to raise funds in support of studies using these technologies, their use will become more routine. Future research should focus on integration of known biomarkers of related systems into comprehensive reproductive assessments and the development of new biomarkers which are sensitive, precise, and employ non-invasive methodologies for the assessment of reproductive health of wildlife species.

Trends in diet and cancer research: A bibliometric and visualization analysis

Giles, E.D. et. al. 2023
Cancers

Diet plays a critical role for patients across the cancer continuum. The World Cancer Research Fund International and the American Cancer Society have published evidence supporting the role of nutrition in cancer prevention. We conducted an analysis of the literature on dietary nutrients and cancer to uncover opportunities for future research. The objective of the bibliometric analysis was to describe trends in peer-reviewed publications on dietary components and cancer and to highlight research gaps. PubMed was queried for manuscripts with diet- and cancer-related keywords and Medical Subject Headings (MeSH) terms. Metadata covering 99,784 publications from 6469 journals were analyzed to identify trends since 1970 on diet topics across 19 tumor types. Publications focused largely on breast, colorectal, and liver cancer, with fewer papers linking diet with other cancers such as brain, gallbladder, or ovarian. With respect to "unhealthy" diets, many publications focused on high-fat diets and alcohol consumption. The largest numbers of publications related to "healthy" diets examined the Mediterranean diet and the consumption of fruits and vegetables. These findings highlight the need for additional research focused on under-investigated cancers and dietary components, as well as dietary studies during cancer therapy and post-therapy, which may help to prolong survivorship.

Role of androgens and androgen receptor in control of mitochondrial function

Ahmad, I. & Newell-Fugate, A.E. 2022
AJP-Cell Metabolism

The effects of androgens have been extensively studied in a variety of organs and cell types with an increasing focus on the sexually dimorphic role androgens play not only with respect to cellular functions but also in metabolism. Although the classical mechanism of androgen action is via ligand-dependent binding with the nuclear transcription factor, androgen receptor (AR), cytosolic AR can also activate second messenger signaling pathways. Given that cytosolic AR can signal in this manner, there has been increased interest in the mechanisms by which androgens may control cellular organelle function. This review highlights the effects that androgens have on mitochondrial structure and function with emphasis on biogenesis, fusion/fission, mitophagy, bioenergetics (oxidative phosphorylation), and reactive oxygen species production. 

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Non-invasive assessment of fecal glucocorticoid, progesterone, and androgen metabolites and microbiome in free-ranging southern white rhinoceros (Ceratotherium simum simum) in South Africa

Kothmann, K.H. et. al. 2022
General and Comparative Endocrinology* 

Aromatase inhibition increases blood pressure and markers of renal injury in female rats

Almutlaq, R.N. et. al. 2022
AJP-Renal Physiology*
 

Ultra-performance convergence chromatography tandem mass spectrometry analysis of adrenal and gonadal steroid hormones in southern white rhinoceros (Ceratotherium simum simum) faeces and serum

Gent, R. et. al. 2022
Journal of Chromatography B*

Virilizing doses of testosterone decrease circulating insulin levels and differentially regulate insulin signaling in liver and adipose tissue of females

Kothmann, K.H. et. al. 2021
AJP-Endocrinology and Metabolism

Increased poaching in northern South Africa has necessitated relocation of large numbers of southern white rhinoceros (Ceratotherium simum simum) to the Eastern Cape Province. The climate and grassland ecology of this province differ from that of northern South Africa which may impact the health of this species. This assessment of fecal steroid levels and microbiome in 10 free-ranging southern white rhinoceros in the Eastern Cape will provide insights into white rhinoceros physiology in this biome. Fecal steroid metabolites were analyzed using enzyme immunoassay (EIA) and ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS). Fecal microbial composition was assessed via next generation sequencing. We detected substantially different FAM and FPM concentrations from those previously reported for both captive and wild white rhinoceros. Comparison of our UPC2-MS/MS and EIA results underscores the fact that most EIAs are highly cross reactive for many steroid metabolites. Our data also demonstrates a distinct effect of stress not only on FGMs but also on the fecal microbiome. This is the first non-invasive assessment of fecal steroid metabolites by UPC2-MS/MS and the fecal microbiome in wild white rhinoceros.

*Collaboration with Amanda Swart, University of Stellenbosch

(click for Google Scholar page)

Aromatase is a monooxygenase that catalyzes the rate-limiting step of estrogen biosynthesis from androgens. Aromatase inhibitors are widely used for the treatment of patients with hormone receptor-positive breast cancer. However, the effects of aromatase inhibitors on cardiovascular and renal health in females are understudied. Given that estrogen is protective against cardiovascular and kidney diseases, we hypothesized that aromatase inhibition elevates blood pressure and induces kidney injury in female Sprague–Dawley rats. Twelve-week-old female rats were implanted with radiotelemetry transmitters to continuously monitor blood pressure. After baseline blood pressure recording, rats were randomly assigned to treatment with the aromatase inhibitor anastrozole (ASZ) or vehicle (Veh) in drinking water. Twenty days after treatment initiation, rats were shifted from a normal-salt (NS) diet to a high-salt (HS) diet for an additional 40 days. Rats were euthanized 60 days after treatment initiation. We found that chronic aromatase inhibition in vivo with ASZ increases blood pressure and markers of renal proximal tubular injury in female Sprague–Dawley rats, suggesting an important role for aromatization in the maintenance cardiovascular and renal health in females. The present findings demonstrate that systemic anastrozole treatment increases blood pressure and renal tubular injury markers in female rats fed a high-salt diet, suggesting an important role for aromatization in preserving cardiovascular and renal health in females.

Steroid hormone analysis is routinely undertaken in the assessment of stress response and reproductive function in the management of both captive and free-ranging wildlife species. Faecal samples have become the preferred sample type for analysis as collection is non-invasive and easily assessable. These investigations are generally aimed at aiding successful translocations, enhanced survival outcomes in captivity and improvement of reproductive rate. Immunoassays are the most common approach in the analysis of hormones, particularly in the case of the southern white rhinoceros (Ceratotherium simum simum). Non-specificity, attributed to structural similarity of steroid metabolites impedes accurate evaluations which can be eliminated by chromatographic techniques which are more specific, selective and provide comprehensive analyses.This study developed and validated three methods using ultra-performance convergence chromatography tandem mass spectrometry for the assessment of classical androgens, progestogens and adrenal steroids, as well as the C11-oxy androgens and C11-oxy progestogens in serum and faeces from white rhinoceros. These methods can be applied independently to assess either androgens, progestogens, or adrenal steroid panels or in combination to assess the cohort of gonadal and adrenal steroids in faeces and/or serum, in southern white rhinoceros as well as other wildlife species. Analysis would enable the accurate assessment of reproductive health and stress responses while also distinguishing between stress and distress thus contributing to the conservation of wildlife species.

*Collaboration with Amanda Swart, University of Stellenbosch
(click for Google Scholar page)

*Collaboration with Eman Gohar,
Vanderbilt Medical Center
(click for Google Scholar page)

Transgender men undergoing hormone therapy are at risk for insulin resistance. However, how virilizing testosterone therapy affects serum insulin and peripheral insulin sensitivity in transgender men is unknown. This study assessed the effect of acute, virilizing testosterone on serum insulin concentrations and insulin signaling in liver, skeletal muscle, and white adipose tissue (WAT) of female pigs as a translational model for transgender men. Females received three doses of intramuscular testosterone cypionate (TEST females; 50 mg/day/pig) or corn oil (control) spaced 6 days apart starting on the day of estrus (D0). Fasting blood was collected on D0, D3, D5, D11, and D13, and females were euthanized on D13. Our results suggest that acute exposure to virilizing concentrations of testosterone suppresses circulating insulin levels and results in increased abundance of proteins in the insulin-signaling pathway in liver and altered phosphorylation of key proteins in control of insulin sensitivity in WAT. These results suggest that insulin resistance in transgender men may be due to suppression of the insulin-signaling pathway and decreased insulin sensitivity in white adipose tissue.

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