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In PCOS, subcutaneous fat has been shown to contribute to hyperandrogenism through increased testosterone (T) production which is accompanied by an increase in the intra-adipose 5\u03b1-dihydrotestosterone (5\u03b1-DHT):T ratio. However, whether 5\u03b1-DHT is produced in isolated adipocytes is uncertain. Here we investigated the ability of subcutaneous human adipocytes to synthesize and inactivate 5\u03b1-DHT in a model of subcutaneous white adipocytes, Simpson-Golabi-Behmel syndrome (SGBS) cells, and primary adipocytes. We quantified the transcripts of genes involved in the biosynthesis of 5\u03b1-DHT (AKR1C3, SRD5A1, SRD5A2, and HSD17B6) and the inactivation of 5\u03b1-DHT (AKR1C1 and AKR1C2). We found that genes that inactivate 5\u03b1-DHT were more abundantly transcribed than genes that biosynthesize 5\u03b1-DHT. This trend was reflected by radioisotope tracing. We developed a radiochromatographic method involving high-performance liquid chromatography and in-line detection of radioactive analytes with precision and accuracy within the 15% tolerance allowable by the US Food and Drug Administration criteria for analytical assays. The lower limit of detection and quantification for 5\u03b1-DHT was 3.4 pg and 15 pg, respectively. The formation of 5\u03b1-DHT was barely detectable when starting with either 10\u2005nM T or 3\u03b1-androstanediol (3\u03b1-diol). Conversely, 5\u03b1-DHT was rapidly metabolized to 3\u03b1-diol but not 3\u03b2-diol. 3\u03b1-Diol was the major metabolite despite comparable levels of AKR1C1 and AKR1C2 transcripts. The same result was observed in both cell lines. Our data reveal that adipocytes do not biosynthesize 5\u03b1-DHT from testosterone. By contrast, 5\u03b1-DHT is rapidly metabolized by AKR1C2 in subcutaneous adipocytes.<\/jats:p>","DOI":"10.1210\/jendso\/bvaf087","type":"journal-article","created":{"date-parts":[[2025,5,16]],"date-time":"2025-05-16T10:19:08Z","timestamp":1747390748000},"source":"Crossref","is-referenced-by-count":2,"title":["Radioactive Tracing of Testosterone Reveals Minimal Formation of 5\u03b1-DHT in SGBS Cells and Human Primary Adipocytes"],"prefix":"10.1210","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4084-3411","authenticated-orcid":false,"given":"Andrea","family":"Andress Huacachino","sequence":"first","affiliation":[{"name":"Department of Biochemistry & Biophysics, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA 19104 ,","place":["USA"]},{"name":"Center of Excellence in Environmental Toxicology, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA 19104 ,","place":["USA"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2955-5304","authenticated-orcid":false,"given":"C\u00e1tia F","family":"Marques","sequence":"additional","affiliation":[{"name":"Center of Excellence in Environmental Toxicology, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA 19104 ,","place":["USA"]},{"name":"Department of Systems Pharmacology & Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA 19104 ,","place":["USA"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5117-2038","authenticated-orcid":false,"given":"Clementina","family":"Mesaros","sequence":"additional","affiliation":[{"name":"Center of Excellence in Environmental Toxicology, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA 19104 ,","place":["USA"]},{"name":"Department of Systems Pharmacology & Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA 19104 ,","place":["USA"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3937-1066","authenticated-orcid":false,"given":"Trevor M","family":"Penning","sequence":"additional","affiliation":[{"name":"Department of Biochemistry & Biophysics, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA 19104 ,","place":["USA"]},{"name":"Center of Excellence in Environmental Toxicology, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA 19104 ,","place":["USA"]},{"name":"Department of Systems Pharmacology & Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA 19104 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Radioactive tracing of testosterone reveals minimal formation of 5\u03b1-dihydrotestosterone in a model of human white adipocytes and primary adipocytes. Figure S1. Figshare Repository. 10.6084\/m9.figshare.29114129.v1"},{"key":"2025052712015888200_bvaf087-B32","unstructured":"Andress Huacachino A, Marques C, Mesaros C, Penning T. Radioactive tracing of testosterone reveals minimal formation of 5\u03b1-dihydrotestosterone in a model of human white adipocytes and primary adipocytes. Figure S2. Figshare Repository. 10.6084\/m9.figshare.29114492.v1"},{"key":"2025052712015888200_bvaf087-B33","unstructured":"Andress Huacachino A, Marques C, Mesaros C, Penning T. Radioactive tracing of testosterone reveals minimal formation of 5\u03b1-dihydrotestosterone in a model of human white adipocytes and primary adipocytes. Figure S3. Figshare Repository. 10.6084\/m9.figshare.29114495.v1"},{"key":"2025052712015888200_bvaf087-B34","unstructured":"Andress Huacachino A, Marques C, Mesaros C, Penning T. Radioactive tracing of testosterone reveals minimal formation of 5\u03b1-dihydrotestosterone in a model of human white adipocytes and primary adipocytes. Figure S4. Figshare Repository. 10.6084\/m9.figshare.29114540.v1"},{"key":"2025052712015888200_bvaf087-B35","doi-asserted-by":"crossref","first-page":"106730","DOI":"10.1016\/j.envint.2021.106730","article-title":"Alternatives for the worse: molecular insights into adverse effects of bisphenol a and substitutes during human adipocyte differentiation","volume":"156","author":"Schaffert","year":"2021","journal-title":"Environ Int"},{"issue":"6","key":"2025052712015888200_bvaf087-B36","doi-asserted-by":"crossref","first-page":"109711","DOI":"10.1016\/j.isci.2024.109711","article-title":"Unveiling the dynamics of acetylation and phosphorylation in SGBS and 3T3-L1 adipogenesis","volume":"27","author":"Aldehoff","year":"2024","journal-title":"iScience"},{"key":"2025052712015888200_bvaf087-B37","unstructured":"Andress Huacachino A, Marques C, Mesaros C, Penning T. Radioactive tracing of testosterone reveals minimal formation of 5\u03b1-dihydrotestosterone in a model of human white adipocytes and primary adipocytes. Figure S5. 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