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Research Team
Research Team
Aiden Atkinson
Aiden Atkinson
Biology
Dr. Paul Harding
Dr. Paul Harding
Biology
Micah Schuster
Micah Schuster
Biology
Zach Ahmed
Zach Ahmed
Biology
Abstract/Introduction
Abstract/Introduction
Obesity and type 2 diabetes are illnesses that affect a significant number of lives worldwide. Brown adipose tissue (BAT) has potential therapeutic applications to treat these sicknesses. Recent findings demonstrate that co-expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and a disintegrin and metalloproteinase 12S (ADAM12S) is capable of cellular reprogramming into BAT-like cells. This study investigates gene expression patterns of key human obesity and insulin resistance genes in reprogrammed BAT-like human primary adipocytes.
HB-EGF/ADAM12S co-infection significantly upregulated multiple genes characteristic of metabolically active BAT, including ADRB1, PPARG, CXCR4, HK2, LIPE, NAMPT, and PDX1. In contrast, MCHR1, a gene involved in appetite regulation, was significantly downregulated. These findings indicate that HB-EGF/ADAM12S-mediated cellular reprogramming promotes a transcriptional profile consistent with thermogenically active BAT, enhanced substrate utilization, and improved metabolic regulation. This strategy may represent a novel therapeutic avenue for combating obesity and insulin resistance.
Methods
Methods
Generation of ADAM12S Adenovirus
The ADAM12S adenovirus was generated using the AdEasy adenoviral system (Agilent Technologies) as previously described14. Briefly, the ADAM12S cDNA (2.1 kb) was cloned into the pShuttle-IRES-hrGFP-1 plasmid for co-expression with a humanized recombinant green fluorescent protein (hrGFP), allowing visual confirmation of infection. The recombinant plasmid was electroporated into BJ5183-AD-1 cells, followed by recombination with the pAdEasy-1 backbone. High-titer viral stocks were produced in AD-293 cells, and viral titers were determined by serial dilution as described in our prior work14.
Generation of ADAM 12S expressing human primary preadipocytes
Human primary subcutaneous preadipocytes (ZenBio Inc.) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin (100 µg/mL), streptomycin (100 µg/mL), and amphotericin B (2.5 µg/mL) until they reached 40% confluence. Cells were then infected with either 15 µL of Ad-ADAM 12S or Ad-MOCK viral particles in the same media, as previously described15. The primary human cultured preadipocytes and adipocytes are pooled lots of cells derived from a wide variety of patients with representation from all levels of adiposity and age including both male and female donors appropriate for basic research in the field of adipogenesis. Successful infection was confirmed by observing hrGFP fluorescence 48 hours post-infection using an Olympus fluorescence microscope (Figure 1 panels C and D). To ensure the cells remained healthy and viable for subsequent experiments, the media was replaced with fresh growth media every 72 hours.
Lipid accumulation in infected cells was observed under brightfield microscopy three weeks post-infection. To quantitatively assess lipid content, Oil Red O staining was performed. Cells were washed with PBS, fixed with 10% formalin for 60 minutes at room temperature, and stained with freshly prepared Oil Red O solution for five minutes. Hematoxylin was used to counterstain the nuclei, and images were captured to compare lipid droplet formation between Ad-ADAM 12S and Ad-MOCK infected cells.
ADAM 12S infection of human primary preadipocytes
Adenovirus expressing ADAM 12S or empty (Mock) were prepared as previously described15. Western blot analysis of ADAM 12S was performed as previously described using cell extracts of Ad-ADAM 12s infected mouse fibroblasts (MLC), human A431epidermoid carcinoma cells, and human embryonic kidney cells (HEK 293), and resulted in immunoreactive proteins of 68kDa while no ADAM 12S immunoreactive proteins were observed in Ad-mock infected cells.
RT-PCR of Human Insulin Resistance and Human Obesity Genes
Total RNA isolation was performed two weeks after infection, and total RNA concentration from Ad-ADAM12S/HB-EGF co-infected human primary adipocytes cells and Ad-MOCK infected human primary adipocyte cells was measured by a NanoDrop machine, and was reverse transcribed into cDNA. RT-PCR was performed using a SYBR Green qRT-PCR Qiagen kit and a CFX connect Real-Time PCR Detection System (BioRad). The relative expressions of human obesity and human insulin resistance marker genes were determined using the 2^-∆∆Ct method, normalizing against the housekeeping genes. For human obesity, each Ad-ADAM12S/HB-EGF co-infected and Ad-MOCK infected array was analyzed six different times (twelve total replicates). For human insulin resistance, the Ad-ADAM12S/HB-EGF co-infected array was analyzed five times and the Ad-MOCK infected array was analyzed six times (eleven total replicates). The data was assessed using Qiagen Gene Globe.
Findings and Discussion
Findings and Discussion
In this study, we investigate the genetic changes occurring during HB-EGF/ADAM12S co-infection and subsequent cellular transdifferentiation into BAT-like cells. Our lab has previously demonstrated that co-expression of HB-EGF and ADAM 12S can generate BAT-like phenotypes, evident by an increased metabolic activity, glycolytic activity, and lipid droplet accumulation. To better understand what is occurring at a cellular level, we measured differential gene regulation for a number of genes that play important roles in human obesity and human insulin resistance.
BAT-like State
To begin, several key genes associated with BAT and its characteristics were upregulated, indicating that transdifferentiation due to cellular reprogramming had produced a BAT-like state on the genetic level. Of significant note is beta-1 adrenergic receptor (ADRB1), which is known to regulate thermogenesis and BAT activity in humans. Its upregulation indicates that these transdifferentiated cells have the capacity for non-shivering thermogenesis. Increased ADRB1 expression is known to increase UCP1 protein levels, thus playing a key role in non-shivering thermogenesis. ADRB1 has also been shown to activate BAT via cAMP-mediated responses. The production of heat via non-shivering thermogenesis is typically accompanied by increases in lipolysis of triglycerides and the oxidation of fatty acids, making it a tool to fight obesity. Another characteristic of BAT is their increased expression of lipase E, hormone sensitive type (LIPE). LIPE functions in adipose tissue to hydrolyze stored triglycerides into free fatty acids. Lipolysis is typically considered a requirement for cold-induced thermogenesis, which is characteristic of BAT. Upregulation of LIPE in our cellular reprogrammed cells reflects an increased lipolytic capacity, consistent with the brown adipocyte phenotype. Finally, PPARG and its receptor, PPARGCR-1 were upregulated with statistical significance, which is highly representative of a BAT-like phenotype. Peroxisome proliferator-activated receptor gamma (PPARγ), encoded by PPARG, is a ligand-activated nuclear receptor that functions as a master transcriptional regulator of adipocyte differentiation and metabolism. In BAT, PPARγ is highly expressed and is essential for the differentiation, development, and metabolic activity of brown adipocytes. It regulates the transcription of genes involved in thermogenesis and mitochondrial function, including uncoupling protein-1 (UCP1), which drives non-shivering thermogenesis and energy expenditure. During brown adipocyte differentiation, PPARγ interacts with cofactors such as PRDM16 and PGC-1α to activate brown fat–specific transcriptional programs and promote mitochondrial biogenesis and thermogenic capacity. Activation of PPARγ, including through pharmacological agonists such as thiazolidinediones, can promote brown or beige adipocyte formation and enhance glucose and lipid metabolism, thereby improving insulin sensitivity. Conversely, impaired PPARγ signaling is associated with reduced BAT activity and compromised thermogenic function, which may contribute to obesity and metabolic disorders.
Increase substrate metabolism/mobilization
The chemokine receptor CXCR4 displays an insightful interplay between the BAT-like state and metabolic adaptation of HB-EGF/ADAM12S co-infected cells. This gene is expressed in adipose tissue and has displayed effects on thermogenic activity, BAT cell stability and increased energy consumption on knockout mice in a study by Yao et al., 2014.The knockout mice displayed reduced UCP-1 expression in BAT and impaired cold tolerance providing strong implications that the upregulation may be indicative of BAT-like phenotype. The mice also showed increased fat mass and an impaired metabolic profile displaying the potential relevance for CXCR4 in protection against obesity and type II diabetes. In recent studies, cellular metabolism increase has been found to be protective against the development of obesity. Hexokinase 2 (HK2) is shown to be upregulated in HB-EGF/ADAM12S co-infected cells, and is responsible for catalyzing the first step of glycolysis, phosphorylating glucose to produce glucose-6-phosphate. Glucose is then removed from circulation, combating hyperglycemia and insulin resistance, which develops when one’s body is exposed to high levels of blood sugar for an extended period of time. In fact, reduced HK2 activity can lead to high blood sugar levels, which can then lead to insulin resistance and diabetes, shown in a recent study. This demonstrates that HK2 serves as a mediator of glucose homeostasis, and indicates that the upregulation of HK2 in HB-EGF/ADAM12S co-infected cells may act to limit the development of obesity, diabetes, and insulin resistance. Further indication that our cellular reprogrammed cells may serve to ameliorate symptoms of obesity and type 2 diabetes, Nicotinamide phosphoribosyltransferase (NAMPT) in adipose tissue mediates NAD+ biosynthesis. In a study by Stromsdorfer et al., NAMPT knockout mice were found to have developed severe insulin resistance and obesity-associated systemic metabolic complications. Adipose tissue dysfunction caused by a lack of NAD+ biosynthesis in knockout mice notably reduces the production of important adipokines adiponectin and adiposin, responsible for sensitizing the body to insulin. The result, hypoadiponectin, is shown to be a key roleplayer in the development of obesity, insulin resistance, and type II diabetes.
Cellular reprogramming via HB-EGF/ADAM12S co-infection produced cells that have an increased capacity to both mobilize and metabolize substrates (glycolysis, lipolysis, NAD⁺ metabolism). LIPE, HK2, NAMPT, and CXCR4 reflect a healthy, thermogenically active BAT-like state that increases substrate use, beneficial for glucose homeostasis.
Conclusions and Future Study
Conclusions and Future Study
Conclusion
The results of this study suggest that BAT-like cellular reprogramming of human primary adipocytes by HB-EGF and ADAM 12S co-infection may reflect the characteristics of wild-type BAT. These reprogrammed cells provide an understanding of the genetic components that are foundational to this transdifferentiation. The human obesity and insulin genes considered here are implicated in a number of biological pathways associated with type-2 diabetes, metabolic rate, glucose regulation, and insulin resistance. These results provide a better understanding of reprogrammed molecular mechanisms, offering insight for potential BAT therapeutic approaches to obesity and insulin resistance.
Future Direction
Pursue evidence of the products of these genes via Western Blot analysis to verify their presence
Analyze the role that DNA methylation plays in gene expression using PacBio long-read sequencing
The following is an image of poster presented at the 2026 Undergraduate Research Forum
References
References
Shi L, et al. (2022). Nat Metab 4: 1573–1590.
Maliszewska K & Kretowski A (2021). Int J Mol Sci 22(4):1530
Cinti S (2006). 16(8): 569-574.
Taylor S.R., et al. (2024). Med Res Arch 13(7)
NACE Career Readiness Competencies
NACE Career Readiness Competencies
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Active listening, respect, and honest discourse have strengthened our relationship as a team and have advanced our progress in an enjoyable manner.
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