Upregulation of Obesity-Related Genes in Cellular Reprogrammed BAT-like Cells

Over one-half of the global population is expected to be obese by 2030 [1]. Obesity is a physically and psychologically debilitating cycle that is positively correlated with depression symptoms and incidental depression, implying that further weight gain will only worsen symptoms [2]. These sorts of mental health implications further the notion that obesity is not only a physically debilitating condition, but a psychological one as well. While causes of obesity and excessive weight gain may vary, the symptoms and biological markers are rather consistent. Grossly excess white adipose tissue composition is the primary factor relating to the function and potential remedial effects of BAT reprogramming [3]. Furthermore, hypertension, inflammation, and insulin resistance should be noted as telling symptoms associated with obesity.

Brown adipocytes are known to host an increased number of mitochondria, especially relative to white adipocytes [4]. The function of the mitochondria in this instance varies slightly from their “quintessential'' function, as these organelles operate to produce heat via non-shivering thermogenesis. Attenuation of ATP synthesis is due to a principal gene, uncoupling protein 1 (UCP-1), one whose products dissipate the coordination of the electron transport chain [5]. By rerouting protons through a channel alternative to ATP synthase, energy produced via oxidative phosphorylation is dissipated as heat as opposed to generating ATP. Furthermore, BAT activity is stimulated by cold temperatures and norepinephrine [6].

Our lab has previously demonstrated that co-expression of HB-EGF and ADAM 12S in mammalian cells results in reprogramming of the cells into brown adipose tissue (BAT)-like cells. Reprogramming white adipose tissue (WAT) into BAT-like cells may serve as a potential combatant for obesity and type II diabetes.

Figure 1. Oil Red O staining of mouse fibroblast, mock, HB-EGF, ADAM 12S, and HB-EGF/ ADAM 12S stably transfected cells (as seen in image on the right).

Figure 2. Panel A. Immunofluorescent analysis and localization of HB-EGF C domain in MLC, HB-EGF, ADAM 12S, and HB-EGF/ADAM 12S were examined using HB-EGF C anti-sera. Cellular nuclei were counter-stained with DAPI (blue). FITC and DAPI stained images were also merged. Panel B. Western blot analysis of cellular extracts from MLC, HB-EGF, ADAM 12S and HB-EGF/ADAM 12S cells were examined using HB-EGF C anti-sera. Panel C. Immunoblot probed with an actin antibody to confirm protein quantity. Data are reflective results from three independent cell lines each (as seen in image on the left).

Figure 3. MitoTracker deep red and DAPI counter stain of MLC, mock, HB-EGF, ADAM 12S, HB-EGF/ADAM 12S stably transfected fibroblasts, WAT, and BAT cells analyzed by fluorescence microscopy (as seen in image on the right).

Research Questions

What mechanisms are involved in transdifferentiation during cellular reprogramming into BAT-like cells?

What genes are differentially regulated in ADAM12-S/HB-EGF co-infected HEK-293 cells (cellular reprogrammed BAT-like cells)?

What do the expression levels of differently regulated genes indicate about the nature of brown adipocytes?

Methods

An HB-EGF and ADAM12-S adenoviral co-expression vector was designed and implemented in HEK-293 co-transfection alongside mock-infected cells; conferring cellular transdifferentiation was done by Oil Red-O staining, nuclear and mitochondrial fluorescence, and analysis of the cells’ proliferative state. Total RNA isolation was performed on the HEK-293 cells after two weeks of infection. Genomic DNA was then eliminated, followed by reverse transcription to synthesize cDNA which was then added to SYBR Green fluorescent dye and aliquoted into a 96-well qPCR Array. Subsequently, real-time polymerase chain reaction (RT-PCR) was performed to determine differential gene expression using Qiagen RT2 Profiler Obesity array.

Results

RT-PCR results demonstrate evidence of the upregulation of obesity marker genes ADRβ-1, DRD1, and GHR in ADAM12-S/HB-EGF co-infected HEK-293 cells.

Upregulation of ADRβ-1 serves as an indicator that BAT-like reprogrammed HEK-293 cells have the capacity for non-shivering thermogenesis.

Upregulation of DRD1 is correlated with increased mitochondrion content as seen in MitoTracker Deep Red staining.

Increased expression of ADRβ-1, DRD1, and GHR implies increased cellular lipolysis, while increased DRD1 and GHR expression implies increased UCP-1 expression, both of which are characteristic of BAT-like cells

Table 1. Differential Gene expression in HEK-293 cells. Total RNA was isolated from Ad-MOCK and Ad-HB-EGF/ADAM-12S adenoviral co-infected HEK-293 cells after two weeks and analyzed for gene expression for human obesity marker genes. RT-PCR was performed to and demonstrated the upregulation of three marker genes: ADRβ-1, DRD1, and GHR.

Future Directions

Western blot analysis utilizing a beta-1 adrenergic receptor polyclonal antibody to provide evidence that upregulation of the key obesity-related gene translates to an increase in production of its respective protein.

Utilizing insight on the molecular mechanisms of BAT-like reprogramming and knowledge of key genes in the process to develop approaches for BAT therapeutic applications to combat obesity and type 2 diabetes.

Career Readiness Skills

Career & Self-Development

We've gained exposure to the world of academic research and its potential implications and benefits for society as a whole. It has opened up future opportunities to continue contributing to scientific knowledge after graduation, most likely during medical school and thereafter as a physician.

Teamwork

We've learned that a collaborative and supportive team is essential for finding success while carrying out research. Additionally, we've learned the importance of working as a structured unit, each piece is critical to the overall function of our team, and clear communication between all members ensures smooth operation.

Critical Thinking

Scientific research often involves intricate, complex processes that require out-of-the-box thinking and approaches to devise new projects and solutions to questions previously left unanswered. The absence critical thinking could lead to ignorance and or ethical problems in the field of research leaving much to be had.

References

[1] Finkelstein E, et al. (2012). AJPM. 42(6): 563-570. [2] Xin Guo Y, et al. (2023). BES. 36(6): 481-489. [3] Shi L, et al. (2022). Nat Metab 4: 1573–1590. [4] Cannon B, et al. (2004). Physiol Rev. 84:277–359. [5] Porter R (2006). 1757(5–6): 446-448. [6] Cinti S (2006). 16(8): 569-574. [7] Ueta C, et al. (2012). J Endocrinol.214(3):359-65. [8] Yu J, et al. (2022). BBRC. 588:83-89. [9] Berryman E. and List O. (2017). Int J Mol Sci. 18(8):1621. [10] Kopchick J, et al. (2020). Nat Rev Endocrinol. 16(3):135-146.

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