Potential Role of Pig UCP3 in Modulating Adipocyte Browning via the Beta-Adrenergic Receptor Signaling Pathway

Abstract

Adipose tissue plays an important role in regulating body temperature and metabolism, with white adipocytes serving as storage units for energy. Recent research focused on the browning of white adipocytes (beige adipocytes), causing thermogenesis and lipolysis. The process of browning is linked to the activation of uncoupling protein (UCP) expression, which can be mediated by the β3 adrenergic receptor pathway. Transcriptional factors, such as peroxisome proliferator activated receptor γ (PPARγ) and PPARγ coactivator 1 alpha, play vital roles in cell fate determination for fat cells. Beige adipocytes have metabolic therapeutic potential to combat diseases such as obesity, diabetes mellitus, and dyslipidemia, owing to their significant impact on metabolic functions. However, the molecular mechanisms that cause the induction of browning are unclear. Therefore, research using animal models and primary culture is essential to provide an understanding of browning for further application in human metabolic studies. Pigs have physiological similarities to humans; hence, they are valuable models for research on adipose tissue. This study demonstrates the browning potential of pig white adipocytes through primary culture experiments. The results show that upregulation of UCP3 gene expression and fragmentation of lipid droplets into smaller particles occur due to isoproterenol stimulation, which activates beta-adrenergic receptor signaling. Furthermore, PPARγ and PGC-1α were found to activate the UCP3 promoter region, similar to that of UCP1. These findings suggest that pigs undergo metabolic changes that induce browning in white adipocytes, providing a promising approach for metabolic research with potential implications for human health. This study offers valuable insights into the mechanism of adipocyte browning using pig primary culture that can enhance our understanding of human metabolism, leading to cures for commonly occurring diseases.

Keywords: adipocyte; animal model; browning; fat primary culture; lipolysis; uncoupling protein.

Figure 1

Gene expressions of pig redifferentiated adipocytes after isoproterenol administration. (A) Gene expression of PGC-1α- and PPARγ-related activation of UCP. (B) Gene expression of UCP1, UCP2, and UCP3. The experiments were conducted twice, and the values of the treatment and control groups were compared. Each color indicates a different concentration of isoproterenol: () control, () 0.01 µM, () 0.1 µM, () 1 µM, () 10 µM, and () 100 µM. The values are shown as mean ± SEM. * p < 0.05 and ** p < 0.01.

Figure 2

Luciferase assay of pig UCP promoter region and alignment analysis. (A) Luciferase assay using Hela cells transfected with PPARγ or/and PGC-1α. Experiments were conducted seven times, and the values are shown as mean ± SEM. A significant difference is indicated by different signs. (B) Comparison of the PPARγ-binding site sequence in the promoter region in UCP promoters among several animal species. Asterisks (*) represent conserved nucleotides among all species. Red boxes show the conserved PPARγ binding sites.

Figure 3

Inhibition of ADRB signaling using propranolol in pig redifferentiated adipocytes. Experiments were conducted thrice, and 1 µM of isoproterenol and 10 µM of propranolol were used. Gene expression was compared to that of the control, and the gene expression values are shown as mean ± SEM. * p < 0.05 and *** p < 0.001.

Figure 4

Number of lipid droplets in pig redifferentiated adipocytes (n = 100). (A) Browning schedule of pig adipocytes. Number of lipid droplets on (B) day 6 and (C) day 9. (D,E) Phenotypes of lipid droplets after the administration of isoproterenol. The scale bar indicates a length of 10 µm. The values are shown as mean ± SEM. *** p < 0.001.

Figure 5

Mitochondrial function in pig redifferentiated adipocytes. (A) Gene expression of citrate synthase related to mitochondria number. (B) Gene expression of COX1, COX2, and COX3 related to the respiratory chain in the mitochondrial membrane. The white and black columns represent the control and 1 µM of isoproterenol administration, respectively. The values are compared to those of the control. The experiments were conducted twice, and the values are shown as mean ± SEM. * p < 0.05.

Figure 6

Schematic representation of the browning pathway in pig adipocytes through ADRB signaling and stimulation of PPARγ and PGC-1α. Isoproterenol-activated PGC-1α and UCP3 gene expressions. UCP3 activation by isoproterenol enhanced mitochondrial function and lipolysis.