Abstract: BACKGROUND: Adipocyte differentiation is a complex biological process involving the transformation of preadipocytes into mature adipocytes. This process plays a key role in the development and progression of obesity and related metabolic diseases. In recent years, the complement C1q/tumor necrosis factor-related protein family, as a new member of the adipokine family, has become a research hotspot in the field of metabolic regulation.
OBJECTIVE: To investigate the effect of complement 1q/tumor necrosis factor-related protein 4 on the differentiation of 3T3-L1 preadipocytes and its underlying mechanism. 
METHODS: The CCK-8 assay was used to determine the effects of different concentrations of complement 1q/tumor necrosis factor-related protein 4 on the viability of 3T3-L1 preadipocytes, and a safe concentration was selected for intervention. 3T3-L1 preadipocytes were divided into a control group, an inducer group, and a complement 1q/tumor necrosis factor-related protein 4 group. 3T3-L1 preadipocytes were first contact-inhibited for 2 days, followed by adipogenic inducer and complement 1q/tumor necrosis factor-related protein 4 treatment. On day 10 of differentiation, lipid droplet formation was observed using Oil Red O staining. The mRNA and protein expression levels of CCAAT/enhancer binding protein α, peroxisome proliferator-activated receptor γ, and fatty acid binding protein 4 were determined by RT-qPCR and western blot assay. Intracellular triglyceride and total cholesterol levels were measured using triglyceride and total cholesterol detection kits.
RESULTS AND CONCLUSION: The safe maximum concentration of complement 1q/tumor necrosis factor-related protein 4 without toxicity to 3T3-L1 preadipocytes was 1 000 ng/mL. Compared with the control group, the inducer group showed increased intracellular lipid droplet formation, elevated triglyceride and total cholesterol levels (P < 0.05), and significantly upregulated mRNA and protein expression levels of CCAAT/enhancer binding protein α, peroxisome proliferator-activated receptor γ, and fatty acid binding protein 4 (P < 0.05). Compared with the inducer group, the complement 1q/tumor necrosis factor-related protein 4 group showed reduced intracellular lipid droplet formation, lower triglyceride and total cholesterol levels (P < 0.05). The mRNA and protein expression levels of CCAAT/enhancer binding protein α, peroxisome proliferator-activated receptor γ, and fatty acid binding protein 4 were significantly downregulated (P < 0.05). These results suggest that complement 1q/tumor necrosis factor-related protein 4 inhibits the differentiation of preadipocytes into mature adipocytes, and its mechanism of action may be related to downregulating the expression of CCAAT/enhancer binding protein α, peroxisome proliferator-activated receptor γ, and fatty acid binding protein 4.

Key words: CTRP4, adipose differentiation, 3T3-L1 preadipocytes, obesity, adipogenesis, metabolic disorders, lipid accumulation, metabolic imbalance