Inhibitory effect of complement 1q/tumor necrosis factor-related protein 4 on 3T3-L1 preadipocyte differentiation

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, oil red O staining was used to observe lipid droplet formation; RT-qPCR and western blot were used to detect the mRNA and protein expression levels of CCAAT/enhancer binding protein alpha, peroxisome proliferator-activated receptor gamma, and fatty acid binding protein 4; and triglyceride and total cholesterol assay kits were used to measure intracellular triglyceride and total cholesterol levels. RESULTS AND CONCLUSION: The highest safe concentration of complement 1q/tumor necrosis factor-related protein 4 with no toxic effect on 3T3-L1 preadipocytes was 1,000 ng/mL. Compared with the control group, the inducer group showed increased lipid droplet formation, elevated triglyceride and total cholesterol levels (P < 0.05), and significantly upregulated mRNA and protein expression of CCAAT/enhancer binding protein alpha, peroxisome proliferator-activated receptor gamma, 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 lipid droplet formation, decreased triglyceride and total cholesterol levels (P < 0.05), and significantly downregulated mRNA and protein expression of CCAAT/enhancer binding protein alpha, peroxisome proliferator-activated receptor gamma, and fatty acid binding protein 4 (P < 0.05). These results indicate that complement 1q/tumor necrosis factor-related protein 4 can inhibit the differentiation of preadipocytes into mature adipocytes, and the mechanism may be related to the downregulation of CCAAT/enhancer binding protein alpha, peroxisome proliferator-activated receptor gamma, and fatty acid binding protein 4 expression.

1. Introduction

Obesity is the most common manifestation of metabolic syndrome and type 2 diabetes mellitus [1]. Adipose tissue, as an important metabolic regulatory organ, expands through two mechanisms in response to caloric excess: hyperplasia (differentiation of new adipocytes) and hypertrophy (enlargement of existing adipocytes) [2]. In obesity, adipose tissue dysfunction leads to disruption of metabolic homeostasis, characterized by increased secretion of pro-inflammatory cytokines (e.g., tumor necrosis factor-alpha, interleukin-6) and decreased anti-inflammatory adipokines (e.g., adiponectin), thereby triggering chronic low-grade inflammation and insulin resistance, ultimately promoting the development of type 2 diabetes mellitus [3-4]. Currently, increasing resources and funds are being used to address obesity, such as low-calorie diet therapy, pharmacological therapy (orlistat, glucagon-like peptide-1 receptor agonists), and surgical treatment [5-6], but the efficacy is suboptimal, so it is crucial to find safe and effective anti-obesity drugs with fewer adverse effects.

The C1q/tumor necrosis factor-related protein (CTRP) family is an important member of the adipokine superfamily, secreted by adipose tissue, and 15 subtypes have been identified [7-8]. Among them, CTRP4 has a unique structure, containing two globular complement 1q domains but lacking a typical collagen domain [9]. The first complement 1q domain tends to maintain a monomeric state, while the second domain can form higher-order oligomers, and this oligomerization significantly enhances receptor binding ability, indicating that the multimeric form is the main active conformation for CTRP4 to exert its biological functions. Furthermore, studies have found that CTRP4 may participate in regulating innate immunity and inflammation-related signaling pathways through synergistic effects with co-receptors such as nucleolin and Toll-like receptor 4 [10]. Basic research has shown that overexpression of CTRP4 in the hypothalamus can effectively reduce local and systemic inflammatory factors (tumor necrosis factor-alpha, interleukin-6), and its anti-inflammatory effect may improve energy metabolism balance by strengthening appetite-suppressing signaling pathways [10-11]. Clinical studies have shown that in patients with both type 2 diabetes mellitus and non-alcoholic fatty liver disease, serum CTRP4 levels are significantly lower than in patients with type 2 diabetes mellitus alone.