New intensive farming methods and selective breeding have improved the growth rates and feed conversion efficiencies of broiler chickens (1, 2). Nevertheless, rapid growth in some breeds is associated with increased deposition of fat, particularly in the abdominal and subcutaneous regions (3). A surplus of fat damages meat quality, raises feed costs, impacts the environment and harms consumer health (4–6). Abdominal adipocytes exhibit higher metabolic activity and greater lipogenic potential (7, 8). Therefore, understanding the molecular pathways responsible for abdominal fat accumulation is crucial.

Guanidinoacetic acid (GAA) is the primary biosynthetic precursor of creatine in vertebrates (9, 10). Creatine is nitrogenous organic acid vital for supplying energy for fast growth and development of muscle tissue via the creatine and phosphocreatine pathway (11–13). Studies show that dietary GAA elevates the concentration of creatine in animal tissues to improve performance, feed conversion efficiency, and enhance antioxidant capacity (12, 14). GAA improves the efficacy of utilizing phosphocreatine-derived energy, a speedy source of ATP for muscle and liver tissues, and also aids its synthesis (11, 15–17). Furthermore, studies indicate that creatine, targeting the PI3K signaling pathway, can block the differentiation of adipocyte at the early stage (18). Thermogenic adipocytes may use creatine to activate futile cycles, contributing to the regulation of energy metabolism.

Molecules like stearoyl-CoA desaturase 1 (SCD1) and transmembrane protein (TMEM) family members (e.g., TMEM35B) are already known to modulate lipid synthesis, adipocyte differentiation and energy metabolism. SCD1 acts as a rate-limiting enzyme for monounsaturated fatty acid production and modulates adipocyte lipid composition and metabolic state directly (19, 20). MicroRNA-103/107 (miR-103/107) family also is critical to metabolism because it targets the transcription factor C/EBPα to inhibit adipogenesis and enhances insulin sensitivity (21). It is, however, unclear whether and how the feed additive GAA influences abdominal fat deposition in poultry through these pathways. Studies on the fat-reducing effects of GAA have focused on growth performance, slaughter traits, and a limited set of serum biochemical markers (22, 23), but have not employed systematic transcriptomic or post-transcriptional network analyses. Thus, elucidating how GAA regulates key molecules such as SCD1, TMEM proteins, and miR-103/107 may provide a strategy to mitigate abdominal fat deposition.

Studies on GAA as a poultry feed additive have focused primarily on production performance and meat quality (24–26). In contrast, its specific impact on abdominal fat deposition remains poorly understood, with only limited research, such as the study by Wu et al. (23), having addressed this aspect. Our previous study showed that dietary supplementation with 1.2 g/kg GAA significantly reduced abdominal fat percentage and adipocyte size in Cobb broilers. While both 1.2 and 3.6 g/kg GAA improved growth performance, the lower dose (1.2 g/kg) proved superior for enhancing breast yield, thigh yield, and meat quality (27). Therefore, the 1.2 g/kg dose was selected for further investigation based on its optimal effects on carcass yield and meat quality. However, the mechanism underlying its fat-reducing action remained unknown. To address this, we performed an integrated analysis of microRNA (miRNA) and mRNA expression using bioinformatics, constructing a regulatory network of miRNAs involved in abdominal fat development in Cobb broilers. This work expands the repertoire of known avian miRNAs in abdominal adipose tissue and offers new insights into miRNA-mediated regulation of fat deposition.

Exogenous GAA administration modulates energy distribution within muscle cells (28, 29), leading to decreased activity of key metabolic enzymes and, consequently, reduced hepatic fat synthesis. Additionally, it promotes the process of β-oxidation of fatty acids, ultimately contributing to a reduction in abdominal fat accumulation in broiler chickens (11, 30, 31). This occurs via either a reduction in adipocyte size or number. Our previous study revealed that a diet supplemented with 1,200 mg/kg of GAA reduced the abdominal fat content and size of adipocytes in broiler chicken. Therefore, we propose that GAA reduces fat storage by enhancing lipid metabolism and promoting the utilization of excess energy, thereby inhibiting its conversion into adipose tissue. To visually summarize the proposed molecular pathways through which GAA may exert its anti-adipogenic effects, we have constructed a schematic model (refer to Figure 7).

To interpret these findings, we explored potential mechanistic insights from existing literature, primarily in mammalian models. TMEM is integral to muscle development and lipid metabolism. Research indicates a significant elevation in the overall level of diacylglycerol (DAG) in cells overexpressing TMEM68, demonstrating that DAG functions as an acyl receptor in the enzymatic reaction catalyzed by TMEM68 for triacylglycerol (TAG) formation, thereby highlighting TMEM8's role in regulating lipid metabolism through the catalysis of TAG synthesis (32, 33). Moreover, TMEM88 is involved in promoting lipid synthesis and cytokine secretion via the Wnt/β-catenin signaling pathway (34, 35). TMEM16F interacts with lipids, as noted by Tang et al. (36). Moreover, studies suggest that the TMEM18 gene is essential for the regulation of adipocyte differentiation via the central nervous system. According to Renström et al. (37) and Wiemerslage et al. (38), this regulation influences obesity and is implicated in the metabolic regulation of triglycerides and other lipids in adipocytes and pre-adipocytes. Although the specific function of TMEM35B in avian lipid metabolism is not yet defined, its downregulation in our study and its targeting by miRNAs suggest a potential, yet hypothetical, role in the GAA-mediated reduction of fat deposition.

Stearoyl-CoA desaturase (SCD) is a crucial fatty acid metabolism enzyme. It introduces a double bond between the ninth and tenth carbon atoms of saturated fatty acids (SFA). The process happens specifically with palmitoyl-CoA (C16:0) and stearoyl-CoA (C18:0) generating monoenoic fatty acids (MEFAs) such as palmitoleic acid (C16:1) as well as oleic acid (C18:1) (39–41). SCD actively participates in the regulation of lipid metabolism.

Lipids are essential molecules that are made and broken down by living things. Monounsaturated fatty acids (MUFA) are vital in the synthesis of complex lipids, including triglycerides (TG), phospholipids, and cholesterol esters. The synthesis of these complex lipids is crucial for maintaining the structural integrity of cellular membranes, storing lipids in fat cells, and regulating energy metabolism (42). Research indicates that SCD plays a significant role in the metabolic pathway of fatty acids and is an essential element in the metabolism of unsaturated fatty acids (43). SCD is responsible for controlling the synthesis of monounsaturated fatty acids, which subsequently influences the fluidity of membranes and the metabolism of lipids within cells (39, 44, 45).

Studies with knockout mice and the model organism Caenorhabditis elegans show that SCD1 knockout (or deficiency) increases SFA and decreases MUFA and shifts fatty acid metabolism from biosynthesis to oxidation (19, 46). Mice that completely lack SCD1, known as global knockout (GKO) mice, show an elevated metabolic rate due to increased lipid oxidation, decreased lipid synthesis, and increased insulin sensitivity. These factors together confer resistance to obesity and fatty liver degeneration that often accompanies high-carbohydrate and high-fat diets in these mice (19). In high-fat diet-fed mice, the SCD1 inhibitor E6446 ameliorates hepatic steatosis, lipid droplet accumulation, and insulin resistance by modulating hepatic lipid metabolism pathways (47). Therefore, the observed downregulation of SCD in our study aligns with a metabolic state that favors reduced lipid synthesis and/or increased oxidation, as seen in other models. However, its direct causal role in the context of GAA action in broilers remains to be functionally validated.

miRNAs are critical mediators of lipid synthesis, fat oxidation and lipoprotein production (48, 49). Among these, miR-103 is particularly notable for its significant role in insulin resistance and lipid metabolism. This specific microRNA inhibits de novo lipogenesis by targeting the fatty acid synthase gene (FASN) and SCD1 in murine liver tissues, thereby contributing to the mitigation of obesity and diet-induced fatty liver disease, as reported by Zhang et al. (50). In contrast, Holik et al. (51) in models utilizing 3T3-L1 mouse pre-adipocytes, it has been noted that miR-103 exerts a unique function by encouraging lipid accumulation. In 3T3-L1 mouse embryonic fibroblasts, miR-103-3p promotes adipocyte differentiation and lipid synthesis by inhibiting the myocyte enhancer factor 2D (MEF2D) and activating the protein kinase-B/mechanistic target of rapamycin (AKT/mTOR) signaling pathway (52, 53). When miR-103/107 is deactivated in adipocytes, noticeable changes occur, including reduced adipocyte size, decreased overall fat mass in mice, improved insulin sensitivity, and enhanced glucose uptake following insulin exposure, as indicated by Trajkovski et al. (21). During porcine adipocyte differentiation, the expression of miR-103-3p (an important subtype of miR-103) is significantly upregulated, while inhibiting its expression can effectively block the differentiation of preadipocytes (54). Additionally, Liu's study reveals that miR-103-3p plays a role in controlling the growth and lipid metabolism in epithelial cells found within bovine mammary tissue (55). Lin and associates showed that a significant rise in pre-miR-103-1 expression in GMECs, attained through adenoviral infection, markedly increases triglyceride levels and lipid droplet formation in these cells (56). Our data indicate that the upregulation of miR-103-3p is associated with the downregulation of the SCD and TMEM35B genes. However, the role of miR-103-3p as a negative regulator of SCD and TMEM35B in broiler adipocytes necessitates further experimental validation.

MiR-107 plays essential roles in a spectrum of biological processes, including the regulation of insulin sensitivity, lipid metabolism, lipogenesis, tumor angiogenesis, and cell proliferation (21, 53, 57). Importantly, miR-107 influences the rise in lipid accumulation within the liver by impacting fatty acid oxidation, as evidenced by Bhatia et al. (58, 59). Furthermore, Ahonen et al. (60) demonstrated that miR-107 promotes adipocyte differentiation and ectopic fat accumulation by interacting with cyclin-dependent kinase 6 (CDK6). Research in bovine adipose tissue has demonstrated that miR-107, which is highly expressed there, inhibits adipocyte differentiation by modulating apolipoprotein C2 (APOC2) (61). It suggests that miR-103/107 induce pre-adipocyte apoptosis specifically through endoplasmic reticulum mechanisms and by stimulating the Wnt3a/β-catenin pathway, pointing to a novel potential strategy against obesity and metabolic syndrome (62). Previous research has also identified miR-107 as a possible regulatory factor associated with abdominal fat accumulating in chickens (63). The expression of gga-miR-107-3p is negatively correlated with the levels of SCD and TMEM35B. This correlation suggests that miR-107-3p may exert the effect of GAA by suppressing SCD and TMEM35B in chicken, however, this regulatory relationship requires further verification through in vitro experiments in broilers.

This study demonstrates that dietary GAA effectively reduces abdominal fat deposition in broilers. Integrated omics analysis revealed a potential mechanism: GAA upregulates gga-miR-103-3p and gga-miR-107-3p, leading to the downregulation of key target genes, SCD and TMEM35B. Given the established role of SCD in lipogenesis and the putative role of TMEM proteins in lipid metabolism, this miR-103/107–SCD/TMEM35B axis provides a novel mechanistic explanation for the anti-adipogenic effect of GAA. Although this study has the above new findings, it still has the following limitations, including the sample size (three biological replicates per group) and the correlative nature of the omics data; thus, the proposed regulatory pathway requires further direct functional validation in avian models. Collectively, our work identifies a possible molecular pathway and provides a strong theoretical foundation for using GAA as a feed additive. These results suggest that GAA supplementation could be a viable strategy to enhance lean meat yield and improve carcass quality in commercial broiler production, potentially offering economic benefits through improved feed efficiency and product value.