4. Discussion
Obesity has become a serious global health issue, and is developed when energy absorption exceeds energy expenditure, resulting in extra energy being stored in adipose tissue in the form of triglycerides. This reduces the ability of lipid-oxidative metabolism to occur, and increases the inflammation development of adipose tissue, the secretion of pro-inflammatory adipokines and insulin resistance [
3,
24]. Evidence has shown that SCFAs affected adipose tissue metabolism [
25], particularly acetate, which was the most abundant SCFAs in the body’s system. Therefore, this study explored the mechanism of acetate regulating lipid metabolism through the treatment of obese mice on a high-fat diet with acetate via gavage.
In addition to body weight, Lee’s index, the adipose tissue coefficient, and the organ index also indirectly reflected the obesity degree of mice [
23]. This study found that the food intake, Lee’s index, adipose tissue coefficient and liver index of obese mice treated with acetate via gavage were higher than those of the control group in a dose-dependent manner, indicating that acetate improved the obesity degree of mice.
The large amount of lipids contained in the high-fat diet was digested by the intestine and entered the blood, causing an increase in the content of free fatty acids, promoting the synthesis of triglycerides, and leading to an increase in blood lipid levels. The clinical significance of determining blood lipids was the reflection of the level of the body’s lipid metabolism [
26,
27]. It was observed that the blood lipid levels in obese mice treated with acetate intragastric administration were increased in a dose-dependent manner, indicating that acetate aggravated abnormal lipid metabolism in obese mice.
The liver was the main site of lipid metabolism. When there is abnormal lipid metabolism in the body, liver tissue might suffer from liver injury or inflammation. The AST, ALT, AKP and GGT levels in serum were important indicators for clinical liver function testing [
28]. We found that the AST, ALT, AKP and GGT levels in obese mice treated with a gavage of acetate gradually increased with the increase in the gavage dose of acetate, indicating that acetate aggravated liver injury and liver inflammation in obese mice.
Inflammatory cytokines are mainly secreted by peripheral immune cells (such as macrophages and lymphocytes), and work together with other cytokines to activate immune responses and mediate normal cellular activity in the body [
29]. There are pro-inflammatory cytokines and anti-inflammatory cytokines in the inflammatory response. Pro-inflammatory cytokines (such as IL-1β, IL-6 and TNF-α) can activate various immune cells and promote inflammation, while anti-inflammatory cytokines (such as IL-4 and IL-10) and activate some other cells and reduce inflammatory reaction [
30]. This study found that the pro-inflammatory cytokines levels in obese mice treated with acetate via gavage increased, and the anti-inflammatory cytokine level decreased in a dose-dependent manner, indicating that acetate aggravated the inflammatory response of high-fat-diet-induced obese mice.
In addition to inflammatory cytokines, adipose tissue also released adipocytokines and extracellular vesicles, with the most representative being LEP and ADPN. The levels of LEP and ADPN were closely related to the obesity degree, and an increase in adipose tissue often lead to an increase in LEP levels and a decrease in ADPN levels [
31,
32]. When the body experienced energy deficiency or decreased body lipids, the LEP level decreased, stimulating the body’s foraging behavior, and inhibiting insulin secretion to reduce energy consumption. On the contrary, when the body had excess energy or increased body lipids, the LEP level increased, inhibiting the body’s food intake, promoting insulin secretion, and accelerating lipid metabolism [
33]. In addition, obese individuals had high levels of leptin, and were prone to the problem of leptin resistance with characteristics such as reduced satiety, and increased food intake and body weight, causing a delay in the satisfaction signal of the hunger hormone, leading to more food intake [
34]. Different from that of LEP, the ADPN level decreased with the increase in adipose tissue. ADPN has metabolic and immune-related activities, can stimulate fatty acid oxidation, reduces the TG level, increases insulin sensitivity, inhibits inflammatory cytokines (IL-1β, IL-6 and TNF-α), and induces anti-inflammatory cytokines (IL-4, IL-10) [
35]. Hyperleptin and hypoadiponectinemia caused by obesity might increase the inflammatory response [
36]. This study found that the LEP level in obese mice treated with acetate via gavage increased and the ADPN level decreased in a dose-dependent manner, suggesting that acetate promoted lipid deposition in obese mice.
Fasting blood sugar and insulin levels truly reflect the body’s glucose metabolism ability and insulin sensitivity with the avoidance of the influence of diet or other factors. This study found that the fasting blood sugar and insulin levels of obese mice treated with acetate via gavage increased; this study’s results corresponded to the proposal of Perry et al. [
13] who reported that acetate aggravated the abnormal glucose metabolism and insulin resistance of obese mice.
The pathological observation of liver tissue and abdominal subcutaneous adipose tissue also directly showed the effect of acetate on lipid metabolism in obese mice. In obese individuals, significant morphological changes occurred in adipose tissue, including an increase in adipocytes and macrophage aggregation. These changes lead to a more significant inflammatory state in adipose tissue, reflected by an increase in the secretion of pro-inflammatory mediators and a decrease in the secretion of insulin-sensitizing protein ADPN in adipose tissue. The increase in the inflammatory status of adipose tissue was believed to lead to the occurrence of systemic insulin resistance [
37]. Macrophages aggregated and infiltrated into adipose tissue, accompanied by a more significant inflammatory state, presenting a pro-inflammatory phenotype, expressing more pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and blocking the effect of insulin [
38]. The liver tissue had also undergone significant morphological changes, with lipid droplets appearing in the liver tissue, forming many lipid voids, causing lipid deposition, and leading to liver function damage and tissue inflammation, which is consistent with the results of the serum AST, ALT, AKP, and GGT levels.
GPR41 and GPR43 are two short-chain fatty acid receptors confirmed in current research, which can be activated by SCFAs to regulate metabolism, immunity and other functions [
39,
40]. However, SCFAs have different activation effects on GPR41 and GPR43. The effects of GPR41 are as follows: valerate = propionate = butyrate > acetate > formate; those of GPR43 are as follows: acetate = propionate > butyrate > valerate = formate [
41]. GPR41 and GPR43 were widely expressed in various tissue cells, such as adipose cells, immune cells, intestinal endocrine L cells, etc. After reaching specific tissues through blood circulation, acetate acted as a ligand to combine with GPR, triggering downstream effects, and regulating the expression of related genes and proteins. Acetate stimulated the secretion of LEP by activating GPR41 and GPR43 in adipose tissue, increasing the body’s food intake, and then promoting lipid deposition [
42]. Lipid deposition was closely related to lipid oxidative decomposition and synthesis. Studies have shown that acetate could regulate a variety of cytokines in adipose tissue, inhibit the oxidative decomposition of fatty acids in the liver, and thus increase lipid deposition [
43]. In this study, we found that acetate gavage treatment significantly increased the mRNA expression levels of adipocyte differentiation factor (PPAR-γ, C/EBP-α, SREBP-1c and AFABP), fatty acid synthesis factor (FAS, ACC-1 and SCD-1), leptin receptor (LEPR), fat synthesis factor (LPL), and short-chain fatty acid receptor (GPR41 and GPR43) genes, while the mRNA expression levels of adipolysis factor (HSL), fatty acid decomposition factor (CPT-1 and CPT-2), adiponectin receptor (AdipoR1 and AdipoR2) and fatty acid metabolism regulation signal protein (AMPK-α) genes were significantly decreased (
p < 0.05). This study’s findings corresponded to the proposal of Huang et al. [
44] who reported that the expression up-regulation of PPAR-γ, C/EBP-α and SREBP-1c genes could activate the expression of FAS, ACC-1 and SCD-1 genes, promoting the de novo synthesis of lipid in the liver and increasing lipid deposition in the liver and adipose tissue. The results indicated that acetate could promote lipid deposition, consistent with evidence from Zhao et al. [
17] who reported that acetate derived from microbiota promoted hepatic lipogenesis. In the study of Wang et al. [
45] suggested that the expression of the AMPK-α gene prevented the transfer of activated HSL to lipid droplets, thereby inhibiting the basal lipolysis of adipocytes.
The differentiation process of adipocytes was regulated by various cytokines, such as PPAR-γ, C/EBP-α, SREBP-1c and AMPK-α, etc. A study reported that acetate could promote the differentiation of 3T3-L1 preadipocytes in mice [
46], which increased the expression levels of PPAR-γ, C/EBP-α and ERK1/2 [
47,
48]. Consistently, with the prolongation of the acetate treatment time, the mRNA expression levels of GPR41, GPR43, PPAR-γ, C/EBP-α and SREBP-1c genes of mouse adipose mesenchymal stem cells gradually increased, while the mRNA expression level of the AMPK-α gene gradually decreased. After being treated with different concentrations of acetate for different times, the size of mouse adipose mesenchymal stem cells increased with the prolongation of the induced differentiation time and the increase in the acetate concentration, and intracellular lipid droplets gradually increased in number and aggregated, showing a dose-dependent pattern. The results indicated that acetate promoted the differentiation and lipid deposition of mouse adipose mesenchymal stem cells.
By analyzing the protein expression levels of genes related to lipid metabolism in the adipose tissue of mice, it was found that the protein expression levels of genes promoting lipid synthesis (PPAR-γ, C/EBP-α, FAS, ACC-1, LPL, GPR41 and GPR43) and the phosphorylation levels of p38 MAPK, ERK1/2, JNK and mTOR signaling pathway proteins in obese mice treated with acetate were significantly increased, while the protein levels and phosphorylation levels of genes promoting lipid decomposition (HSL and AMPK-α) were significantly reduced (
p < 0.05). It has been reported that acetate could increase the phosphorylation levels of p38 MAPK by activating GPR43, activating the adipocyte differentiation transcriptional regulation factor (PPAR-γ and C/EBP-α), and then promoting the differentiation of adipocytes [
49,
50]. Likewise, Kwon et al. [
51] observed that suppressed protein levels of the phosphorylated MAPK-related factors ERK, JNK, and
p-38 indicated the inhibition of PPAR-γ pathway-linked adipogenesis. The mTOR signaling pathway was also an important regulator of lipid synthesis metabolism, which could positively regulate the activity of lipid synthesis-related proteins. Some studies have shown that the activation of the mTOR pathway in 3T3-L1 preadipocytes could up-regulate the level of SREBP-1c, a transcription factor that promotes lipid synthesis, and promote lipid deposition while inhibiting the mTOR pathway could inhibit the de novo synthesis of lipids [
52,
53,
54].