HF consumption is blameworthy for the development and progression of metabolic diseases, and overconsumption of fructose can lead to obesity, fat accumulation, and dyslipidemia. However, the underlying biological mechanisms remain unclear as most studies was only to examine one time point or investigate the additive effects of high-fructose and high-fat diets 15,16. Hence, in this study, we investigated the effects of long-term consumption of HF on mice at different stages and found that damage to the colon and adipose tissues might be an early event and that liver damage may be a subsequent effect. Furthermore, our study demonstrates that a long-term HF diet alters the gut microbiota diversities, leading to T cells alterations in the liver and MLN, and resulting in hepatic steatosis.
Previous studies have demonstrated that the chronic HF diets can impair glucose tolerance and insulin tolerance and lead to NAFLD 17. While the effect of HF on body weight remains controversial. Several studies found that HF diets increase body weight 18,19. Do MHet al. 20 suggested that a high-glucose or HF diet can cause metabolic disorders in mice without body weight change. Our study also found that chronic HF diet intake did not result in being overweight from the 1stweek until the end of treatment but led to oral glucose intolerance and insulin resistance after8 weeks. Excessive fructose intake can affect the liver 21.As expected, fructose markedly increased the levels of plasma ALT and AST and hepatic TG and TCHO. It also led to severe hepatic steatosis and aggregation of lipid droplets after 12 weeks. However, the liver of mice in HF diet group did not have apparent vacuolar degeneration and lipid droplet aggregation at 4 weeks, suggesting that the short-term HF diet did not affect the liver. Interestingly, the white fat and brown fat cells were significantly enlarged, the Inflammation foci was appeared in white adipose tissue, the proportion of oversized adipocytes was significantly increased, the islet cells were disordered, and the length of the colon was significantly shortened in the HF diet group compared with the controls from the 4th week. Therefore, we indicated that colon, pancreas, and adipose tissue lesions may be an early event after an HF diet, and liver damage may be a subsequent effect. Next, we will further discuss the specific mechanism of the metabolic disorder caused by HF.
Phylogenetic and metagenomic analyses of gut microbiota have been extensively studied in the context of metabolic disorders. A growing body of evidence has demonstrated that obesity and its related diseases, such as dyslipidemia, inflammation and impaired glucose tolerance, are highly correlated with changes in gut microbiota 22,23. Rong Tan et al. 16 reported that an HF diet could increase Firmicutes/Bacteroidetes ratio. Firmicutes have been implicated in the development of diabetes and obesity 24,25. In contrast, Bacteroidetes have normal effects on intestinal development, which produce molecules that mediate healthy immune responses and protect the host from inflammatory disease 26,27. In this present study, we observed that an HF diet significantly reduced the richness and diversity of the gut microbiota. At the genus level, we observed more specific shifts, including the relative abundance of Firmicutes, such as Blautia, Lachnoclostridium, and Oscillibacter were significantly higher in the HF than in the ND-fed group. These data confirm and extent previous findings.
The gut microbiota regulates metabolism and plays an important role in the breakdown and absorption of nutrients. Numerous studies have shown a high correlation between the gut microbiota and gut barrier function 28,29. As the intestine consumes a considerable amount of energy, modulating the intestinal volume and cellular architecture is deemed an important adaptation to fluctuations in nutrient availability 30,31. Previous studies have suggested that an HF diet can alter the intestinal function. Samuel et al. 32 reported that the length of intestinal villi was longer in HF-fed mice. Whereas our data showed that the length of small intestine villus, as well as the total length of the colon, decreased in the HF-fed group. These inconsistent results may be attributed to the differences in the animal models, such as the HF feeding time and age of the mice. Furthermore, it has been found that fructose metabolism in the gut leads to disruption of tight junction proteins 33, which may account for the increased gut permeability and disruption of gut barrier function observed after fructose ingestion 34,35. In this study, we demonstrated that the HF diet increased intestinal permeability and altered intestinal barrier characterized by decreased expression of tight junction proteins, such as ZO-1, occludin, and claudin-1. Impaired TJs can also lead to loose intercellular junctions, increased intestinal permeability, and even shedding of epithelial cells, leading to luminal macromolecular epithelial layer invasion and activation of inflammatory responses. The immune system is controlled by Treg cells, which express the transcription factor forkhead box P3 (Foxp3). Their primary function is to minimize excessive effector-T-cell activation and resultant tissue damage during infection-induced immunological responses 36,37. Th1 cells are proinflammatory cells that are involved in adipose tissue inflammation associated with obesity-related pathologies 38.We have found an increase in the proportion of pro-inflammatory Th1 cells and a decrease in the proportion of anti-inflammatory Treg cells in the intestinal lymph nodes of the HF-fed group. This imbalance in the ratio of Th1 and Treg cells may also be caused by changes in the gut microbiota. Additionally, compared with the control group, the jejunum, as the main part of fructose absorption, was found to have more CD4+ and CD8+ T cell infiltration in the HF-fed group. These results suggest that changes in the gut microbiota induced by an HF diet impair the intestinal mucosal barrier. Destruction of the integrity of the intestinal barrier increases the chances of various metabolites contacting the immune system, which may be the primary cause of hepatic steatosis caused by HF.
Importantly, NAFLD is no longer considered an exclusively hepatic disease, as multiple other organ systems participate in the pathogenesis 39. Being on the most import one hand the gut, through dysregulation of the microbiome 40.Increased intestinal permeability leads to the entry of gut-derived bacterial LPS into the portal vein, which is an essential trigger for fatty liver development 41.Hepatic lipid accumulation upregulates hepatic pro-inflammatory cytokines by directly activating the TLR-4 pathway 42. A recent report suggested that TNF-α plays a casual role in the onset of fructose-induced NAFLD/NASH and insulin resistance in mice 43. IL-6 can induce B cell differentiation, antibody production, and T cell activation, proliferation, and differentiation 44. IL-6 is known to be involved in the body’s immune response and initiates inflammation 45. In our study, consistent with the histological and biochemical data, the levels of hepatic and plasmatic TNF-α, IL-6, and IL-1β were significantly elevated in fructose-exposed mice. Increased intestinal permeability can activate Kupffer cells through Toll-like receptor 4 (TLR-4) on the cell membrane, leading to liver inflammation 46. We found that HF can significantly increase the proportion of CD3+CD4+ T cells, CD3+CD8+ T cells, and M1 macrophages in the liver. In addition, we found that the expression levels of FASN and ACC1, which are considered critical enzymes in adipogenesis, were significantly increased in the HF-fed group. CD36, ChREBP, and SREBP1c were increased in the HF-fed group compared to the ND-fed group. Fareeba et al. 47 suggested that CD36 is essential in hepatic fat absorption and triglyceride storage. Therefore, an HF diet can induce higher expression of inflammatory cytokines and upregulate the protein expression levels of FASN, ACC1, CD36, ChREBP, and SREBP1c, thereby promoting the development of the fatty liver.
Next, to further investigate the mechanistic involvement of the observed alterations in T cells and macrophages in the pathogenesis of NAFLD in an HF diet mouse model, we sought to correct these disruptions using gut microbiota reconstitution. FMT effectively decreased CD3+CD4+ T cells, CD3+CD8+ T cells and M1 macrophages in liver and Th1 cells in MLN, whereas it increased Treg cells, thereby decreasing plasma ALT, AST, TG and TCHO, as well as histologically attenuating hepatic steatosis and lipid accumulations, thus improving NAFLD. The fact that the most pronounced effect on NAFLD was achieved by correcting an immune disruption at the level of the liver and MLN underlines the immense importance of gut microbiota in the pathogenesis of HF diet-induced hepatic steatosis and should encourage further exploration of the modulation of correcting gut microbiota in the treatment of metabolic disease.
In conclusion, we demonstrated that an HF diet-induced alterations in the gut microbiota are associated with the impairment of the gut barrier function, and the link between gut microbiota and innate immune system-mediated chronic inflammation in the liver. Intestinal structure impaired and intestinal inflammation may be an early event, and liver inflammation and hepatic steatosis may be a subsequent effect in HF-fed mice. Furthermore, FMT effectively improve systemic metabolic disorders by maintaining liver and intestinal immune homeostasis.