Ketosis is one of the most common metabolic diseases in perinatal cows [11]. Furthermore, ketosis-related diseases, including fatty liver, abomasum displacement, infectious diseases and other productive diseases, have long plagued agricultural development [12, 13]. Perinatal cows are physically characterized by NEB [14]. The initial stage of NEB in cows is characterized by low glucose and high NEFA levels [15]. Cows under NEB conditions mobilize a large amount of fat to provide more ATP to oxidize NEFAs in the cow liver [16]. However, high NEFA concentrations in the blood of perinatal cows trigger some NEB-related metabolic disorders and diseases, such as liver lipid accumulation, ketosis, abomasum displacement and retained fetal membranes [17]. Clinical data show that fatty liver in cows often manifests as insulin resistance. Cows with type I ketosis often exhibit low insulin levels, and those with type II often exhibit high insulin levels with decreased insulin sensitivity. After recovery, the effects of insulin cannot correct NEB-mediated liver lipid metabolic disorder [18]. Therefore, specific liver regulation of the insulin signaling pathway is crucial to explaining the pathogenesis of cow ketosis and fatty liver. Postpartum cows often exhibit insulin resistance mainly caused by inadequate energy intake. However, the intake of too much energy may lead to obesity and insulin resistance, which are also closely related. TLRs are pattern recognition receptors that can affect microbial pathogens by promoting inflammatory signaling pathways such as the NF-κB pathway in the innate immune system [19]. The NF-κB pathway can promote the synthesis of inflammatory cytokines and chemokines [20]. Obesity is one of the most important factors that affects insulin resistance. Insulin resistance caused by obesity is associated with cytokines secreted by fat cells such as adiponectin and resistin and inflammatory factors such as TNF-α and IL-6 [21]. Obesity is characterized by long-term inflammatory pathway activation, and inflammatory diseases are associated with insulin resistance in obesity. However, the mechanism of this inflammatory pathway activation is not clear in obese patients. The levels of blood FFAs in obese patients tend to be increased, and a variety of hypotheses regarding insulin resistance caused by FFAs have now been proposed. High NEFA and BHBA levels are clinicopathological characteristics of NEB; however, it is unclear whether insulin resistance in perinatal cows with NEB is associated with high blood NEFA levels.
NEFAs affect the NF-κB inflammatory signaling pathway through TLR4
Studies in humans and mice have shown a very close connection between inflammation and insulin resistance [22–24]. Rats in which TLR4 was specifically knocked out fed a high-fat diet exhibit fatty liver, but this high-fat diet can obviously increase insulin sensitivity, reducing fat accumulation and inflammation in the blood [25]. The metabolic characteristics of cows are different from those of humans and rats, and the relationship between insulin resistance and inflammation in bovines is not clear. In this study, we assumed that NEFAs can bind TLR4 on the cell membrane of hepatocytes and influence the NF-κB and insulin signaling pathways.
We found that NEFAs obviously increased the initial calf hepatocyte TLR4 protein expression level, and with increasing concentrations of NEFAs, the P65 phosphorylation level also gradually increased (Fig. 1). Because the activation of NF-κB is mainly achieved through the phosphorylation of p65, NEFAs can activate the TLR4 and NF-κB pathways. After activation of the NF-κB pathway, a large number of inflammatory factors such as IL-6, IL-1β, and TNF-α are produced. To detect whether NEFAs affect the NF-κB inflammatory pathway by binding TLR4, we added a TLR4 inhibitor group and the corresponding control group (Fig. 3). Our experimental results showed that the protein expression of TLR4 and p-P65 in the group mixed with a TLR4 inhibitor and NEFAs was significantly lower than that in the NEFAs group, and nuclear expression of the nuclear transcription factor p65 decreased significantly after the addition of inhibitor (Fig. 4). Similarly, the mRNA expression and supernatant concentration of the proinflammatory factors IL-6 and TNF-α, were significantly increased in the NEFAs group but decreased after the addition of TLR4 inhibitors (Fig. 5, 6). This indicates that the inhibitor of TLR4 could inhibit not only TLR4 but also P65, a key factor in the inflammatory pathway of NF-κB, and can also inhibit the expression and secretion of proinflammatory factors. Overall, these findings indicate that NEFAs can activate the NF-κB pathway through TLR4, promote the release of proinflammatory factors such as IL-6 and TNF-α, and then promote the inflammatory response.
NEFAs affect insulin pathway through TLR4
In patients with diabetes, metabolically active cells cannot use glucose, so they heavily accumulate in the blood [26]. Glucose is mainly used in targeted tissues through two pathways-the PI3K and AMPK pathways [27]. The PI3K/AKT pathway promotes the expression of GLUT4 in muscle cells and adipocytes and plays an important role in glucose metabolism [28]. AMPK is activated mainly through drug stimulation, such as stimulation with metformin, or through exercise, effectively promoting glucose utilization and insulin sensitivity [29]. Therefore, the PI3K/AKT and AMPK pathways may be potential targets for regulating insulin resistance to glucose metabolism in type 2 diabetes and obesity. In this study, we studied the PI3K/AKT pathway. IRS1 is a key regulatory factor downstream of the insulin receptor in insulin signaling. IRS1-deficient mice showed normal glucose tolerance and insulin resistance [30]. Nakamura A (2012) et al indicated that although severe hyperinsulinemia occurred in IRS1-/- rats fed a high-fat diet, there was no liver lipid degeneration [31]. The activation of PI3K in the PI3K pathway depends on the binding of two SH2 regions to the tyrosine-phosphorylated IRS protein [32]. Knockout of the PI3K regulatory subunits p85a and p85β increased insulin sensitivity [33, 34]. Previous studies have shown that liver-specific knockout of PRSS8 reduced the phosphorylation of AKT in the liver, resulting in insulin resistance [35]. GSK-3β is a key enzyme that regulates insulin signaling through glycogen synthase (GS) [36]. GSK-3β can be activated after phosphorylation of AKT, and AKT/GSK-3β signaling plays an important role in insulin resistance [37].
In this study, we first examined whether NEFAs could affect the insulin pathway in primary calf hepatocytes. High concentrations of NEFAs increased the protein expression of p-IRS1 (Ser307), p-IRS2, IRS2 and GLUT4 but decreased the expression of PI3K, p-AKT and p-GSK (Fig. 2). Therefore, we determined that high concentrations of NEFAs can induce insulin resistance in primary calf hepatocytes. To determine whether NEFAs affect the insulin pathway through TLR4, we detected changes in the expression of key proteins in the insulin pathway after adding TLR4 inhibitors and prepared a non-insulin group as a control. After the addition of insulin, the expression levels of key proteins in the insulin pathway were greater than those in the non-insulin group; for example, the decrease in PI3K and p-AKT was more pronounced than that in the non-insulin group (Fig. 7, 8). In general, the addition of TLR4 inhibitors affected the insulin pathway, indicating that NEFAs can affect the insulin pathway by binding TLR4. PEPCK and G6-Pase are two key enzymes involved in gluconeogenesis. Previous studies have shown that the activation of AKT in mouse liver can induce insulin-mediated gluconeogenesis, accompanied by downregulation of PEPCK and G6-Pase expression [38]. To further verify the effect of TLR4 inhibitors on genes downstream of the insulin pathway, we detected the mRNA expression levels of PEPCK and G6-Pase in hepatocytes. In the absence of insulin, the mRNA expression levels of PEPCK and G6-Pase were increased in the NEFAs group and decreased in the inhibitor mixed group, but after the addition of insulin, G6-Pase showed the opposite change (Figs. 9, 10). Because PEPCK and G6-Pase are downstream of the insulin pathway, their sensitivity to insulin is not particularly accurate, so this finding is not enough to explain its effect on the insulin pathway.
Consequently, these results indicate that NEFAs can regulate the insulin pathway through the TLR4/PI3K/AKT metabolic axis.