In the current study, AD model mice (APP/PS1) were treated with a HFD to induce insulin resistance and the effect of diet with different n-6/n-3 PUFAs rations on insulin resistance phenotype and AD-like pathology was explored. Our data showed that HFD efficiently induced insulin resistance in APP/PS1 mice, which was indicated by the dramatic increase in body weight, blood glucose, lipids and insulin levels, glucose tolerance, and severe hepatic lipidosis as compared with the normal diet-fed control mice. We also found that a reduction of fat-derived energy (as demonstrated with a 45% HFD) was efficient in reversing the insulin resistant phenotype that was initially caused by the 60% HFD in APP/PS1 mice.
Dietary n-6/n-3 PUFA ratio-dependent regulatory effect of HFD on insulin resistance has been previously described, in which the author reported that rats fed with HFD containing n-3 PUFAs (n-6/n-3 = 1:1) showed significantly higher insulin sensitivity than rats treated with high n-6 PUFA HFD (n-6/n-3 = 4:1) , suggesting that an increase in dietary n-3 PUFAs proportion could improve insulin sensitivity and prevent HFD-induced insulin resistance. It has also been postulated that the visceral adipose tissue (e.g., fatty liver) induced increased circulating free fatty acids (FFAs) and triglycerides, released a significant amount of pro-inflammatory cytokines, and these cytokines disrupt the insulin action, consequently leading to decreased insulin sensitivity and resistance . Consistent with these studies, our data indicated that diets with different n-6/n-3 PUFAs rations discrepantly affected insulin resistance phenotype in AD model mice, which was demonstrated by the significant decrease in body weight, serum TC, LDL-c levels and hepatic lipid deposition in the initial HFD-induced APP/PS1 mice later treated with high n-3 PUFA diet (n-6/n-3 = 1:1). These results align well with the previous report, which showed that n-6 PUFA rich diet-fed animals were more likely to manifest liver steatosis , while n-3 PUFAs intervention significantly reduced serum TC and LDL-c levels , and significantly relieved the signs of HFD-mediated hepatic steatosis. Unexpectedly, in our study, we did not observe the different reflection of serum glucose and insulin levels to diets with different n-6/n-3 PUFA ratios, demonstrating that the regulation of lipids metabolism may partly account for the discrepant insulin resistance relieving effect of the high n-3 PUFA diet (n-6/n-3 = 1:1) in AD model mice.
A study has shown that an HFD resulted in a significant increase in plasma IL-6 and TNF-α levels in APP/PS1 mice . Consistent with this report, we observed significantly increased levels of serum IL-6 and TNF-α in 60% HFD-fed mice, and the elevation of serum pro-inflammatory markers were dramatically lowered following treatment with 45% HFD. Moreover, dietary n-6/n-3 ratio-dependent inhibition of serum TNF-α level was also observed, which was indicated by the lowest serum TNF-α level in n-3 PUFA rich diet-fed APP/PS1 mice. High dietary n-6/n-3 PUFAs ratio has been reported to correlate with elevated plasma concentration of IL-6 and TNF-α in rats . High n-6/n-3 PUFAs ratio (4:1) HFD-fed SD rats also displayed higher serum IL-6 and TNF-α than low n-6/n-3 ratio (1:1) HFD-fed animals . Reduced IL-6 and TNF-α gene expression have been reported in skeletal muscle tissue of diabetic rat-supplemented with n-3 PUFA . All these results suggested that n-3 PUFA enriched diet might antagonize HFD-induced pro-inflammatory response, and dietary n-6/n-3 PUFAs ratio might have potent modulatory effects on inflammatory reaction in experimental animals.
Reduced n-3 PUFA levels have been found in the brain (mainly in the hippocampus) of AD patients, and the increase of n-6/n-3 PUFAs ratio in the brain has been considered to be associated with the increased risk of AD . In the current study, we did not observe a significant difference in cortical PUFA between C57BL/6J control mice and APP/PS1 control mice, indicating that the mutation of APP gene did not alter the cortical PUFAs profile in the AD model mice. Of note, the treatment of HFD caused an increased trend in cortical PUFAs profile in APP/PS1 mice, although the difference was not statistically significant when compared with control mice. Importantly, our data indicated the potential effect of n-6 PUFA rich diet on elevating cortical n-6 PUFA level and n-6/n-3 PUFAs ratio in APP/PS1 mice, Moreover, this increase of cortical n-6 PUFA level and n-6/n-3 PUFAs ratio effect could be antagonized by 45% HFD (n-6/n-3 = 5:1) and 45% HFD-N3 (n-6/n-3 = 1:1) diets. All these results indicated a strong correlation between cerebral PUFAs profile and dietary fatty acids status, indicating a potential modifying role of dietary n-6/n-3 PUFAs ratio on cerebral n-6 and n-3 PUFA levels.
The senile plaque remains the pathological hallmark sign in AD process, and results from clinical studies have also shown that n-3 PUFA interventions can alleviate AD pathological changes and prevent synaptic degeneration . Data from experimental animals also found that n-3 PUFAs rich diet has been effective in inhibiting the formation of amyloid plaque in the hippocampus and cortex in APP transgenic mice . In our study, although the number of plaques in 45% HFD-N3 diet-fed APP/PS1 mice showed a slightly lesser number of senile plaques than mice treated with control diet, 60% HFD and 45% HFD-N6 diet, no statistical significance was observed between groups. We further detected cortical soluble and insoluble Aβ1−40 and Aβ1−42 levels. Consistent with a previous report , we found that HFD treatment increased cortical soluble Aβ1−40 and Aβ1−42 levels in APP/PS1 mice, indicating that HFD might promote the generation of neurotoxic soluble Aβ1−42 and potentially accelerate synaptic degeneration and AD pathological process. It was reported that the excessive increase of lipids (especially cholesterol) in the brain could modify the composition and function of lipid raft in neuronal membranes and enhance β-secretase-mediated APP processing, ultimately promoting over-generation of Aβ . Thus, we further examined cortical cholesterol levels in mice treated with different diets. The dramatic increase in cortical LDL-c was observed in 60% HFD-fed APP/PS1 mice, but HFD fortified with PUFAs significantly reduced cortical LDL-c levels, especially in HFD-N6 diet-fed group. This finding is consistent with a previous study, that identified n-6 PUFA as a potent down-regulator of LDL-c level and alleviating depressed LDL receptor activity while promoting the diminishment of circulating LDL-c . We, therefore, speculated that the disturbance of cerebral lipid hemostasis in HFD-fed animals might contribute to the increased cortical soluble Aβ content observed in the AD model mice. Ettcheto et al. demonstrated that HFD-fed APP/PS1 mice showed significantly higher cerebral insoluble Aβ1−42 content than ND-fed control animals . On the contrary, we found that 60% HFD treatment decreased cortical insoluble Aβ1−40 and Aβ1−42 levels. A study conducted in FAT-1 mice has found that n-3 PUFA enriched diet could promote the clearance of Aβ from the brain through the cerebral lymphatic system . In our study, we observed an n-6/n-3 ratio-dependent effect of HFD on regulating cortical soluble and insoluble Aβ levels, which was demonstrated by lower soluble and insoluble Aβ levels in mice treated with 45% HFD-N3 and 45% HFD-N6 diets. Also, consistent with the change in LDL-c level as documented earlier, we found that the diet with the highest dietary n-6/n-3 ratio caused the most significant decrease in soluble and insoluble Aβ levels in the cortex, suggesting the important role of n-6/n-3 PUFA ratio in modulating cerebral Aβ1−40 and Aβ1−42 levels. Our findings also imply that specific PUFAs (n-3 or n-6) may be critically required by cerebral components to execute optimal brain function(s) and that could account for the observed discrepant levels of cortical Aβ and LDL-c in response to HFD fortified with high n-6/n-3 PUFAs ratio. Further studies are needed to uncover the underlying mechanisms behind the observed discrepant response of Aβ levels to dietary n-6/n-3 PUFA ratio.
It is well known that BACE1 catalyzes the formation of the amyloid-beta peptide from the APP. An increase in BACE1 activity enhanced the production of Aβ and triggered the deposition of Aβ plaque in the brain . In our study, we only observed a decreased trend of APP in mice treated with different n-6/n-3 PUFA ratios without affecting the protein expression of BACE1. This finding suggests that the APP was modulated by PUFAs in HFD, and this modulatory effect was exclusive to BACE1 gene activity. The mismatched changes in APP and BACE1 protein expressions and cerebral Aβ content suggest the involvement of other potential molecular mechanisms in the homeostasis of Aβ in the brain.
The IR is a kind of receptor from the tyrosine kinase family. After binding with insulin, IR activates intracellular tyrosine kinase and initiates intracellular signaling pathways involving a series of physiological responses. Reduced IR sensitivity and hyperphosphorylation of IR have been reported in AD patients , potentially suggesting that impaired insulin responsiveness in the brain may be intrinsic to AD pathogenesis. In the current study, we found that the mice treated with n-3 and n-6 PUFA rich diets showed a significant decrease in cortical p-IR/IR ratio. The decrease of p-IR/IR ratio indicates the inhibition of insulin metabolic pathway, which in turn inhibits the glucose uptake. Vishal Kothari et al. also reported that the HFD-fed C57BL/6J mice exhibited a lower p-IR/IR ratio than the normal diet-fed control animals . Contrary to that finding, in our study, 60% and 45% HFD did not cause a significant decrease of cortical p-IR/IR ratio, however, a decrease of cortical p-IR/IR ratio was observed, especially in mice treated with n-6 PUFA rich 45% HFD, suggesting that dietary PUFAs have the potential in regulating phosphorylation of IR in HFD-fed APP/PS1 mice. It has been reported that increasing pro-inflammatory cytokines (such as TNF-α and IL-6) can potentially disorient insulin metabolic action(s) and receptor functioning . Excessively high n-6 PUFAs was reported to accelerate oxidative stress and oxidized LDL-c . All, these results could partially explain the decrease of cortical p-IR/IR ratio which was especially observed in mice treated with n-6 PUFA rich 45% HFD.
Insulin mRNA expression has been found in both human and mouse brain samples. Rhea et al. also reported that insulin can transport across the blood-brain barrier (BBB) in vivo with either loss or inhibition of the signaling-related insulin receptor, but the binding of insulin to the brain endothelial cells would be decreased . In our study, APP/PS1 mice showed similar cortical insulin levels with C57BL/6J control mice, indicating that the AD pathological changes did not affect the insulin content in the AD model mice. Also, we did not observe any alteration in cortical insulin level in mice treated with HFD. Stanley et al. reported that peripheral hyperinsulinemia could not affect cerebral insulin signaling and insulin levels . Moreover, a study has proved that cells from the hippocampus and olfactory bulb can produce insulin . Our study further confirms that the cerebral insulin homeostasis cannot be affected by peripheral hyperinsulinemia in APP/PS1 mice, and we reasonably hypothesized that this restriction to the hyper-permeability of insulin to the cortex may be tightly regulated by the BBB. The inconsistent changes of serum and cortical insulin level in response to HFD treatment indicated a discrepant regulating mechanism of peripheral and central insulin metabolism. Further investigations are needed to elucidate the underlying mechanism attributing to the discrepant peripheral and central insulin metabolism.
Glucose uptake in the brain is dependent on IR-stimulated translocation of glucose transporter in the plasma membrane. GLUT3 is extensively expressed in the cortex and plays an important role in neuronal glucose uptake. Evidence suggests that HFD has the potential of reducing cerebral GLUT3 protein expression and causing impaired cerebral glucose uptake . Conversely, Kothari and colleagues’ study reported a compensatory overexpression of GLUT3 in the brains of diabetic rats . In the present study, APP/PS1 mice showed lower cortical expression of GLUT3 protein than C57BL/6J control mice, which indicated an AD-like pathology-mediated decrease of cerebral glucose uptake in the AD-model mouse. We also found that 60% HFD-fed APP/PS1 mice displayed a significant increase of cortical GLUT3 expression, suggesting that HFD was efficient in reversing the downregulated glucose uptake in the brain in AD model mice. These results indicated that the increased cortical expression of GLUT3 protein expression could be a compensatory genetic positive-feed response to the insulin resistance initially induced by the HFD, and potentially due to a decreased threshold to cortical insulin sensitivity (since the primary energy source to the brain is obtained from glucose). Prolonged exposure to HFD not only induces pro-inflammatory cytokines and insulin resistance but may also be involved in adaptive genetic feedback response to engage essential proteins and transporters to compensate and improve glucose or nutrient uptake into cells as we have observed with GLUT3 in these HFD-induced insulin resistant APP/PS1 mice. Relative lower cortical GLUT3 expression was found in APP/PS1 mice from 45% HFD-N3 group as compared with mice treated with 45% HFD and 45% HFD-N6 diets, indicating a dietary PUFAs-dependent optimal response of cerebral GLUT3 expression. On the contrary, we detected higher GLUT3 expression in the liver and GLUT4 expression in muscle in APP/PS1 control mice in comparison with C57BL/6J mice (Supplementary Fig. 2A&B). This observation suggests that there may be an overlap of peripheral (hepatic and skeletal) insulin resistance phenotype and AD-like pathological cascade in these ND-fed APP/PS1 mice, implying that AD pathologic process (particularly, APP gene dysfunction) may be a risk factor for peripheral insulin resistance. Further study is required to ascertain this observation. We, however, observed a significant decrease of these GLUTs in 60% HFD-fed APP/PS1 mice in liver and muscle tissues (potentially due to a slightly increased threshold to peripheral insulin sensitivity) further indicating a discrepant peripheral and central glucose metabolism in response to HFD intervention in APP/PS1 mice. Additionally, the divergent sensitivities of liver or muscle GLUT3/4 expression in mice treated with the different diets also hint the role of dietary PUFA rations on glucose uptake in peripheral tissues.
IRSs are known to be major substrates of receptor kinases, mediating insulin-like growth factors (IGFs)/insulin signals to direct anabolic bioactivities, such as energy and glucose homeostasis, growth and adipocyte differentiation . Postmortem studies have described a reduction in brain expression of IR adapter protein IRS-1 in AD subjects . In comparison with C57BL/6J mice, significantly upregulated IRS-1 protein expression was found in the hippocampus of APP/PS1 mice . The IDE is responsible for insulin breakdown in various tissues. Results from other studies have indicated that HFD-fed APP/PS1 mice showed significantly lower brain IDE protein expression than normal diet-fed control animals . We found that the IRS-1 and IDE cortical protein expression remained unchanged in APP/PS1 mice treated with different diets. These results indicate that the effect of n-6/n-3 PUFA ratios in HFD on cerebral glucose homeostasis and insulin signaling pathway in APP/PS1 mice are not entirely attributable to the regulation of IRS-1 and IDE expression. The other mechanisms, such as the regulation of enzyme activity, interactions or binding between substrates and enzymes, might contribute to the HFD-mediated changes of central insulin metabolism, warranting further exploration.
The GSK3β is a key molecule involving in neural progenitor cell proliferation, neuronal polarity, and neuroplasticity . Insulin could stimulate phosphorylation of GSK3β and consequently result in bidirectional (phosphorylation and inactivation) effect on glycogen synthase, an enzyme responsible for glycogenesis. Elevated total GSK3β protein expression indicates hyper-activity of GSK3β signaling . In the pathology of AD, GSK3β has been shown to promote Tau phosphorylation through the insulin/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway . In our study, APP/PS1 mice showed a relatively higher cortical GSK3β expression but lower p-GSK3β/GSK3β ratio than C57BL/6J control mice, indicating a potential activation of the GSK3β signaling pathway in this AD model mice. HFD treatment has been reported to cause a decrease in cortical p-GSK3β expression and p-GSK3β/GSK3β ratio in APP/PS1 mice, suggesting a regulatory effect of HFD on the phosphorylation of GSK3β and in modifying the activity of GSK3β. Moreover, a decreased trend of cortical GSK3β expression was found in 45% HFD-N3 and 45% HFD-N6 diets treated mice. Additionally, significant decreases of cortical p-GSK3β and p-GSK3β/GSK3β ratio were also found in 45% HFD-N6 diet-fed APP/PS1 mice, indicating hyper-activation of GSK3β in these high n-6/n-3 PUFA ratio diet-fed APP/PS1 mice. Together with the relatively high cortical GLUT3 protein expression observed in these animals, we speculate that diet with a high n-6/n-3 PUFA ratio might affect brain glucose metabolism through regulating the phosphorylation of GSK3β, therefore affecting the uptake of glucose in the brain. Altogether, our findings indicated that the discrepant regulation of GSK3β activation might contribute to the dietary n-6/n-3 PUFA ratio-associated modifying effect in insulin signaling pathway in the brain of APP/PS1 mice.
NF-κB is a nuclear transcription factor that belongs to the Rel protein family, and accumulating evidence suggests its critical role in the pathophysiology of AD. Activated NF-κB p65 in the brain could regulate the expression of many genes, most of which encode immune proteins with pro-inflammatory activities . Consistent with a previous study , we detected that cortical NF-κB p65 translocated from the cytoplasm to nuclei in the cortex in mice-treated with 60% HFD, indicating the activation of NF-κB p65 signaling pathway. While, the treatment of PUFAs rich HFD treatment inhibited the intracellular translocation of NF-κB p65, especially in n-3 PUFAs rich HFD-fed animals. We also found that APP/PS1 mice showed significantly up-regulated cortical TNF-α, COX2, IL-1β and IL-6 mRNA expressions, but lower iNOS mRNA expression than C57BL/6J mice, demonstrating an enhanced neuro-inflammatory response in these AD model mice. Anthony Pinçon et al. reported that administration of a high-cholesterol diet caused 38% increase of cortical TNF-α mRNA expression in APP/PS1 mice as compared with control mice without affecting IL-6 and IL-1β mRNA expressions . Inconsistent with this report, we observed a significant decrease in cortical TNF-α, COX2 and IL-6 mRNA expression, but an increase in iNOS mRNA expression in HFD-fed APP/PS1 mice. The discrepancy between studies might attribute to the diversity of intervention diet or the study design. Moreover, we observed that the 60% and 45% HFDs did not affect cortical IL-1β mRNA expression. In contrast, n-3 PUFAs-rich 45% HFD significantly increased cortical IL-1β mRNA level, and a decrease expression was observed in mice treated with n-6 PUFAs-rich 45% HFD. Although it is commonly recognized that n-6 PUFA has pro-inflammatory functions as prostaglandin and leukotriene precursors, n-6 PUFA can also be converted into anti-inflammatory mediators or modulate the gene expression of the pro-inflammatory cytokine to exert anti-inflammatory effects. Alashmali et al. reported that adequate dietary n-6 PUFA intake restored hippocampal inflammation cytokine gene expression caused by intra-cerebroventricular lipopolysaccharide (LPS) injection . We, therefore, speculate that the AD-like pathology in APP/PS1 mice might have caused hyperactivated neuroinflammation in the brain, which could be antagonized by the treatment with HFD. The inconsistent expressional pattern of pro-inflammatory cytokines in the cortexes of HFD-fed mice further indicated the potentially varied regulatory effect(s) of dietary fatty acids on the cerebral inflammatory response. Thus, further studies are needed to elucidate the relationship between HFD (especially with different PUFAs ratios) and neuroinflammation in modulating AD pathology process.
A few limitations remain in this study. First, due to the young age of the mice and the short duration of the intervention, no significant differences were found in learning and memory abilities. Therefore, a much longer period of dietary intervention was required of to explore the persistent effects of dietary PUFAs on these behavioral indices. Second, we only used male APP/PS1 mice for experiment, while recent studies have shown that APP/PS1 transgenic mice showed a gender difference in behavior and AD-like pathology in brain . Thus, it is essential to further investigate the sex-based effects of dietary PUFAs on APP/PS1 mice. Also, due to the limited brain species, we fall to detect the level of pro-inflammatory cytokine in the brain, thus, we could not provide a more accurate cerebral neuro-inflammatory profile in the experimental animals.