*3.5. CY-09 Attenuated Insulin Resistance in 3*×*Tg-AD Mice*

GLUT4 is regulated by the insulin signaling pathway to participate in glucose metabolism. Insulin resistance manifests itself as insensitivity to insulin and a higher level of insulin and glucose in the blood, eventually leading to impaired insulin signaling pathways and glucose metabolism. It had been confirmed to exist in AD.

To evaluate the effect of NLRP3 inflammasome activation on insulin resistance in AD, we first detected blood glucose and blood insulin levels. As shown in Figure 4, higher fasting and basal blood glucose and fasting insulin level were found in 3×Tg-AD mice compared with NTg mice, with the *p*-values all lower than 0.05 and 0.01, respectively. Results of the GTT and ITT showed an increased glucose tolerance and decreased insulin tolerance in 3×Tg-AD mice. However, after CY-09 treatment, blood glucose and insulin levels were decreased and better insulin sensitivity was found in CY-09-treated 3×Tg-AD mice. Thus, the results indicated that inhibiting NLRP3 inflammasome activation can attenuate insulin resistance in the 3×Tg-AD mice.

**Figure 2.** 18F-FDG positron emission tomography (PET) images of NTg, NTg + CY-09, 3×Tg-AD, and 3×Tg-AD + CY-09 mice. (**a**) Body weight of mice, before and after CY-09 treatment; (**b**) static PET images of the four groups of mice; (**c**–**e**) standard uptake values in the whole brain, cortex, and hippocampus in the four groups of mice; (**f**) dynamic PET images of the four groups of mice; (**g**) cerebral time–activity curves (TAC) and (**h**) cerebral metabolic rate of glucose (CMRglu) in the four groups of mice. (n = 6, mean ± SD, one-way ANOVA and Bonferroni post hoc test; \* *p* < 0.05 vs. NTg mice, # *p* < 0.05 vs. 3×Tg-AD mice).

**Figure 3.** Increased expression of glucose transporters in CY-09-treated 3×Tg-AD mice. (**a**) Representative Western blots and quantification of (**b**) glucose transporters 1 (GLUT1), (**c**) GLUT3, and (**d**) GLUT4 expression in NTg, NTg + CY-09, 3×Tg-AD and 3×Tg-AD + CY-09 mice (n = 6, mean ± SD, one-way ANOVA and Bonferroni post hoc test; \* *p* < 0.05, \*\* *p* < 0.01 vs. NTg mice, & *<sup>p</sup>* < 0.05 vs. NTg + CY-09 mice, # *<sup>p</sup>* < 0.05 vs. 3×Tg-AD mice); (**e**) distribution of GLUT4 in the CA3 region of the four groups of mice (scale bar: 50 μm).

Then, to further explore the underlying mechanism by which NLRP3 inflammasome activation affects insulin resistance, we detected the expression and distribution of the IR-AKT-AS160 insulin signaling pathway-related proteins in NTg, NTg + CY-09, 3×Tg-AD, and 3×Tg-AD + CY-09 mice. As shown in Figure 5a,b, compared with NTg mice, expression of p-IR-Tyr1150 (phosphorylation protein of IR at Tyr1150) relative to IR was significantly decreased in 3×Tg-AD mice, with a *p*-value lower than 0.01, but the expression was increased after the CY-09 treatment, with a *p*-value lower than 0.05. The distribution of p-IR-Tyr1150 was found to be lower in the 3×Tg-AD mice and higher in the 3×Tg-AD + CY-09 mice. By contrast, the expression and distribution of IR were not different among the four groups of mice (Figures 5i and S3). These findings suggested that inhibiting NLRP3 inflammasome activation can enhance the self-phosphorylation of IR in the 3×Tg-AD mice.

**Figure 4.** CY-09 relieved the insulin resistance in 3×Tg-AD mice. (**a**–**c**) Fasting blood glucose, fed blood glucose, and fasting blood insulin were measured in NTg, NTg + CY-09, 3×Tg-AD and 3×Tg-AD + CY-09 mice; (**d**,**e**) glucose tolerance tests (GTT) and (**f**,**g**) insulin tolerance tests (ITT) were conducted to determine the insulin sensitivity of the four groups of mice. (n = 6, mean ± SD, one-way ANOVA and Bonferroni post hoc test; \* *p* < 0.05, \*\* *p* < 0.01 vs. NTg mice, & *p* < 0.05, && *p* < 0.01 vs. NTg + CY-09 mice, # *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001 vs. 3×Tg-AD mice).

Self-phosphorylation of IR recruits IRS and starts the IRS-AKT-AS160 insulin signaling pathway. In parallel, we detected the expression of IRS, AKT, and AS160 and their phosphorylated levels in the four groups of mice. The results reported in Figure 5c–h show a notable reduction of p-AKT-Ser473 and p-AS160-T642, while significantly increased p-IRS-Ser1101 was found in the 3×Tg-AD mice, with the *p*-value lower than 0.01, 0.01, and 0.05, respectively. Treatment with CY-09 helped to reverse the expression of these proteins in 3×Tg-AD + CY-09 mice. There was also a significant reduction in IRS between the NTg + CY-09 mice and 3×Tg-AD + CY-09 mice, with a *p*-value less than 0.01. No differences were found in the expressions of AKT and AS160 among the four groups of mice. All the results showed that inhibiting NLRP3 inflammasome activation can restore the IR-IRS-AKT-AS160 insulin signaling pathway to alleviate insulin resistance in the 3×Tg-AD mice.

**Figure 5.** CY-09 restored the insulin signaling pathway in 3×Tg-AD mice. (**a**–**h**) Western blot analysis of insulin receptors (IR), p-IR-Tyr1150, insulin receptor substrate (IRS), p-IRS-Ser1101, AKT, p-AKT-Ser473, AS160 and p-AS160-Thr642 in NTg, NTg + CY-09, 3×Tg-AD, and 3×Tg-AD + CY-09 mice. (n=6, mean ± SD, one-way ANOVA and Bonferroni post hoc test; \* *p* < 0.05, \*\* *p* < 0.01 vs. NTg mice, & *<sup>p</sup>* < 0.05, && *<sup>p</sup>* < 0.01 vs. NTg + CY-09 mice, # *<sup>p</sup>* < 0.05 vs. 3×Tg-AD mice). (**i**) Distribution of IR and p-IR-Tyr1150 in the brains of the four groups of mice (scale bar: 100 μm; arrows indicate p-IR in 3×Tg-AD + CY-09 mice).

## *3.6. CY-09 Increased the Expression and Distribution of Metabolic Enzymes in 3*×*Tg-AD Mice*

Increased glucose transport and improved insulin resistance were found in CY-09 treated 3×Tg-AD mice; we also detected the expressions and distribution of HK, which is the first enzyme that phosphorylates glucose when associated with VDAC1 in the mitochondria. We previously reported that the expression of cHK1 (HK1 in the cytoplasm) was significantly increased in 3×Tg-AD mice, while the expression of mHK1 (HK1 in the mitochondria) was remarkably decreased. However, increased HK1 expression and HK activity were found in CY-09-treated N2a-sw cells (a model cell of AD). These results prompted us to examine the expression and activity of HK in CY-09-treated 3×Tg-AD mice. Here, we isolated the mitochondria and cytoplasm from NTg, NTg + CY-09, 3×Tg-AD, and 3×Tg-AD + CY-09 mice to detect the expression and distribution of HK1.

As shown in Figure 6, consistent with our previous results, increased cHK1 and significantly decreased mHK1 and mHK2 were found in the 3×Tg-AD mice, with the *p*-value lower than 0.01 and 0.05. However, the expression of mHK1 was increased, while cHK1 was decreased in the CY-09-treated 3×Tg-AD mice, and the differences were significant compared to the untreated 3×Tg-AD mice, with the *p*-values lower than 0.01 and 0.05. cHK2 was also found to be notably decreased in the CY-09-treated 3×Tg-AD mice, with a *p*-value lower than 0.05. Although HK activity was remarkably decreased in 3×Tg-AD + CY-09 mice than in NTg + CY-09 mice, it was notably increased in the CY-09 treated 3×Tg-AD mice compared to the non-treated 3×Tg-AD mice, with a *p*-value lower than 0.05 (Figure 6b). Besides, we also detected the expressions of pyruvate dehydrogenase α 1 (PDHE1α) and cytochrome c oxidase subunit IV (COX4). They were all significantly reduced in 3×Tg-AD mice, with *p*-values lower than 0.05. Meanwhile, the expression of PDHE1α was remarkably increased in CY-09-treated 3×Tg-AD mice. In short, the data demonstrated an increase in the expression and distribution of HK by inhibiting NLRP3 inflammasome activation in the 3×Tg-AD mice.
