*3.1. Activation of Specific Ser Residues on Hippocampal Insulin Receptor Substrate 1 (IRS1) Is Associated with T2DM-Induced Memory Impairment*

We recently reported that T2DM-related cognitive declines in 45-week-old DIO mice fed a 60% HFD for 42 weeks were accompanied by an increased phosphorylation of hippocampal IRS1 at mSer1097 [20]. While mice fed a 60% HFD for 17 days and mice or rats fed a 40% HFD for 6–8 weeks exhibited memory impairment accompanied by increased phosphorylation at hSer616/mSer612 or hSer312/mSer307 [21,22], 12-week-old DIO mice fed a 60% HFD for 9 weeks exhibited normal memory function under our experimental conditions (Figure S1), suggesting that changes in neural IRS1 Ser residues are variable on a temporal scale in DIO mice. Therefore, we investigated the phosphorylation levels of hippocampal IRS1 at Ser sites and the activities of downstream factors involved in memory impairment in 35-week-old (middle-aged) DIO mice fed a 60% HFD for 32 weeks (Figure 1A,C). At 35 weeks of age, DIO mice displayed significant weight gain, hyperglycemia, and hyperinsulinemia (Figure 1B, Figure S2A), as observed at other time points [20–22,26].

**Figure 1.** Changes in specific Ser sites on hippocampal insulin receptor substrate 1 (IRS1) in type 2 diabetes (T2DM)-induced memory impairment. (**A**) Schematic diagram of the experimental procedure. (**B**) Graphs of body weight and blood glucose level in wild-type (WT) and diet-induced obesity (DIO) mice (35 weeks of age, n = 5 mice per group). (**C**) Evaluation of learning memory function in middle-aged WT and DIO mice (n = 14 mice per group) using the water T-maze test. (**D**) Quantitative analysis of T-PER (Tissue Protein Extraction Reagent)-extractable Aβ40 and Aβ42 levels in the hippocampi of middle-aged WT and DIO mice using the human/rat/mouse β amyloid (1–40 and 1–42) enzyme-linked immunosorbent assay (ELISA) (35 weeks of age, n = 5 biologically independent samples per group). (E) Western blot analysis of phosphorylated insulin receptor substrates 1 mouse Ser307 [p-IRS1 (mSer307)], p-IRS1 (mSer612), p-IRS1 (mSer632/635), p-IRS1 (mSer1097), IRS1, and β-tubulin in the hippocampi of middle-aged WT and DIO mice (35 weeks of age, n = 5 biologically independent samples per group). Arrow indicates the p-IRS1 mSer612-corresponding band (lower band) in (**E**). Quantitative analysis of the phosphorylation of IRS1 at mSer307, mSer612, mSer632/635, and mSer1097 normalized to total protein. (**F**) Western blot analysis of phosphorylation levels of Akt Ser473, p70S6K Thr389, AMP-activated protein kinase (AMPK) Thr172, and glycogen synthase kinase 3 beta (GSK3β) Ser9 as well as total protein levels of Akt, p70 S6K, AMPK, GSK3β, and ß-tubulin in the hippocampi of middle-aged WT and DIO mice (35 weeks of age, n = 5 biologically independent samples per group). Quantitative analysis of the phosphorylation of Akt Ser473, p70S6K Thr389, AMPK Thr172, and GSK3β Ser9 normalized to the respective total protein contents. Results are presented as mean ± standard error of the mean (SEM), \* *p* < 0.05; \*\* *p* < 0.01.

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Furthermore, we examined whether memory impairment in middle-aged DIO mice is linked to increased levels of Aβ42, a pathological feature of AD. Biochemical analysis with specific kits (see Materials and Methods) demonstrated that, compared with age-matched wild-type (WT) mice, there was no change in Aβ40 and Aβ42 levels in the T-PER fractions obtained from the hippocampi of 35-week-old DIO mice (Figure 1D).

In contrast to DIO mice at 45 weeks of age, the phosphorylation of IRS1 at mSer307, which increases in insulin resistance, diabetes, and obesity [5], with phosphorylation at mSer1097 significantly increased in the hippocampus of DIO mice at 35 weeks of age, although there were no significant differences in phosphorylation at mSer612 and mSer632/635 between DIO and age-matched WT mice (Figure 1E, Figure S4A). Consistent with previous studies [27,28], the basal phosphorylation level of p70S6K slightly but significantly increased in the hippocampus of middle-aged DIO mice compared with that in the hippocampus of age-matched WT mice, whereas the basal phosphorylation of Akt and GSK3β and activation of AMPK and atypical protein kinase C ζ/λ (aPKC ζ/λ), downstream factors of insulin signaling, in the hippocampus were comparable between middle-aged WT and DIO mice (Figure 1F, Figures S3A, S4B and S5A). Additionally, the basal phosphorylation of JNK, an inflammationand stress-related factor, remained unchanged in the hippocampus between the two groups (Figures S3A and S5A), although a relationship between the phosphorylation of IRS1 at mSer307 and activation of these factors in yeast cells, culture cells, and muscles has been reported [5]. These results indicate that T2DM-induced memory decline is provoked by an Aβ-independent mechanism and that the concomitant activation of IRS1 mSer307 and mSer1097 with p70S6K activation in the hippocampus is associated with memory deficits in 35-week-old DIO mice.
