*3.3. Phosphorylation of IRS1 at Age-Specific Ser Residues with the Activation of Downstream Kinases Is Linked to Age-Related Memory Deficits*

Although memory decline associated with physiological aging can occur naturally, in contrast to pathogenic memory deficits, whether the onset processes of age-related memory impairment are related to the mechanisms underlying pathogenic memory deficits in T2DM and AD remains unclear. We examined the modification of IRS1 at Ser residues and the activity of downstream factors in the hippocampi of 21-month-old mice (aged mice). Aged mice displayed memory impairments without an increase in blood glucose and plasma insulin levels, despite an increase in body weight (Figure 3A,B, Figure S2C). Meanwhile, the Aβ40 and Aβ42 levels in the T-PER fractions of the hippocampus were comparable between young and aged WT mice (Figure 3C).

In common with DIO mice, the activation of mSer307, but not of mSer1097, on IRS1 accompanied by p70S6K activation and unchanged AMPK was observed with increased phosphorylation of IRS1 at mSer612 and mSer632/635 in the hippocampi of aged mice (Figure 3D, Figure S4E). Nonetheless, unlike in DIO mice, the basal phosphorylation levels of Akt and GSK3β prominently increased in the hippocampus of aged mice (Figure 3E, Figure S4F); however, the basal activities of aPKC ζ/λ, and JNKs remained unchanged, similar to those in DIO mice (Figure S3C and S5C). These results suggest that aging causes the concomitant phosphorylation of hippocampal IRS1 at mSer307, mSer612, and mSer632/635 with the activation of canonical downstream kinases, which may be associated with Aβ-unrelated physiological decline in memory function.

**Figure 3.** Age-related memory deficits accompanied by increased phosphorylation of IRS1 Ser sites in the hippocampus. (**A**) The graphs of body weight and blood glucose levels in young (12 weeks of age, n = 7 mice per group) and aged (84 weeks of age, n = 7 mice per group) wild-type (WT) mice. (**B**) Evaluation of learning memory functions in young WT mice (n = 16 mice per group) and aged WT mice (n = 18 mice per group) using the water T-maze test. (**C**) Quantitative analysis of T-PER-extractable Aβ40 and Aβ42 levels in the hippocampi of young (12 weeks of age, n = 6 biologically independent samples) and aged (84 weeks of age, n = 6 biologically independent samples) WT mice using the human/rat/mouse β amyloid (1–40 and 1–42) ELISA. (**D**) 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 ß-tubulin in the hippocampi of young WT mice (12 weeks of age, n = 6 biologically independent samples) and aged WT mice (84 weeks of age, n = 6 biologically independent samples). Quantitative analysis of the phosphorylation of IRS1 at mSer307, mSer612, mSer632/635, and mSer1097 normalized to total protein. (**E**) Western blot analysis of phosphorylation levels of Akt Ser473, p70S6K Thr389, AMPK Thr172, and GSK3β Ser9 as well as total protein levels of Akt, p70S6K, AMPK, GSK3β, and ß-tubulin in the hippocampi of young WT mice (12 weeks of age, n = 6 biologically independent samples) and aged WT mice (84 weeks of age, n = 6 biologically independent samples). Arrows indicate the p-IRS1 mSer612-corresponding band (lower band) and the p-IRS1 mSer632/635-corresponding band (lower band) in (**D**). 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 ± SEM, \* *p* < 0.05; \*\* *p* < 0.01.
