*3.2. Type 1 Diabetes Mellitus (T1DM)-Induced Memory Deficits Occur Independently of IRS1 Activity*

To investigate whether the alteration of IRS1 through Ser phosphorylation is associated with type 1 diabetes mellitus (T1DM)-induced memory deficits, we generated STZ-induced insulin-deficient T1DM mice. Two weeks after STZ injection, STZ mice exhibited weight loss, elevated blood glucose levels (>400 mg/dL), and low insulin levels (Figure 2A, Figure S2B). While there were no significant differences in hippocampus-dependent spatial memory between WT and STZ mice, STZ mice displayed hippocampus- and prefrontal cortex-related memory decline (Figure 2B), consistent with the findings reported in previous studies [29–32]. However, STZ-induced T1DM had no effect on Aβ40 and Aβ42 levels in the T-PER fractions of the hippocampus (Figure 2C).

Subsequently, we examined the impact of STZ-induced T1DM on the phosphorylation of IRS1 at Ser residues and downstream components in the hippocampus. In STZ mice that had already developed memory impairment, the phosphorylation levels of hippocampal IRS1 at Ser residues in STZ mice were comparable to those in WT mice (Figure 2D, Figure S4C). Although the activation of p70S6K and the monotonous activities of Akt, AMPK, aPKC ζ/λ, and JNK were observed in STZ mice as well as in middle-aged DIO mice regardless of the presence or absence of Ser phosphorylation on hippocampal IRS1, the phosphorylation of GSK3β at Ser9 significantly increased in the hippocampus of STZ mice (Figure 2E, Figure S3B, S4D and S5B). These data indicate that T1DM-induced memory deficits accompanied by increased phosphorylation of GSK3β arises independently of the modification of IRS1 signaling via Ser phosphorylation and independently of Aβ elevation.

**Figure 2.** No alteration of IRS1 Ser phosphorylation in the hippocampus of T1DM mouse models. (**A**) Graphs of body weight and blood glucose level in wild-type (WT) and streptozotocin (STZ)-induced insulin-deficient type 1 diabetes (T1DM) model mice (10 weeks of age, n = 5 mice per group). (**B**) Evaluation of hippocampus-dependent learning and memory function and hippocampus/prefrontal cortex associated working memory function in WT (10 weeks of age, n = 19 mice per group) and STZ mice (10 weeks of age, n = 18 mice per group) using the water T-maze test and reverse water T-maze test. (**C**) Quantitative analysis of T-PER-extractable Aβ40 and Aβ42 levels in the hippocampi of WT and STZ mice using the human/rat/mouse β amyloid (1–40 and 1–42) ELISA (10 weeks of age, n = 5 biologically independent samples per group). (**D**) In WT and STZ-induced type 1 diabetes mice (10 weeks of age, n = 5 biologically independent samples per group), 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 was performed. 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, ß-tubulin in WT and STZ mice (10 weeks of age, n = 5 biologically independent samples per group). 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, p70 S6K 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.
