Fig 2: Simultaneous removal of AKT1 and AKT3 isoforms from the brain promotes memory deficits in both male and female mice.Male and female mice with conditional Akt1 removal in the background of Akt3 deficiency (Akt1 cKO Akt3 KO) were assessed in anxiety-related behavior, spatial memory, and associative fear memory tests. No differences were detected between Akt1 cKO Akt3 KO mutant mice and WT controls for both sexes in (A) OFA activity, measured as percent time spent exploring the center zone, and (B) EPM activity, measured as percent time spent in the open arms. Distance moved during OFA or EPM assays were similar between genotypes for both sexes. N = 15 WT-M, 13 cKO/KO-M, 16 WT-F, 15 cKO/KO-F. (C) Male and (D) female Akt1 cKO Akt3 KO mice had similar latencies to escape the MWM during training, reversal, and visible platform phases compared to their respective WT controls but showed significantly reduced target quadrant time and platform crossings. N = 13 WT-M, 9 cKO/KO-M, 12 WT-F, 12 cKO/KO-F. (E) Male and (F) female Akt1 cKO Akt3 KO mice displayed normal fear learning but impaired contextual and cued LTM compared with their respective WT controls. N = 11 WT-M, 11 cKO/KO-M, 14 WT-F, 10–11 cKO/KO-F. *p<0.05.Figure 7—source data 1.Figure 7 source data.

Fig 3: Genome-wide cDNA microarray and Wb analyses of HHAT expression in eSAS cells(A) List of fold-changes in BPIFA1, HHATA, and ARHGEF3 mRNA levels, showing that theses gene are conspicuously upregulated in eSAS cells compared with SAS cells. (B) Scatter plot of the average expression values of eSAS samples versus SAS samples from DNA microarray analysis, indicating that the mRNA levels of HHATA and ARHGEF3 are differentially expressed in eSAS cells versus SAS cells and that these differences are statistically significant. The mRNA level of the BPIFA1 gene was very low in the SAS group, which resulted in erroneous fold-change values. (C) Wb analysis indicating that the expression of ARHGEF3 at the protein level was not significantly different between eSAS and SAS cells. (D) Wb analysis demonstrated that HHAT protein levels were higher in eSAS cells than in SAS cells at both low (L) and high (H) cell densities (blue arrowhead). (E) Addition of RU-SKI 43 to eSAS and SAS cells under SFM/SFP conditions. Typical microscopic image (i), and line graphs showing the average percentage value for spheroid formation of three independent experiments at each concentration of RU-SKI 43 (ii). (F) Wb analysis of the spheres from SAS and eSAS cells with (+) or without (−) 10 μM RU-SKI 43 for 4days using the antibodies against HHAT, mTOR, phosphorylated-mTOR (pS2448), AKT, and phosphorylated-AKT (pS308). Arrows show the bands of HHAT protein. α-tubulin is a loading control. (G-i) Wb analysis of the lysates from eSAS cells transfected with siRNA against HHAT (siHHAT) or luciferase (siGL2) as a negative control. Arrow shows the band of HHAT. α-tubulin is a loading control. (G-ii) Wb analysis of the spheres from eSAS cells knocked-down HHAT. Arrows show the band of stem cell markers. α-tubulin is a loading control. (H) Bar graphs indicate the percentages of sphere formation for eSAS cells transfected with siHHAT or siGL2 at day 4. Bar graphs were constructed based on results of three independent experiments. (I, K) SAS cells were transfected with 6Myc-tagged HHAT or 6Myc-vector alone. At 48 hours after transfection (TF), the cells were cultured for 4 days under SFM/SFP condition to generate spheroids. Wb analysis of the lysates from TF or spheres using the antibodies against Myc-tag and HHAT (I) or stem cell markers (K). Asterisks show non-specific bands. (J) Bar graphs indicate the percentages of sphere formation for SAS cells transfected with 6Myc-tagged HHAT or 6Myc-vector alone at day 4. Bar graphs were constructed based on results of three independent experiments.

Fig 4: Incremental gene expression following AKR1D1 knockdown followed by subsequent treatment with CA, CDCA, FXR agonst or LXR antagonist treatment to attempt to rescue the cellular phenotype. CA replacement (50 μM, 24 h) in the cell culture media failed to prevent upregulation of carbohydrate, lipid or insuling signalling gene expression in AKR1D1 knocked down HepG2 cells (a). In contrast, CDCA replacement (50 μM, 24 h) significantly impaired upregulation of the expression of CYP7A1, FASN, SCD1, SREBF1, AKT1 and GLUT1, in AKR1D1 knocked down HepG2 cells (b). Pharmacological manipulation of the bile acid receptor FXR, using the FXR agonist GW4064 (5 μM, 24 h), also normalised the expression of CYP7A1, FASN, ACC1, SREBF1, SCD1, AKT1, GLUT1 and GLUT9 to levels seen in scrambled controls (figure c–h). In addition, treatment with the LXR antagonist 22-S Hydroxycholesterol (22-S HC - 10 μM, 24 h) and the LXRβ antagonist GKS2033 (100 nM, 24 h) partially restored the expression levels of FASN, ACC1 and SREBF1, but had no impact on the induction of expression of the other genes caused by AKR1D1 knockdown (c–h). qPCR data were normalised to 18SrRNA. Data are presented as mean ± se of incremental gene expression of n = 3–6 experiments, performed in triplicate, *p < 0.05, **p < 0.01, ***p < 0.001 and compared to vehicle treated controls. (KD = AKR1D1 knockdown).

Fig 5: Akt deficiency affects spatial memory formation in an isoform- and sex-specific fashion.Spatial learning and memory were assessed using the Morris Water Maze (MWM) in male and female mice with single-isoform deletions of Akt1, Akt2, or Akt3. Left graph: Learning measured by latency of mice to escape the MWM during training to locate a hidden escape platform (days 1–8), reversal training to locate the hidden escape platform moved to the opposite quadrant (days 9–11), and visible platform training to locate the escape platform marked by a visual cue (days 12–13). Memory measured by percent time spent in the target quadrant where mice were trained to locate the hidden escape (left bar graph) and number of platform location crossings in the target quadrant (right bar graph) during a probe test. MWM performance in (A) Akt1 KO male, (B) Akt1 KO female, (C) Akt2 KO male, (D) Akt2 KO female, (E) Akt3 KO male, and (F) Akt3 KO female mice. Akt1 KO male mice showed a significant reduction in platform crossings compared with WT controls during the probe test. No other differences were detected between Akt isoform KO and WT mice. *p<0.05. N = Akt1 (WT-M = 9 KO-M = 9, WT-F = 9, KO-F = 9); Akt2 (WT-M = 9, KO-M = 11, WT-F = 11, KO-F = 9); Akt3 (WT-M = 10, KO-M = 11, WT-F = 10, KO-F = 9).Figure 2—source data 1.Figure 2 source data.