Fig. 1Experimental schedule for evaluating the effect of a growth hormone (GH)-releaser diet on the cognitive dysfunction in klotho mutant mice. Mice received a GH-releaser diet or a vehicle diet during the entire experimental period. JB-1, an insulin-like growth factor-1 (IGF-1) receptor antagonist, was infused (1 µg/5 µL/5 min, once daily for 25 days) into the lateral ventricle. Twenty days after the GH releaser diet began, a novel object recognition test (NORT) and a conditioned fear learning test (CFL) were performed. Mice were sacrificed for Western blot, reverse transcription-polymerase chain reaction (RT-PCR), or neurochemical analyses at 15 minutes after the CFL.
Fig. 2Effect of a growth hormone (GH)-releaser diet on the mRNA expression of insulin-like growth factor-1 (IGF-1) and IGF-1 receptor in the hippocampus of klotho mutant mice. Each value is the mean±SEM of six animals. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; aCSF, artificial cerebrospinal fluid; Veh, vehicle diet; GHR, GH-releaser diet. aP<0.01 vs. wild-type mice treated with aCSF and vehicle diet; bP<0.05; cP<0.01 vs. klotho mutant mice treated with aCSF and vehicle diet; dP<0.05; eP<0.01 vs. klotho mutant mice treated with aCSF and GH releaser diet (three-way analysis of variance followed by multiple pair-wise comparisons with Bonferroni's correction).
Fig. 3Effect of JB-1, an insulin-like growth factor-1 (IGF-1) receptor antagonist, on the growth hormone (GH)-releaser diet-mediated attenuation of changes in the expression of (A) phospho-Akt (p-Akt), (B) phospho-glycogen synthase kinase3β (p-GSK3β), and (C) phospho-calcium/calmodulin-dependent kinase II (p-CaMKII) in the hippocampus of klotho mutant mice. Each value is the mean±SEM of six animals. aCSF, artificial cerebrospinal fluid; Veh, vehicle diet; GHR, GH-releaser diet. aP<0.01 vs. wild-type mice treated with aCSF and vehicle diet; bP<0.01 vs. klotho mutant mice treated with aCSF and vehicle diet; cP<0.05; dP<0.01 vs. klotho mutant mice treated with aCSF and a GH-releaser diet (three-way analysis of variance followed by multiple pair-wise comparisons with Bonferroni's correction).
Fig. 4Effect of JB-1, an insulin-like growth factor-1 (IGF-1) receptor antagonist, on the growth hormone (GH)-releaser diet-mediated attenuation of changes in the expression of (A) phospho-extracellular signal-related kinase (p-ERK), (B) phospho-c-jun N-terminal kinase (p-JNK), (C) phospho-p38 (p-p38), and (D) phospho-cAMP responsive element binding protein (p-CREB) in the hippocampus of klotho mutant mice. Each value is the mean±SEM of six animals. aCSF, artificial cerebrospinal fluid; Veh, vehicle diet; GHR, GH-releaser diet. aP<0.01 vs. wild-type mice treated with aCSF and vehicle diet; bP<0.05; cP<0.01 vs. klotho mutant mice treated with aCSF and vehicle diet; dP<0.05; eP<0.01 vs. klotho mutant mice treated with aCSF and a GH-releaser diet (three-way analysis of variance followed by multiple pair-wise comparisons with Bonferroni's correction).
Fig. 5Effect of JB-1, an insulin-like growth factor-1 (IGF-1) receptor antagonist, on the growth hormone (GH)-releaser diet-mediated attenuation of changes in the expression of (A) Bax, (B) cleaved caspase-3, (C) Bcl-2, and (D) Bcl-xL in the hippocampus of klotho mutant mice. Each value is the mean±SEM of six animals. aCSF, artificial cerebrospinal fluid; Veh, vehicle diet; GHR, GH-releaser diet. aP<0.01 vs. wild-type mice treated with aCSF and vehicle diet; bP<0.01 vs. klotho mutant mice treated with aCSF and vehicle diet; cP<0.05; dP<0.01 vs. klotho mutant mice treated with aCSF and a GH-releaser diet (three-way analysis of variance followed by multiple pair-wise comparisons with Bonferroni's correction).
Fig. 6Effect of JB-1, an insulin-like growth factor-1 (IGF-1) receptor antagonist, on the growth hormone (GH)-releaser diet-mediated attenuation of lipid peroxidation, as measured by (A) the malondialdehyde (MDA) level, and protein oxidation, as measured by (B) the protein carbonyl level in the hippocampus of klotho mutant mice. Each value is the mean±SEM of eight animals. DNPH, 2,4-dinitrophenylhydrazine; aCSF, artificial cerebrospinal fluid; Veh, vehicle diet; GHR, GH-releaser diet. aP<0.05; bP<0.01 vs. wild-type mice treated with aCSF and vehicle diet; cP<0.05; dP<0.01 vs. klotho mutant mice treated with aCSF and vehicle diet; eP<0.05 vs. klotho mutant mice treated with aCSF and a GH-releaser diet (three-way analysis of variance followed by multiple pair-wise comparisons with Bonferroni's correction).
Fig. 7Effect of JB-1, an insulin-like growth factor-1 (IGF-1) receptor antagonist, on the growth hormone (GH)-releaser diet-mediated attenuation of cognitive dysfunction, as evaluated by (A) a novel object recognition test and (B) a context-dependent conditioned fear learning test in klotho mutant mice. Each value is the mean±SEM of 10 animals. aCSF, artificial cerebrospinal fluid; Veh, vehicle diet; GHR, GH-releaser diet. aP<0.01 vs. wild-type mice treated with aCSF and vehicle diet; bP<0.01 vs. klotho mutant mice treated with aCSF and vehicle diet; cP<0.05 vs. klotho mutant mice treated with aCSF and a GH-releaser diet (three-way analysis of variance followed by multiple pair-wise comparisons with Bonferroni's correction).