Ketogenic diets initiated in late mid-life improved measures of spatial memory in male mice

Abstract

Studies have shown ketogenic diets (KD) started from early middle-age improved health span and longevity in mice. KDs started later in life or administered intermittently may be more feasible and promote compliance. Therefore, this study sought to test if continuous or intermittent KDs started in late-middle-aged mice would improve cognition and motor function at advanced age. Eighteen-month-old male C57BL/6JN mice were assigned to an isocaloric control (CD), KD, or intermittent ketogenic (IKD, 3-day KD/week) diet. A panel of behavior tests were performed to assess cognitive and motor functions with aging. Y-maze alternation rate was higher for both IKD and KD mice at 23 months of age and for KD mice at 26 months indicating an improved spatial working memory. Twenty-six-month-old KD mice also showed better spatial learning memory in Barnes maze when compared to the CD. Improved grid wire hang performance was observed in aged IKD and KD versus CD mice indicating better muscle endurance under isometric contraction. A reduced level of circulating proinflammatory cytokines in aged KD (IL-6 and TNF-α) and IKD (IL-6) mice may contribute to the phenotypic improvements observed with these interventions. This study demonstrates that when initiated at late-middle age, the KD improved measures of spatial memory and grid wire hang performance in aged male mice, with IKD showing results intermediate to the CD and KD groups.

Keywords: Aging; Health span; Ketogenic diet.

Fig. 1

Differential adaptation of 18-month male mice to a KD or IKD. (A) Body weights of CD, IKD, and KD mice throughout the study (n = 19–20/group). (B–C) Percent fat (B) and lean (C) mass measured using EchoMRI at 27 months of age (n = 10–12/group). (D–F) Relative mass of gastrocnemius (D), tibialis anterior (E), and soleus (F) muscle to total lean mass (n = 10–12/group). (G) Circulating level of β-hydroxybutyrate (n = 19–20/group). (H–I) Content of liver total cholesterol (H) and triglyceride (I) (n = 8/group). Diets: CD—control, IKD—intermittent ketogenic, KD—ketogenic. ∗ p < 0.05, ∗  ∗ p < 0.01, ∗  ∗  ∗  ∗ p < 0.0001, #p < 0.05 between fasted vs. fed ketone levels, based on repeated measures two-way ANOVA or one-way ANOVA followed with Tukey’s multiple comparisons tests

Fig. 2

Levels of circulating proinflammatory cytokines and markers of metabolism at 27 months of age in male mice. Circulating levels of (A) IL-6, (B) TNF-α, (C) KC/GRO, (D) total triglyceride (TG), (E) free fatty acids (FFA), (F) total cholesterol (TC), (G) HDL-C, (H) LDL-C and VLDL-C, (I) insulin, (J) fibroblast growth factor-21 (FGF21), and (K) insulin like growth factor-1 (IGF1) (n = 10/group). Diets: CD—control, IKD—intermittent ketogenic, KD—ketogenic. ∗ p < 0.05, ∗  ∗ p < 0.01, ∗  ∗  ∗ p < 0.001 based on one-way ANOVA followed with the Tukey’s multiple comparisons test or the Brown-Forsythe and Welch ANOVAs followed with the Dunnett’s T3 multiple comparisons test where appropriate

Fig. 3

Behavior tests of motor function and anxiety in male mice at 20, 23, and 26 months of age. (A) Grid wire hang test: maximum hanging impulse. (B) Grip strength test: relative peak force of forelimbs exerted on the force meter. (C) Rearing score: number of wall-contact rears by the front paws of the animal. (D) Open field test: total distance moved as a measure of locomotor activity. (E) Composite score of motor function tests (data from A–D). (F) Open field test: time spent in the center region as a measure of anxiety (not included in the composite score of motor function tests). 20 months: n = 18–20; 23 months: n = 17–18; 26 months: n = 14–17/group. Diets: CD—control, IKD—intermittent ketogenic, KD—ketogenic. Data were analyzed by repeated measures two-way ANOVA followed with the Tukey’s or the Bonferroni multiple comparisons tests. ∗ p < 0.05 compared to the CD group at the same age. Different letters of a, b, or c denote difference (p < 0.05) between ages

Fig. 4

Behavior tests of cognitive function in male mice at 23 and 26 months of age. (A) Y maze spontaneous alternation test: percent of alternations. (B) Novel object test: percent time exploring the novel object. (C) Barnes maze test: latency to the target hole in the probe trial (lower the better). (D) Barnes maze test: time spent in the target quadrant in the probe trial (higher the better). (E) Composite of cognitive tests. 23 months: n = 17–18; 26 months: n = 14–17/group. Diets: CD—control, IKD—intermittent ketogenic, KD—ketogenic. Data were analyzed by repeated measures two-way ANOVA followed with the Tukey’s or the Bonferroni multiple comparisons tests. ∗ p < 0.05, ∗  ∗  ∗  ∗ p < 0.0001. Different letters of a and b denote difference (p < 0.05) between ages