2-month ketogenic diet preferentially alters skeletal muscle and augments cognitive function in middle aged female mice

Abstract

The effect of a ketogenic diet (KD) on middle aged female mice is poorly understood as most of this work have been conducted in young female mice or diseased models. We have previously shown that an isocaloric KD started at middle age in male mice results in enhanced mitochondrial mass and function after 2 months on diet and improved cognitive behavior after being on diet for 14 months when compared with their control diet (CD) fed counterparts. Here, we aimed to investigate the effect of an isocaloric 2-month KD or CD on healthy 14-month-old female mice. At 16 months of age cognitive behavior tests were performed and then serum, skeletal muscle, cortex, and hippocampal tissues were collected for biochemical analysis. Two months on a KD resulted in enhanced cognitive behavior associated with anxiety, memory, and willingness to explore. The improved neurocognitive function was associated with increased PGC1α protein in the gastrocnemius (GTN) muscle and nuclear fraction. The KD resulted in a tissue specific increase in mitochondrial mass and kynurenine aminotransferase (KAT) levels in the GTN and soleus muscles, with a corresponding decrease in kynurenine and increase in kynurenic acid levels in serum. With KAT proteins being responsible for converting kynurenine into kynurenic acid, which is unable to cross the blood brain barrier and be turned into quinolinic acid-a potent neurotoxin, this study provides a potential mechanism of crosstalk between muscle and brain in mice on a KD that may contribute to improved cognitive function in middle-aged female mice.

Keywords: Alzheimer's disease; acetylation; cognitive behavior; mitochondria; skeletal muscle.

FIGURE 1

Blood ketone levels, body weight, heart, liver, and muscle mass after 2 months of a KD. (a) 3‐h postprandial and 12‐h fasted blood β‐hydroxybutyrate levels in middle‐aged female fed a control (CD) or ketogenic diet (KD). (b) Body weight, (c) percent fat measured using NMR relaxometry, (d) rearing score: Number of wall‐contact rears. Total distance travelled in (e) Y maze, (f) elevated plus maze, and (g) open field. Percent time spent (h) in center region of the open field and (i) on the open arms of the elevated plus maze. (j) Grip strength test: Relative to body weight. (k) Grid wire hang: Maximum hanging impulse. (l) Rotarod: Maximum time on the rod. (m) Time spent in the target quadrant and (n) latency to find the target hole in the banes maze probe trial. (o) Percent alternation in the Y maze spontaneous alternation test. (p) Percent time exploring the novel object in the novel object recognition time. Of female mice fed an isocaloric CD or KD. *indicates p < 0.05, **p < 0.01. All values are presented as mean ± SEM (n = 16).

FIGURE 2

Increased acetylation in liver is not associated with mitochondrial biogenesis after 2‐months on a ketogenic diet. Quantification of (a) acetylated lysine, (b) BHB‐lysine, (c) acetylated p300, (d) acetylated p53, (e) SIRT1, (f) phosphorylated AMPK^thr172, (g) total oxidative phosphorylation, (h) SOD2, (i) IRE1, BiP, CHOP, and phosphorylated eIF2α, (j) KAT1, (k) KAT3, and (l) KAT4 levels. (m) Representative images for all Western blot data are shown. CD, control and KD, ketogenic diet animals. *p < 0.05, **p < 0.01. All values are presented as mean ± SEM (n = 8).

FIGURE 3

Acetylation and BHBylation increase in response to a 2‐month KD in gastrocnemius muscle. (a) Acetylated lysine, (b) BHB‐lysine, (c) acetylated p300, (d) acetylated p53, (e) SIRT1, (f) SIRT3, and (g) IRE1, BiP, CHOP, and phosphorylated eIF2α levels in the GTN muscle following 2 months on a KD. (h) Representative images for all graphs. CD, control and KD, ketogenic diet animals. *indicates p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All values are presented as mean  SEM (n = 8).

FIGURE 4

A 2‐month KD increases mitochondrial proteins, PGC‐1α, phosphorylation of AMPK, and KAT1 in gastrocnemius muscle. (a) Total OxPhos protein, (b) PGC‐1α, SIRT1, TFAM, and mitofusin expression, (c) PGC‐1α protein, (d) phosphorylated AMPK^thr172, (e) nuclear PGC‐1α and (f) AMPK, (g) KAT1, (h) KAT3, (i) KAT4, (j) circulating kynurenine (KYN), and (k) kynurenic acid (KYNA) levels, and (k) representative images for all Western blot data is shown. CD, control and KD, ketogenic diet animals. *p < 0.05, **p < 0.01. All values are presented as mean ± SEM (n = 8).

FIGURE 5

A 2‐month KD increases acetylation and KAT4 levels in soleus muscle. (a) Acetylated lysine, (b) PGC‐1α, (c) SIRT3, (d) KAT1, (e) KAT3, (f) KAT4 levels, and (g) representative im. CD = control and KD = ketogenic diet. *p < 0.05, **p < 0.01, ****p < 0.0001. All values are presented as mean ± SEM (n = 8).

FIGURE 6

Improvements in cognitive behavior are independent of acetylation or mitochondrial levels in the cortex and hippocampus. (a) Acetylated lysine, (b) BHB‐lysine, (c) Total Oxphos, and (d) representative Western blots from the cortex. (e) Acetylated lysine, (f) BHB‐lysine, (g) Total Oxphos, and (h) representative Western blots from the hippocampus. CD, control and KD, ketogenic diet animals. All values are presented as mean ± SEM (n = 8).