Cognitive impairment caused by compromised hepatic ketogenesis is prevented by endurance exercise

Abstract

Extensive research has demonstrated endurance exercise to be neuroprotective. Whether these neuroprotective benefits are mediated, in part, by hepatic ketone production remains unclear. To investigate the role of hepatic ketone production on brain health during exercise, healthy 6-month-old female rats underwent viral knockdown of the rate-limiting enzyme in the liver that catalyses the first reaction in ketogenesis: 3-hydroxymethylglutaryl-CoA synthase 2 (HMGCS2). Rats were then subjected to either a bout of acute exercise or 4 weeks of chronic treadmill running (5 days/week) and cognitive behavioural testing. Acute exercise elevated ketone plasma concentration 1 h following exercise. Hepatic HMGCS2 knockdown, verified by protein expression, reduced ketone plasma concentration 1 h after acute exercise and 48 h after chronic exercise. Proteomic analysis and enrichment of the frontal cortex revealed hepatic HMGCS2 knockdown reduced markers of mitochondrial function 1 h after acute exercise. HMGCS2 knockdown significantly reduced state 3 complex I + II respiration in isolated mitochondria from the frontal cortex after chronic exercise. Spatial memory and protein markers of synaptic plasticity were significantly reduced by HMGCS2 knockdown. These deficiencies were prevented by chronic endurance exercise training. In summary, these are the first data to propose that hepatic ketogenesis is required to maintain cognition and mitochondrial function, irrespective of training status, and that endurance exercise can overcome neuropathology caused by insufficient hepatic ketogenesis. These results establish a mechanistic link between liver and brain health that enhance our understanding of how peripheral tissue metabolism influences brain health. KEY POINTS: Decades of literature demonstrate endurance exercise to be neuroprotective. Whether neuroprotective benefits are mediated, in part, by hepatic ketone production remains unclear. This study provides the first set of data that suggest hepatic ketogenesis is required to maintain cognition, synaptic plasticity and mitochondrial function. These data indicate endurance exercise can protect against cognitive decline caused by compromised hepatic ketogenesis. These results establish a mechanistic link between liver and brain function, prompting further investigation of how hepatic metabolism influences brain health.

Keywords: HMGCS2; cerebral cortex; cognitio; exercise; ketogenesis; liver; mitochondria; proteomics.

Figure 1.. Hepatic HMGCS2 knockdown reduced circulating ketones after acute and chronic exercise

A, hepatic HMGCS2 protein expression normalized to total protein stain. B, AcAc, BHB and TKB plasma concentration (μM) 1 h after an acute bout of exercise (n = 6). Exercise groups were compared to SCR-SED values from chronic training group measurements. C, hepatic HMGCS2 protein expression normalized to total protein stain (n = 10). D–F, AcAc (D), BHB (E) and TKB (F) plasma concentration (μM) 48 h after the last bout of exercise following chronic exercise training (5 weeks after AAV injection) (n = 5–6). G–J, 3-hydroxybutyrate dehydrogenase 1 (BDH1) (G), 3-hydroxymethyl-3-methylglutaryl-CoA, lyase (HMGCL) (H), succinyl-CoA:3-ketoacid CoA transferase (SCOT) (I) and acetyl-CoA acetyltransferase 1 (ACAT1) (J) protein expression in the liver 48 h after the last bout of exercise during chronic exercise training (5 weeks after AAV injection) (n = 9–10). Representative image of western blots and their respective weight (kDA) are shown. K, schematic of ketogenesis and ketolysis pathway and key enzymes involved. Image generated in BioRender. Main effect of HMGCS2 KD represented by ^##P ≤ 0.01. Values are presented as mean ± SE. EX, exercise. KD, knockdown. Values presented as mean ± SD. Arbitrary units, AU; acetoacetate (AcAc); β-hydroxybutyrate (BHB); total ketone bodies (TKB).

Figure 2.. Hepatic HMGCS2 knockdown increases markers of mitochondrial dysfunction 1 h after an acute bout of exercise

A, volcano plot for comparison between HMGCS2-EX and SCR-EX (n = 6). Red dots represent DEPs (Q-value < 0.05). B, top upregulated and downregulated canonical pathways from DEPs (n = 6). C, predicted upstream regulators from protein clustering analysis. Log₂-fold change of DEPs used for predictive calculation are shown below each protein. D, image of proteins from each mitochondrial complex used for calculation of z-score in the oxidative phosphorylation canonical pathway. Orange indicates a positive z-score in the numerator and blue a negative z-score (absolute z-score = 2 for significance). IPA graphical figure key is shown to the right of each panel. Differentially expressed protein, DEP.

Figure 3.. Four weeks of treadmill running confers mitochondrial endurance exercise adaptations in the gastrocnemius

A–E, basal (A), state 2 (B), state 3 Complex I (C), state 3 Complex I + II (D) and uncoupled respiration (E) in mitochondrial isolates from the gastrocnemius muscle 48 h after the last bout of exercise during chronic exercise training (5 weeks after AAV injection). O₂ flux normalized to total protein (n = 8–9). F–H, complete (F), incomplete (G) and total (H) [1-¹⁴C] palmitate oxidation in mitochondrial isolates from the gastrocnemius muscle 48 h after the last bout of exercise during chronic exercise training (5 weeks after AAV injection). Palmitate oxidation normalized to total protein (n = 8–9). Main effect of exercise represented by *P ≤ 0.05 or **P ≤ 0.01. Values presented as mean ± SD. Oxygen, (O₂).

Figure 4.. Hepatic HMGCS2 knockdown (in vivo) or neuronal OXCT1 (in vitro) impairs mitochondrial function

A–E, basal (A), state 2 (B), state 3 Complex I (C), state 3 Complex I + II (D) and maximal uncoupled respiration (E) in mitochondrial isolates from the frontal cortex 48 h after the last bout of exercise during chronic exercise training (5 weeks after AAV injection). O₂ flux normalized to total protein (n = 8–9). F, transcript expression of OXCT1 normalized to PPIB in PC12 cells (n = 3). G, PC12 cell OCR at baseline, after oligomycin (1.0 μM), carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) (1.5 μM) and rotenone with antimycin A (0.5 μM). OCR was measured three times in 3 min intervals before the initial port injection and after each port injection and normalized to total protein (n = 5). H, average OCR across three time points in PC12 cells (n = 5). Basal mitochondrial respiration was determined by taking OCR before oligomycin injection minus OCR after rotenone with antimycin A. Maximal mitochondrial respiration was determined by taking OCR after FCCP injection minus OCR after rotenone with antimycin A. Spare capacity was determined by taking OCR after FCCP injection minus OCR before oligomycin injection. Non-mitochondrial respiration was determined by the OCR after rotenone with antimycin A. Main effect of HMGCS2 KD represented by ^#P ≤ 0.05. Values presented as mean ± SD. OCR, oxygen consumption rate; oxygen, O₂; peptidyl-prolyl cis-trans isomerase, PPIB; RQ, relevant quotient.

Figure 5.. Endurance exercise ameliorates cognitive impairment induced by hepatic knockdown of HMGCS2

A–D, volcano plots for comparison between SCR-EX and SCR-SED (A), HMGCS2 KD-SED and SCR-SED (B), HMGCS2 KD-EX and HMGCS2 KD-SED (C) and HMGCS2 KD-EX and SCR-EX (D) 48 h after the last bout of exercise during chronic exercise training (5 weeks after AAV injection) (n = 6). E, time spent in the novel arm (seconds) during the testing phase (n = 7–8). Testing was performed in the final (4th) week of endurance training with any animal that did not leave the starting arm excluded from analyses. Representative tracings of each group are shown to the right of the graph. F, BDNF protein expression in the frontal cortex expressed as raw intensity (n = 6). G and H, top upregulated and downregulated proteomic (G) and phospho-proteomic (H) canonical pathways related to synaptic plasticity in HMGCS2 KD-SED compared with SCR-SED (n = 6). I and J, top upregulated and downregulated canonical pathways related to synaptic plasticity in HMGCS2 KD-EX compared to HMGCS2 KD-SED (I), and HMGCS2-KD vs. SCR- EX (J) (n = 6). Orange indicates a positive z-score in the numerator and blue a negative z-score (absolute z-score = 2 for significance). Z-score generated in IPA. Main effect of HMGCS2 KD represented by ^#P ≤ 0.05. Values presented as mean ± SD. Differentially expressed protein, DEP.

Figure 6.. AAV-mediated knockdown of hepatic HMGCS2 causes mitochondrial calcium overload inversely correlated with state 3 mitochondrial respiration

A and B, canonical signalling pathways revealed from proteomic (A) and phosphor-proteomic (B) IPA analyses. Blue indicates a decrease and orange an increase in HMGCS2-KD compared with SCR-SED rats. C, ADP/ATP translocase 2 (ANT2) protein expression in the frontal cortex expressed as raw intensity (n = 6). D, single-pass membrane protein with aspartate-rich tail (SMDT1) protein expression in the frontal cortex expressed as raw intensity (n = 6). E, correlation between State 3 Complex I + II mitochondrial respiration and SMDAT1 raw intensity in HMGCS2 KD-SED and SCR-SED rats. Z-score generated in IPA. Values presented as mean ± SD.