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https://www.frontiersin.org/articles/10.3389/fnagi.2018.00225/full
Caloric Restriction and Ketogenic Diet Enhance Brain Vascular Functions and Shift Metabolism in Young Mice
In the early 1930s, Clive McCay demonstrated that restricting calorie intake without malnutrition can prolong both the mean and maximal lifespan in rats when compared to animals on ad libitum diet (AL; free eating) (McCay et al., 1989; Park, 2010). Since then, CR is perhaps the most studied anti-aging manipulation within a broad range of species (Colman et al., 2009; Choi et al., 2011; Rahat et al., 2011). This is further supported by other studies that display lower incidences of age-related neurodegenerative disorders found in animals treated with CR (Park et al., 2005; Duan and Ross, 2010).
Recently, our efforts have been focused on understanding how CR impacts brain function in the early stage. In particular, we would like to know how brain vascular and metabolic functions might be impacted with CR in young mice. We imaged mice at 5–6 months of age, either on 40% CR diet or AL, and found that CR significantly enhanced CBF (>20%) both globally and in the hippocampus, compared to their AL littermates (Parikh et al., 2016). The increase in CBF was associated with reduced mTOR and increased eNOS levels that were similar to what we observed with rapamycin. In addition, CR-fed mice had significantly increased P-glycoprotein (P-gp) transport activity levels at the BBB, which facilitates clearance of Aβ out of the brain. These findings are consistent with literature showing that CR reduces AD-like pathology and the onset of cognitive impairment (Mouton et al., 2009; Schafer et al., 2015).
We used 1H-MRS to determine energy metabolites in the hippocampus (Guo et al., 2015). We observed that CR mice displayed significantly increased levels of total creatine (tCr), a high-energy substrate, in comparison to AL mice. Given that tCr is the sum of creatine and phosphocreatine, we posited that CR increases adenosine triphosphate (ATP) production in young CR mice since phosphocreatine acts in a central role as an intracellular buffer during ATP production in mitochondria. This finding is consistent with literature that CR enhances ATP production by activating AMP-activated protein kinase (AMPK) and sirtuins pathways, which in turn suppresses the mTOR pathway (Blagosklonny, 2010). It has been found that the level of glucose regulates the AMPK pathway. With low levels of glucose and metabolic stress that accompany CR, there is a depletion of energy (low ATP: AMP ratio), which in turn activates AMPK (Salt et al., 1998; Hardie, 2014). AMPK, when activated, can be seen as an indicator of cellular energy status, turning on catabolic pathways that generate ATP while inhibiting cellular processes that consume ATP such as the mTOR pathway.
We also found significantly elevated levels of taurine in the CR mice when compared to the AL mice. Since taurine is correlated with neuromodulation, higher levels imply that young CR mice might have augmented excitability compared to the age-matched AL mice. Interestingly, both globally and in the hippocampus and frontal cortex (regions related to cognitive functions), CR mice displayed significantly reduced brain glucose uptake as determined by PET imaging (Guo et al., 2015). Our imaging findings are consistent with Western blot data showing that CR reduces glucose transporter 1 (GLUT-1) in brain capillaries of the mice (Parikh et al., 2016). As a result, we found a mismatch of CBF-CMRglc coupling induced by CR, opposite to tight coupling of CBF-CMRglc in a normal brain at rest (Fox et al., 1988; Lin et al., 2010).
These reduced glucose uptake results led us to hypothesize that in order to sustain essential mitochondrial activity and neuronal functions, the brain may utilize alternative fuel substrates as an energy source. As the brain would also use ketone bodies as energy source (Akram, 2013), we measured ketone body levels in the brain and blood and found that CR rodents had a significantly higher concentration of ketone bodies in comparison to AL animals (Guo et al., 2015; Lin et al., 2015). The findings indicated that CR may, at a very early stage in the brain, induce a metabolic switch from glucose to ketones.
To verify the impact of elevated ketone bodies on vascular functions, we fed young, age-matched WT mice with the KD for 16 weeks. Similar to what we observed with young CR mice, mice fed with KD also had significant increases in CBF and P-gp transport activity levels in brain capillaries compared to control mice (Ma et al., 2018). These neurovascular enhancements were also associated with reduced mTOR and increased eNOS protein expressions. The result is consistent with previous reports that ketogenesis is associated with the down-regulation of mTOR (McDaniel et al., 2011). In line with this, two other studies indicate that an acute increase in ketone body concentration (via infusion of β-hydroxyl butyrate) elevated CBF independent of overall cerebral metabolic activity. This suggests that ketone bodies can directly increase CBF via the cerebral endothelium (Hasselbalch et al., 1996; Roy et al., 2013).
Caloric Restriction Preserves Brain Vascular and Metabolic Functions in Aging Rodents
Healthy aging is accompanied by CBF reduction, BBB impairment and Aβ retention (Lin et al., 2015; Parikh et al., 2016; Hoffman et al., 2017). To identify CR effects on the aging brain, we included old CR and AL mice (18–20 months of age) in the same CR experiments and compared them with young mice (5–6 months of age). We found that CR enhanced CBF in young mice; moreover, CR also reduced the CBF decline in aging (Parikh et al., 2016). As a result, when compared to young AL mice, old CR mice had comparable levels of CBF, indicating that CR preserves CBF with age. Similar results were found in rats, showing that old rats with chronic CR diet had much higher CBF compared to the age-matched animal, and had comparable CBF level compared to the young AL rats (Lin et al., 2015). These results support that CR has an early enhancement effect on CBF that is preserved with aging. In addition, the preserved CBF in the hippocampus and frontal cortex were highly associated with the preserved memory and learning, as well as the reduced anxiety (Parikh et al., 2016). Our results suggest that dietary intervention initiated at a young age (e.g., young adults) may prove beneficial in the preservation of cognitive and mental abilities in aging.
A similar trend was also observed in hippocampal tCr concentration. As mentioned above, tCr was enhanced in young CR mice. Although tCr dropped dramatically as the CR mice getting older, tCr levels remained comparable to those in young AL mice and were higher than those in old AL mice. This suggests that CR increases ATP production in young CR mice while preserving ATP production in old CR mice (Guo et al., 2015).
Using advanced MRS techniques like POCE, we were able to trace in vivo mitochondrial oxidative metabolisms in neurons and neurotransmitter cycling between neuronal and glial cells (Lin et al., 2014). We found that, compared with the young AL rats, old CR rats had similar levels of neuronal glucose oxidation and neurotransmitter cycling, suggesting CR preserved mitochondrial functions and neuronal activity with age. In contrast, old AL rats had much lower levels in both measures. When calculating the ATP production rates for the three groups we found that old CR and young AL animals also had comparable levels of ATP production. We also observed metabolic shifts in aging animals. When compared to age-matched AL rats, old CR rats had significantly lower glucose uptake values in the various brain regions but had significantly higher levels of ketone bodies, β-hydroxyl butyrate (BHB) in the brain (Lin et al., 2015). The metabolic shift may play a critical role in sustaining brain energetics in aging.
Taken together, using multi-modal imaging methods we demonstrated that CR enhances vascular and metabolic functions in early life stages and decelerates the decline with age. Maintaining a healthy brain homeostasis may be due to the metabolic shift from glucose to ketone bodies (Lin et al., 2017b).
https://www.frontiersin.org/articles/10.3389/fnagi.2018.00225/full
Caloric Restriction and Ketogenic Diet Enhance Brain Vascular Functions and Shift Metabolism in Young Mice
In the early 1930s, Clive McCay demonstrated that restricting calorie intake without malnutrition can prolong both the mean and maximal lifespan in rats when compared to animals on ad libitum diet (AL; free eating) (McCay et al., 1989; Park, 2010). Since then, CR is perhaps the most studied anti-aging manipulation within a broad range of species (Colman et al., 2009; Choi et al., 2011; Rahat et al., 2011). This is further supported by other studies that display lower incidences of age-related neurodegenerative disorders found in animals treated with CR (Park et al., 2005; Duan and Ross, 2010).
Recently, our efforts have been focused on understanding how CR impacts brain function in the early stage. In particular, we would like to know how brain vascular and metabolic functions might be impacted with CR in young mice. We imaged mice at 5–6 months of age, either on 40% CR diet or AL, and found that CR significantly enhanced CBF (>20%) both globally and in the hippocampus, compared to their AL littermates (Parikh et al., 2016). The increase in CBF was associated with reduced mTOR and increased eNOS levels that were similar to what we observed with rapamycin. In addition, CR-fed mice had significantly increased P-glycoprotein (P-gp) transport activity levels at the BBB, which facilitates clearance of Aβ out of the brain. These findings are consistent with literature showing that CR reduces AD-like pathology and the onset of cognitive impairment (Mouton et al., 2009; Schafer et al., 2015).
We used 1H-MRS to determine energy metabolites in the hippocampus (Guo et al., 2015). We observed that CR mice displayed significantly increased levels of total creatine (tCr), a high-energy substrate, in comparison to AL mice. Given that tCr is the sum of creatine and phosphocreatine, we posited that CR increases adenosine triphosphate (ATP) production in young CR mice since phosphocreatine acts in a central role as an intracellular buffer during ATP production in mitochondria. This finding is consistent with literature that CR enhances ATP production by activating AMP-activated protein kinase (AMPK) and sirtuins pathways, which in turn suppresses the mTOR pathway (Blagosklonny, 2010). It has been found that the level of glucose regulates the AMPK pathway. With low levels of glucose and metabolic stress that accompany CR, there is a depletion of energy (low ATP: AMP ratio), which in turn activates AMPK (Salt et al., 1998; Hardie, 2014). AMPK, when activated, can be seen as an indicator of cellular energy status, turning on catabolic pathways that generate ATP while inhibiting cellular processes that consume ATP such as the mTOR pathway.
We also found significantly elevated levels of taurine in the CR mice when compared to the AL mice. Since taurine is correlated with neuromodulation, higher levels imply that young CR mice might have augmented excitability compared to the age-matched AL mice. Interestingly, both globally and in the hippocampus and frontal cortex (regions related to cognitive functions), CR mice displayed significantly reduced brain glucose uptake as determined by PET imaging (Guo et al., 2015). Our imaging findings are consistent with Western blot data showing that CR reduces glucose transporter 1 (GLUT-1) in brain capillaries of the mice (Parikh et al., 2016). As a result, we found a mismatch of CBF-CMRglc coupling induced by CR, opposite to tight coupling of CBF-CMRglc in a normal brain at rest (Fox et al., 1988; Lin et al., 2010).
These reduced glucose uptake results led us to hypothesize that in order to sustain essential mitochondrial activity and neuronal functions, the brain may utilize alternative fuel substrates as an energy source. As the brain would also use ketone bodies as energy source (Akram, 2013), we measured ketone body levels in the brain and blood and found that CR rodents had a significantly higher concentration of ketone bodies in comparison to AL animals (Guo et al., 2015; Lin et al., 2015). The findings indicated that CR may, at a very early stage in the brain, induce a metabolic switch from glucose to ketones.
To verify the impact of elevated ketone bodies on vascular functions, we fed young, age-matched WT mice with the KD for 16 weeks. Similar to what we observed with young CR mice, mice fed with KD also had significant increases in CBF and P-gp transport activity levels in brain capillaries compared to control mice (Ma et al., 2018). These neurovascular enhancements were also associated with reduced mTOR and increased eNOS protein expressions. The result is consistent with previous reports that ketogenesis is associated with the down-regulation of mTOR (McDaniel et al., 2011). In line with this, two other studies indicate that an acute increase in ketone body concentration (via infusion of β-hydroxyl butyrate) elevated CBF independent of overall cerebral metabolic activity. This suggests that ketone bodies can directly increase CBF via the cerebral endothelium (Hasselbalch et al., 1996; Roy et al., 2013).
Caloric Restriction Preserves Brain Vascular and Metabolic Functions in Aging Rodents
Healthy aging is accompanied by CBF reduction, BBB impairment and Aβ retention (Lin et al., 2015; Parikh et al., 2016; Hoffman et al., 2017). To identify CR effects on the aging brain, we included old CR and AL mice (18–20 months of age) in the same CR experiments and compared them with young mice (5–6 months of age). We found that CR enhanced CBF in young mice; moreover, CR also reduced the CBF decline in aging (Parikh et al., 2016). As a result, when compared to young AL mice, old CR mice had comparable levels of CBF, indicating that CR preserves CBF with age. Similar results were found in rats, showing that old rats with chronic CR diet had much higher CBF compared to the age-matched animal, and had comparable CBF level compared to the young AL rats (Lin et al., 2015). These results support that CR has an early enhancement effect on CBF that is preserved with aging. In addition, the preserved CBF in the hippocampus and frontal cortex were highly associated with the preserved memory and learning, as well as the reduced anxiety (Parikh et al., 2016). Our results suggest that dietary intervention initiated at a young age (e.g., young adults) may prove beneficial in the preservation of cognitive and mental abilities in aging.
A similar trend was also observed in hippocampal tCr concentration. As mentioned above, tCr was enhanced in young CR mice. Although tCr dropped dramatically as the CR mice getting older, tCr levels remained comparable to those in young AL mice and were higher than those in old AL mice. This suggests that CR increases ATP production in young CR mice while preserving ATP production in old CR mice (Guo et al., 2015).
Using advanced MRS techniques like POCE, we were able to trace in vivo mitochondrial oxidative metabolisms in neurons and neurotransmitter cycling between neuronal and glial cells (Lin et al., 2014). We found that, compared with the young AL rats, old CR rats had similar levels of neuronal glucose oxidation and neurotransmitter cycling, suggesting CR preserved mitochondrial functions and neuronal activity with age. In contrast, old AL rats had much lower levels in both measures. When calculating the ATP production rates for the three groups we found that old CR and young AL animals also had comparable levels of ATP production. We also observed metabolic shifts in aging animals. When compared to age-matched AL rats, old CR rats had significantly lower glucose uptake values in the various brain regions but had significantly higher levels of ketone bodies, β-hydroxyl butyrate (BHB) in the brain (Lin et al., 2015). The metabolic shift may play a critical role in sustaining brain energetics in aging.
Taken together, using multi-modal imaging methods we demonstrated that CR enhances vascular and metabolic functions in early life stages and decelerates the decline with age. Maintaining a healthy brain homeostasis may be due to the metabolic shift from glucose to ketone bodies (Lin et al., 2017b).
