Early Shifts of Brain Metabolism by Caloric Restriction Preserve White Matter Integrity and Long-term Memory in Aging Mice
Janet Guo1, Ailing Lin1,2, and Vikas Bakshi1

1Department of Pharmacology & Nutritional Sciences, University of Kentucky, Lexington, KY, United States, 2Department of Biomedical Engineering, Lexington, KY, United States

Synopsis

Caloric restriction (CR) has been shown to increase healthspan in various species; however, its effects on preserving brain functions in aging remain largely unexplored. We used multimodal neuroimaging (PET/MRI/MRS) and behavioral testing to determine in vivo brain glucose metabolism, energy metabolites, and white matter structural integrity in young and old mice fed with either control or 40% CR diet. Blood glucose and ketone bodies were measured. Our findings suggest CR could slow brain aging, partly due to early shift of energy metabolism caused by lower caloric intake. These results provide rationale for CR-induced sustenance of brain health with extended longevity.

Purpose

In this study, our goal was to use non-invasive neuroimaging to identify the impact of caloric intake on brain integrity over time. We used multi-metric imaging methods (PET/MRI/MRS) to determine CMRglc, brain metabolites and structural connectivity, and identified associations of their changes with cognitive functions.

Methods

Young control (5-6 mo), young calorie-restricted (5-6 mo), old control (18-20 mo), and old calorie-restricted C57BL/6 mice (18-20 mo) were obtained from the National Institute on Aging Caloric Restricted Colony. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Kentucky according to NIH guidelines.

Brain structural and metabolic integrity determination using MRI Brain structural and metabolic integrity were measured using a 7T Clinscan MR scanner. We used MRI-based diffusion tensor imaging (DTI) to measure fractional anisotropy (FA) in CC. Following DTI, brain metabolite levels were determined with proton (1H) MR spectroscopy (MRS) using a point-resolved spectroscopy sequence.

We used fluorodeoxyglucose (18FDG) positron emission tomography (PET) to measure CMRglc. The PET experiments were conducted at the University of Texas Health Science Center at San Antonio. The experimental procedure was approved by the IACUC of UTHSCSA.

The RAWM protocol consisted of a 2-day testing paradigm. A staggered training schedule was used, running the mice in cohorts of ten mice, while alternating the different cohorts through the trials over day 1 and day 2 of the test.

1-2 µl of blood sample was used to measure blood glucose level. Another 10 µl of blood sample was used for ketone bodies level measurement.

Results

There are five key findings from the studies reported here. Firstly, there was an early onset of glucose reduction induced by CR. In contrast, increased ketone bodies were found in the young CR mice. These changes were age-independent — old CR mice had similar levels on those indices when compared to their young litter-mates. The findings are consistent with literature, indicating CR induced a metabolic shift from utilizing glucose to ketone bodies 1 2.Secondly, we found that CR increases ATP production in young CR mice and preserves ATP production in old CR mice, relative to controls, based on the TCr data. Thirdly, we observed a preservation of white matter structural integrity with age in the CR mice. Fourthly, the mice fed with chronic CR diet preserved long-term memory. Finally, we found associations between glucose level, body weight, and lifespan. Taken together, we observed distinct patterns between normal aging and CR aging on brain functions. Normal aging shows reductions in brain glucose metabolism, white matter integrity, and long-term memory, resembling human brain aging. CR aging, in contrast, displays early onset changes in brain metabolism and preservations of energy production, white matter integrity, and long-term memory in aging mice.

Discussion

Our findings suggest that the benefits found in the CR mice might be in part due to the early shift of energy metabolism caused by lower caloric intake. Because of reduced glucose availability, CR mice adapted to use ketone bodies metabolism at a very early age. These metabolic alterations remained stable with age. Moderate ketosis has been shown to have many beneficial properties for brain functions, including sustaining neuronal activities 3, preserving energy substrates 4, enhancing memory 5 6, reducing insulin resistance, and alleviating damage caused by oxidative stress and hypoxia 4 7 8. However, whether feeding ketogenic diet will mimic CR’s beneficial effects on brain functions remains an object of study 9.

It has to be pointed out that we used a long-lived rodent model in the present study to investigate CR effects. Recent studies have shown that the lifespan response to a single level of CR (e.g., 40% CR) varies widely in mice from different genetic backgrounds 10. It will be important in the future to use neuroimaging to determine if CR also has adverse effects on brain metabolic functions in rodent strains where deleterious effects on lifespan or cognitive functions are observed.

Conclusion

In conclusion, we successfully used non-invasive neuroimaging to identify CR effects on brain physiology in aging mice. Specifically, we found an early shift in brain metabolism in mice with low caloric intake, which was associated with preserved energy production, brain structural integrity, and long-term memory. Understanding nutritional effects on brain function may have profound implications in human aging and other age-related neurodegenerative disorders. Using multimodal neuroimaging methods, we will be in a position to identify effective nutritional interventions, and the treatment efficacy thereof, to slow sown brain aging and/or prevent dementia for humans.

Acknowledgements

We thank Max Baker for assisting the experiments.

References

1. Lin AL, Zhang W, Gao X, et al. Caloric restriction increases ketone bodies metabolism and preserves blood flow in aging brain. Neurobiol Aging 2015;36(7):2296-303.

2. Shimazu T, Hirschey MD, Newman J, et al. Suppression of oxidative stress by beta-hydroxybutyrate, an endogenous histone deacetylase inhibitor. Science 2013;339(6116):211-4.

3. Masino SA, Kawamura M, Wasser CD, et al. Adenosine, ketogenic diet and epilepsy: the emerging therapeutic relationship between metabolism and brain activity. Curr Neuropharmacol 2009;7(3):257-68.

4. Sullivan PG, Rippy NA, Dorenbos K, et al. The ketogenic diet increases mitochondrial uncoupling protein levels and activity. Ann Neurol 2004;55(4):576-80.

5. Nordli DR, Jr., Kuroda MM, Carroll J, et al. Experience with the ketogenic diet in infants. Pediatrics 2001;108(1):129-33.

6. Pulsifer MB, Gordon JM, Brandt J, et al. Effects of ketogenic diet on development and behavior: preliminary report of a prospective study. Dev Med Child Neurol 2001;43(5):301-6.

7. Cahill GF, Jr., Veech RL. Ketoacids? Good medicine? Trans Am Clin Climatol Assoc 2003;114:149-61; discussion 62-3.

8. Maalouf M, Sullivan PG, Davis L, et al. Ketones inhibit mitochondrial production of reactive oxygen species production following glutamate excitotoxicity by increasing NADH oxidation. Neuroscience 2007;145(1):256-64.

9. Brownlow ML, Benner L, D'Agostino D, et al. Ketogenic diet improves motor performance but not cognition in two mouse models of Alzheimer's pathology. PLoS One 2013;8(9):e75713.

10. Liao CY, Rikke BA, Johnson TE, et al. Genetic variation in the murine lifespan response to dietary restriction: from life extension to life shortening. Aging Cell 2010;9(1):92-5.

Figures

Association of caloric restriction with blood glucose, body weight and lifespan.

(A) A positive correlation between blood glucose and body weight among the CR and control mice (r = 0.55, p < 0.001). (B) An inverse association between blood glucose level (measured in the current study) and the lifespan (reported by Froster et al., 2003) of the CR and control mice.


Caloric restriction induced early onset of glucose reduction and ketone bodies increase.

(A) CMRglc visual map of the four mice groups. The color code indicates the CMRglc (in SUV) in a linear scale. (B) Quantitative global CMRglc. (C) Quantitative hippocampal CMRglc. (D) Blood glucose. (E) Blood ketone bodies. Data are presented as Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.


Caloric restriction increased production of brain energy metabolite and preserved it with age.

(A) The voxel replacement on the hippocampus and (B) the representative 1H-MRS spectrum, showing total choline (TCho), total creatine (TCr), taurine (Tau), glutamate-glutamine complex (Glx), myo-inositol (mI), N-acetylaspartate (NAA), in parts per million (ppm). (C) TCr levels of the four groups. Data are presented as Mean ± SEM.*p < 0.05; **p < 0.01; n.s.: non-significant.


Caloric restriction preserved white matter structural integrity.

(A) The region showing corpus callosum (CC) on MRI diffusion–weighted images. (B) The quantitative measurements of fractional anisotropy (FA) in CC. Data are presented as Mean ± SEM. ***p < 0.001 and ****p < 0.0001.


Caloric restriction prevented age-dependent long-term memory deterioration.

(A) Average errors corrected over 6 blocks of the control mice, with significant difference observed on Blocks 3 and 4. (B) Average errors corrected over 6 blocks of the CR mice, with significant difference observed on Block 3. (C) Comparison of the errors made by the four groups on Block 3. (D) Comparison of the errors made by the four groups on Block 4. Data are presented as Mean ± SEM. *p < 0.05; **p < 0.01.




Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)
0056