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Nutritional ketosis increases NAD+/NADH ratio in healthy human brain: an in vivo study by 31P-MRS1Center for Biomedical Imaging, École polytechnique fédérale de Lausanne, Lausanne, Switzerland, 2Clinical Development Unit, Nestlé Research Center, Lausanne, Switzerland, 3Nestlé Institute of Health Sciences SA, Lausanne, Switzerland, 4Nestlé Health Science, Epalinges, Switzerland, 5Department of radiology, University of Lausanne and Geneva, Lausanne and Geneva, Switzerland
Synopsis
Ketones represent an important alternative fuel for the brain under glucose hypo-metabolic conditions induced by neurological diseases or aging, however their metabolic consequences in healthy brain remain unclear. Here we report that ketones can increase the redox NAD+/NADH ratio in the resting brain of healthy young adults. As NAD is an important energetic and signaling metabolic modulator, these results provide mechanistic clues on how nutritional ketosis might contribute to the preservation of brain health.
Introduction
Ketone is an alternative brain energy source. Interestingly, while brain glucose utilization decreases in mild cognitively impaired elderly and in Alzheimer’s disease patients, ketone metabolism remains intact1. Interventions using ketones or their precursors have shown therapeutic potential in several neurometabolic disorders2, demonstrating the possible value of ketones as an alternative source of brain energy. Remarkably, under healthy homeostatic conditions, an increase in brain ketones proportionally decreases brain glucose utilization3. It is not clear if a shift in energy substrate from glucose to ketones has any further benefit or metabolic consequence in healthy human brain beyond providing energy. For example, the energy production through ketones metabolism toward acetyl-CoA generation is distinct from using glucose and requires a lower utilization of the oxidized form of nicotinamide adenine dinucleotide (NAD+)(Fig.1). Therefore, one can speculate that an increase in ketolysis may spare NAD+ and lead to a greater brain NAD+/NADH ratio, a potential mechanism that is becoming recognized4,5. When healthy rats were fed with a ketogenic diet, regional increase of brain NAD+/NADH was observed5. In mice model of ischemic stroke, injection of ketones after ischemia induced by transient middle cerebral artery occlusion improved neurological and mitochondria functions, and increased brain NAD+/NADH ratio6. As NAD+ is also a critical co-substrate for enzymes that play key roles in many biological processes, preserving NAD+ concentration may contribute to maintaining health. Therefore, to test if ketones can increase the brain NAD+/NADH ratio in human or affect other energy metabolic pathways, we conducted a 31P-MRS study at 7T, where brain energy related metabolites, redox state and enzymatic activities were assessed before and after a nutritional ketogenic intervention.Methods
25 healthy individuals (26.6±6.0years) provided written informed consent and consumed 250mL Peptamen® (Nestlé Health Science SA), a liquid nutrition product containing 10g of medium chain triglycerides(MCT), which are efficient ketone precursors. Pharmacokinetic profile of plasma ketones suggests the maximal values around 30 min and then an additional 15 min was estimated for the ketones to achieve maximum brain concentration. Therefore, 31P MRS was conducted before and 45min after the uptake of the product.
MR experiments were performed on a 7T/68cm MR scanner (Siemens Medical Solutions, Erlangen, Germany) with a 1H quadrature surface coil (10cm-diameter) and a single-loop 31P coil (7cm-diameter) for the occipital lobe. Four 31P acquisitions were performed:1) a pulse acquire sequence without the saturation transfer; 2) saturation pulses7 applied at g-ATP(-2.5ppm) with a saturation time τsat of 8.25ms (stead-states measurement) 3) saturation pulses applied at 12.2 ppm (control measurement for Pi) 4) saturation pulses applied at 2.5 ppm (control measurement for PCr). Acquisition 1 is used to measure all 31P resonance signals including NADH, NAD+. Acquisition 2-4 are used to measure forward rate constants of creatine kinase(kf,CK) and ATP synthase(kf, ATPase).
All spectra were analyzed by LCModel8 and NAD+, NADH concentrations were calculated assuming [α-ATP] of 2.8mM9,10. kf,CK and kf, ATPase were calculated from Mss=Mc/(1+kf·T1int) using T1int at 7T11. Mss and Mc are signal intensities of Pi or PCr obtained respectively from steady-state and control measurements. Statistical analysis done using a mixed model with Peptamen® as fixed effect and subject as random effect.
Results
After the intake of Peptamen®, the main observation was a significant change in NAD metabolites levels: NAD+ was increased by 3.4% while the level of NADH was reduced by 13% resulting in a 18% increase of the redox ratio NAD+/NADH (p = 0.01; Fig.2). To further demonstrate that the variations in NAD levels were independent of the fitting model, a non-flat difference spectrum was shown in Fig.3, supporting an elevated signal for NAD+ and a diminished signal for NADH. No change could be detected in PCr, Pi, their ratios or metabolic fluxes for ATP production.Discussion and conclusion
This is the first interventional study showing that ketones can have a NAD+ sparing effect in healthy human brain. That no other metabolic change could be detected supports previous brain studies in healthy humans demonstrating that ketones can replace a portion of the glucose utilized by the brain while maintaining brain energy homeostasis12.
A decrease in the NAD+/NADH ratio has been reported in elderly13 and in psychiatric disorders together with a reduction in total NAD14. Our study shows that ketones can increase the brain NAD+/NADH ratio by about 18% in humans, at least transiently. Therefore, we conclude that a nutritional ketone intervention, beyond providing an alternative source of energy to the brain, offers the potential to boost the NAD+/NADH redox state and may provide additional benefits to the brain such as protection from oxidative stress and inflammation.
Acknowledgements
We thank Centre d’Imagerie BioMédicale (CIBM) of the UNIL, UNIGE, HUG, CHUV, EPFL, the Leenaards and Jeantet Foundations. Funding was provided by the Center for Biomedical Imaging and Nestlé.References
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