Ketogenic Diet Modulates NAD+-Dependent Enzymes and Reduces DNA Damage in Hippocampus

Abstract

The ketogenic diet's (KD) anti-seizure effects have long been documented. Recently, its therapeutic potential in multiple neurodegenerative and neurodevelopmental disorders has emerged. Yet experimental evidence for a fundamental mechanism underlying beneficial effects across numerous diseases remains lacking. We previously showed that feeding rats a KD produced an early (within 2 days) and persistent elevation of hippocampal nicotinamide adenine dinucleotide⁺ (NAD⁺), an essential metabolic coenzyme and signaling molecule. NAD⁺ is a marker of cellular health and a substrate for enzymes implicated in longevity and DNA damage repair such as sirtuins and poly-ADP ribose polymerase-1 (PARP-1). As a result, activation of NAD⁺-dependent enzymes' downstream pathways could be the origin of KD's broad beneficial effects. Here rats were fed ad libitum regular chow or KD for 2 days or 3 weeks and the levels of hippocampal sirtuins, PARP-1, and the oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine were quantified. We found a significant immediate and persistent increase in the collective activity of nuclear sirtuin enzymes, and a significant augmentation of Sirt1 mRNA at 2 days. Levels of PARP-1 and 8-hydroxy-2'-deoxyguanosine decreased after 2 days of treatment and further declined at 3 weeks. Our data show that a KD can rapidly modulate energy metabolism by acting on NAD⁺-dependent enzymes and their downstream pathways. Thus, therapy with a KD can potentially enhance brain health and increase overall healthspan via NAD⁺-related mechanisms that render cells more resilient against DNA damage and a host of metabolic, epileptic, neurodegenerative, or neurodevelopmental insults.

Keywords: PARP-1; hippocampus; ketone bodies; longevity; metabolism; nicotinamide adenine dinucleotide; oxidative stress; sirtuin.

FIGURE 1

Hippocampal changes in NAD and sirtuins after ketogenic diet (KD) treatment. (A,B) NAD⁺/NADH ratios and NAD⁺ levels in the hippocampus after standard chow diet (CD; n = 5), 2 day (2d; n = 7), or 3 weeks KD treatment (3w; n = 7). (C) Collective deacetylation activity of Sirt1, Sirt6, and Sirt7 enzymes. A significant increase was observed at 2d and remained elevated at 3w. n = 6–8 animals (n = 2 per animal). (D–F) Real-time PCR analysis of Sirt1,–6,–7 gene expression in hippocampus. Sirt1 expression was increased only at 2d of treatment. CD, n = 6; 2d KD, n = 8; 3w KD, n = 8 (n = 2 per animal).

FIGURE 2

Effect of KD treatment on hippocampal PARP-1 levels and 8-OHdG levels. (A) Quantification of normalized PARP-1 protein levels in hippocampi obtained from animals fed standard chow (CD), or KD for 2d or 3w. Representative image of PARP-1 Western blot included. Each lane represents an individual animal. Blots were repeated 2–3 times. (B) Significant and progressive decrease in 8-OHdG levels was observed in hippocampi obtained from KD treated animals. CD, n = 6; 2d KD, n = 8; 3w KD, n = 8 (n = 2 for each animal).