METABOLIC EFFECTS OF DIETARY APPROACHES:KETONE BODIES & KETOGENIC DIET

One of the dietary approach that has attracted particular attention in recent years is the “ketogenic diet” (KD). This is a dietary program designed in the 1920s as a therapy for drug-resistant epilepsy, which then gained popularity in the 1970s as a weight-loss diet (Atkins). Indeed, many current diets are based on the KD model, and, recently, this model was proposed for a number of neurological disorders like Alzheimer’s and Parkinson’s diseases. The KD is a high-fat, low-carbohydrates diet planned to achieve ketosis, characterized by increased levels of circulating KB. Under normal conditions and with a balanced diet, KB are produced in small quantities, but, under a KD, their synthesis is induced as a result of a very low carbohydrate intake and high fat intake. The low glucose supply stimulates the catabolism of fats to obtain energy, leading to the accumulation of acetyl-CoA and synthesis of KB by the liver, which are sent to the peripheral tissues where they are oxidized to produce energy. The overall aim of the present Ph.D. thesis was to investigate the metabolic effects of KB and KD, through both in vitro and in vivo approaches. Thus, the research project has been subdivided in two different parts. In the first part we focused our attention on the vascular risk represented by KD-induced oxidative stress. We evaluated the effects of physiological levels of KB by measuring markers of oxidative stress using an in vitro model of endothelium. With this aim, we decided to investigate: a) the DNA oxidative damage by comet assay in order to study the genotoxicity due to KB; b) the ability of KB to modulate DNA susceptibility toward a secondary oxidative insult and c) the cell adaptive metabolic response to KB exposure, by activation of the Nrf2, which is a transcriptional factor involved in the cellular response to a stress, by western blot, immunofluorescence and real time PCR. The results obtained in this in vitro study demonstrate the activation of a cellular metabolic response caused by the effect of KB exposure. Ketones, by inducing moderate oxidative stress, activate the transcription factor Nrf2, which translocates into the nucleus. By binding to the ARE, Nrf2 activates the transcription of target genes, among which HO-1. As a consequence, the metabolic response caused by KB exposure makes cells more protected against a secondary insult, such as H2O2, leading to a reduction in DNA oxidative damage. Every time a KD is applied it should be considered that this diet could have an impact on cell metabolism in different tissue functions. The second part was devoted to verify the impact of the KD on human intestinal environment, by an in vivo study. This project was in collaboration with the Human Nutrition and Eating Disorder Research Center of the University of Pavia, where the subjects were recruited. After the collection of the samples, three different activities were scheduled: a) analysis of the composition of gut bacteria (NGS, qPCR) performed in collaboration with the Department of Health Sciences of University of Milan; b) assessment of markers of bacterial metabolism (SCFA) and c) evaluation of fecal water citotoxicity and genotoxicity (Trypan blue, Comet assay). We found that, first of all, the microbiota gut composition of patients affected by refractory epilepsy or GLUT1-DS undergoing a KD as a therapy, was significantly different than healthy control subjects, thus it is possible that KD may influence human gut. Moreover, KD group showed an intestinal dysbiosis, highlighted by reduced biodiversity and richness in microbial populations. In addition, we found an altered ratio Firmicutes/Bacteroidetes, due to a significant decrease of Bacteroidetes phylum in KD group, consistent with the status of dysbiosis. This study was the first to evaluate the SCFA concentrations in relation with the consumption of a ketogenic dietary protocol; we found lower levels of SCFA in KD group compared to CTR, but also analyzing the same subjects before and after the diet. These data underline how KD is able to affect gut health by altering microbiota composition and its metabolic activity. This can mainly be due to reduction in carbohydrates content, and in bacterial genera able to do fermentation, like Fecalibaterum prausnitzii and Bacteroides. Both citotoxicity and genototixicy of fecal water analyzes have shown that ketogenic treatment does not adversely affect human colon mucosa, as the results indicated medium level of toxicity, comparable with levels found in other studies of healthy population. In conclusion, this study allow us to conclude that KD has an impact on the human gut, highlighting the need for further research to avoid long-term effects and optimize the therapy. Considering our findings, it may be reasonable to suggest a supplementation of probiotics/prebiotics to potentially restore the microbiota stability.