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Increasing life expectancy in the past decades has led to the emergence of age-related chronic diseases and disabilities. A deeper understanding in the molecular events of the aging process is essential to provide evidence-based guidance how lifestyle interventions will be more efficient in delaying age-related disease phenotypes. Calorie restriction (CR) is by far the best nutritional strategy to achieve longevity in animal models. Although potentially also effective for humans, most people experience this rigorous diet as not feasible. To search for a practicable alternative we explored, using a C57BL/6J mice cohort, the effects of intermittent (INT) diet, a weekly alternating diet regimen between 40E% CR and ad libitum medium-fat feeding. We hypothesized that the weekly fluctuating energy availability provides beneficial challenges to the body.
In this thesis we focused on the effects induced by the INT diet on the liver, the central metabolic organ in the body. Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease that develops with age and is considered as the hepatic phenotype of the metabolic syndrome. NAFLD is a disease that develops slowly over the years; its prevalence has been shown to increase at old age (>60 years). In chapter 2 we investigated whether the INT dietary regimen was able to reverse the unfavourable effects of a medium-fat (25%E fat; MF) diet on the liver and its implication on NAFLD development. We showed that, at the age of 12 months, the INT diet prevented NAFLD development. INT-exposed animals retained healthy physiological features as displayed by continuous exposure to CR; maintenance of glucose tolerance, normal insulin levels and low plasma alanine and aspartate aminotransferases. Furthermore, they did not exhibit signs of hepatic steatosis and fibrosis, indicated by the reduced hepatic TG levels and morphological observations. The results presented in chapter 3 show that, at the age of 24 months, INT-fed mice displayed normal plasma ALT levels, no liver inflammation or fibrosis. These mice, however, display mild steatosis with IHTG levels significantly lower than the MF-exposed mice. To summarize, long-term exposure to a MF diet seriously impaired metabolic homeostasis and was a risk factor for NAFLD development. Applying every-other-week 40E% CR largely reversed the adverse health effects induced by the MF diet. Although the livers of the INT-exposed mice were still protected for the advanced stages of NAFLD, it is noteworthy that, in the long run, liver fat accumulation still occurred.
The second part of chapter 3 describes the obesity-counteracting effects of the INT diet. Part of the mice that had been exposed to the MF diet till 12 months of age was switched to the INT diet until the age of 24 months. The switch to the INT diet successfully improved glucose clearance, survival and liver health, but failed to improve IHTG levels. Within the diet switch experiment, we also investigated the plasticity of adaptive response to the switch by means of transcriptome analysis. Most of the genes differentially expressed between the INT- and MF-exposed mice (~95% of 2,667 genes) switched to the INT-expression profile. There was only a small subset of 148 genes which expression levels persistently remained similar to the MF diet-induced expression levels, instead of adapting to INT’s expression profile. Pathway analysis pointed out that this subset of 148 genes contains genes involved in lipid and xenobiotic metabolism, with PXR as the strongest upstream regulator. This suggests that MF-induced deregulated PXR activity persistently affects lipid and xenobiotic metabolism in the liver of the old diet switch mice. Therefore, we suggested that, despite the strong improvement of overall and liver-specific phenotypes, these persistently regulated genes might have potentially adverse effects on health.
The adaptive response to the diet switch at an old age was further investigated in chapter 4, but then in the reverse order: switching from a healthy to an unhealthy diet. Our results showed that, despite the long-term exposure to CR regimen, mice in the CR-MF group displayed hyperphagia, rapid weight gain, and hepatic steatosis. However, no hepatic fibrosis/injury or alteration in CR-improved survival was observed in the diet switch group. The liver transcriptomic profile of CR-MF group largely shifted to a profile similar to the MF-fed animals but leaving ~22% of the 1578 differentially regulated genes between the CR and MF diet groups comparable with the expression of the life-long CR group. Therefore, although the diet switch was performed at an old age, the CR-MF-exposed mice were still able to rapidly gain weight to similar level as life-long MF mice with the same age, but without developing severe liver pathologies.
In chapter 5, the data from the different dietary interventions and age time points were combined to further explore the molecular mechanisms underlying the NAFLD development. Hereby, we focussed our analysis on the association with Fgf21, an emerging non-invasive biomarker for NAFLD. We demonstrated that plasma Fgf21 levels strongly reflected liver fat accumulation, confirming its potential as NAFLD marker. Transcriptomics analysis of the liver was performed and revealed that the link between plasma Fgf21 and IHTG levels was associated with differentially regulated PPARα and NRF2 targets during NAFLD. This suggested that the elevated Fgf21 levels in NAFLD was a measure to maintain homeostasis against the adverse effects of lipotoxicity, oxidative stress and endoplasmic reticulum stress in NAFLD. The PPARα challenge test, which was performed by administrating PPARa agonist Wy-14,643 to the mice, confirmed the dysregulation of PPARα signalling in NAFLD, including the hepatic expression of Fgf21.
To conclude, the results presented in this thesis adds to our understanding the effects of different diets have on genotype-phenotype relationships, which translate into different health states and are essential for identifying healthy aging strategies. We investigated the role of different dietary regimen on the phenotypes of genetically identical mice, particularly on an intermittent (INT) diet, which alternates weekly between the ad libitum medium-fat (MF) and calorie restriction (CR) diet. We found that the INT dietary regimen provided a remarkable protection against the severe health outcomes of the long-term medium-fat diet consumption, which may improve life quality by reducing the burden of chronic disease. Although it is too early to conclude that the INT dietary regimen (or modulation of the energy intake) is beneficial and safe to be applied in human population, this study is a proof-of-concept of intervening a chronic overnutrition status with a metabolic challenge of energy stress. Further investigation of this novel dietary regimen is needed to allow it to be safely applied in humans. By switching the diets at a defined time point during the study, we demonstrated that, even at middle and old age, the liver is still a highly flexible organ that rapidly adapts its transcriptional program to the different dietary challenges. We also demonstrated that the strong link between the diet-induced NAFLD and Fgf21 denoted a dysregulation of PPARa signalling pathway during the development of the liver disease.
|Qualification||Doctor of Philosophy|
|Award date||29 Aug 2016|
|Place of Publication||Wageningen|
|Publication status||Published - 2016|
- liver diseases
- energy restricted diets
- fatty liver