Role of nutritional intervention on metabolic health and autophagy

Abstract

Obesity is an independent risk factor for a number of chronic diseases including type 2 diabetes and cardiovascular diseases. Weight loss strategies via nutritional interventions remain first line non-pharmacological therapy to mitigate the risk. The nutritional interventions include: 1) moderate caloric restriction that curbs daily food intake by 20-40%, 2) intermittent fasting that employs 1-3 days per week of fasting interspersed with ad libitum intake on the non-fasting days, or 3) time-restricted feeding that restricts daily food intake to 6-12h of the day. However, the cellular processes and mechanism governing this remains controversial in many aspects. This thesis examines the impact of these nutritional interventions at the molecular level in humans and mice, and its role in ameliorating the metabolic outcomes. Firstly, I utilised samples from a single-centred randomised controlled trial in humans with obesity who were randomly assigned to one of the four diets: 1) CR70 (calorie restriction at 70% of baseline requirement per week), 2) IF70 (intermittent fasting at 70% of the baseline energy requirement), 3) IF100 (intermittent fasting at 100% of the baseline energy requirement per week), and 4) control (100% of baseline energy requirements). A subset of participants from CR70 and IF70 groups who had lost >5% of body weight and were not under lipid lowering medication were included for the assessment of anti-inflammatory property and cholesterol efflux capacity of high-density lipoprotein (HDL). The IF70 group displayed greater weight loss including total cholesterol and low-density lipoprotein (LDL) vs CR70, but the change in HDL was not different between groups. We did not observe within or between group effect in changes in mRNA markers of inflammation assessed including the cholesterol efflux capacity of HDL. In conclusion, the findings of the study suggest that short-term CR or IF with clinically significant weight loss did not alter the functions of high-density lipoprotein. I went on to explore the role of intermittent fasting in autophagy, a cellular catabolic housekeeping process, in liver of mouse and muscle of both human and mouse. Starvation is known to activate autophagy at cellular level in wide range of cells that includes HeLa, yeast, fat, muscle and ovaries cells of Dorsophila melanogaster and in most tissue of mouse that includes liver, fat, pancreatic -cells, neural and muscle cells. In the mouse study, ten-weeks-old C57BL/6J male mice were fed ad libitum (AL) of either high-fat diet (HFD) (43% energy from fat) or chow diet for 8-weeks before randomisation to continue either AL or IF (24h fast initiated at Zeitgeber time 11) on 3 non-consecutive days/week for further 8-weeks. Tissues were collected in both fed-state and in 22h fast. Vastus lateralis muscle tissue was also collected following a 12h and 24h fast from the IF study described above and both mRNA transcripts and proteins of autophagy markers were assessed. The findings of this study show that markers of autophagy in liver, but not in skeletal muscle, were elevated in response to IF in mice. This effect was blunted in mice with obesity. In humans, there was no evidence that fasting stimulated basal levels of autophagy markers in muscle, and in fact this was reduced from baseline, likely in response to weight loss. Circadian desynchrony in peripheral metabolic organs results from eating out of the phase of circadian rhythmicity and is associated with increased incidence of chronic diseases. Time-restricted feeding (TRF), a form of IF, limits the ad libitum food intake to 6-12h of the day, while maintaining the total fasting length of >12h per day. TRF improves metabolic outcomes in both humans and mouse alike. Most of the studies conducted so far have tested TRF initiated at the onset of the dark (active) phase, and there is a lack of knowledge about the impacts of TRF in delayed setting, which may be more feasible to implement in a human population. For this study, 8-weeks-old C57BL/6J mice (n=192) were subjected to AL feeding of chow or HFD for 4-weeks before randomising them to one of the following diets: 1) continued AL feeding, 2) 10h TRF initiated at lights off (TRFe), or 3) 10h TRF initiated 4h after lights off (TRFd) for further 8-weeks. The findings of this study suggest that both forms of TRF reduced weight, fat gain, hepatosteatosis, and increased glucose tolerance, metabolic flexibility, amplitude of genes involved in circadian regulation, and markers of NAD metabolism vs AL. TRFd limited weight and fat gain benefit and induced a phase delay in body temperature, clock gene expression including markers of NAD metabolism in liver compared to TRFe, but was not different to TRFd in improving glucose tolerance and the amplitude of genes involved in circadian regulation. In conclusion, this study suggests that delaying the TRF, akin to breakfast skipping, provides equal metabolic benefits as that of TRFe. The circadian rhythmicity of autophagy is influenced by both nutritional and clock signals and may be dampened in diet-induced obese mice. Whether TRF acts by stimulating autophagy at cellular level is unknown. We utilised the liver samples at 6 time points of mice under TRFe and TRFd to study markers autophagy. The mRNA markers of autophagy showed circadian rhythmicity. Both forms of TRF increased both the mean and amplitude of hepatic markers of autophagy vs AL in both diet groups, but no difference was observed between TRFe and TRFd. However, only TRFe increased the mean of Tfeb vs AL and TRFd in chow-fed mice. TRFe mice had a phase advance in Map1lc3b and Tfeb mRNA transcript vs TRFd in both diet groups. In conclusion, this study suggests that both forms of TRF are equally effective in increasing the mRNA markers of autophagy in liver. In conclusion, the findings of these studies suggest that nutritional interventions such as IF and TRF can effectively increase the hepatic autophagy in mice and improve the metabolic outcomes. However, IF does not provide a greater improvement in HDL biochemical properties compared to CR although both IF and CR can equally reduce clinically significant weight loss. This thesis indicates that although IF, TRF and CR may provide a similar metabolic outcome at physiological level, they affect differentially at cellular level.