Circadian Rhythms, Intermittent Fasting, Metabolic Health

Abstract

Lifestyle induced metabolic diseases such as obesity, type 2 diabetes (T2DM) and cardiovascular diseases (CVD) are often associated with increased energy intake, and reduced levels of physical activity. The feeding and fasting cycle is one of the strong time cues to entrain circadian rhythms in peripheral organs without altering the central clock in the brain. Recently, insulin was shown to be a time cue and thus an important reset signal for peripheral circadian clocks in vitro, in mice and in humans. Therefore, mistimed and erratic eating patterns, known to occur in modern society, may induce circadian disruption, and could contribute to the development of metabolic diseases. The aim of this thesis was to provide the evidence that mistimed eating will associate with markers of obesity and T2DM and intermittent fasting on 3 non-consecutive days per week may impact peripheral clocks, while time restricted eating (TRE) will decrease the risk factors of T2DM via restoring the 24-hour rhythms of blood metabolite and glucoregulatory hormones, as well as the transcriptome in subcutaneous adipose tissue (SAT). To examine the impact of erratic eating patterns on metabolic health, I enriched the existing concept of eating architecture, to include variations in meal frequency, size, timing and regularity. I first carried out a cross-sectional study to determine the relationship between components of eating architecture on body fat and markers of glycaemic control in 73 healthy adults at increased risk of T2DM. I aligned the datasets from the food diary, accelerometers and continuous glucose monitors in real-time for 1 to 2 weeks collected under free-living conditions. The results from the multivariable linear regression analysis show that lower day-to-day variability in first meal consumption was associated with lower body fat and improved glucose control in adults at increased risk of T2DM. Intermittent fasting (IF) is a practice of alternating periods of eating and fasting, which has emerged as an effective therapeutic dietary strategy for combating obesity and improving markers of cardiometabolic diseases. IF regimes include zero or minimal calories consumed 1 to 4 days per week on fasting days, followed by ad libitum eating on the remaining days, leading to about 10% calorie restriction overall per day of fasting (e.g., 3 days of fasting is ~30% calorie restriction). However, the day-to-day variation in the patterning of food intake under IF intervention could also lead to circadian disruption, but this is not well tested to date. Therefore, I next examined the effects of eight weeks of IF on mRNA levels of genes involved in circadian regulation in skeletal muscle and SAT. In this study, breakfast was consumed before a 24-hour fast on 3 non-consecutive days/week. The results indicate no universal effect of IF to alter peripheral clocks, which may partly be due to the alignment of the fasting/feeding cycle with the biological clock by initiating the fasting day at breakfast. TRE, is a circadian rhythm-reinforcing lifestyle that recommends restricting energy intake within a daily shortened period of time during the active phase (6-10 hours in human studies, 8-12 hours in animal studies), alternatively lengthening the daily fasting period, without altering calorie intakes or diet quality. In animal models of obesity and aging, TRE reduced diet-induced weight gain and fat accumulation, improved glucose tolerance and insulin sensitivity, prevented the age- and diet-induced reduction in cardiac contractile function and muscle function, as well as rescued the metabolic consequences induced by clock gene mutants. In humans, TRE also reduced body weight and fat mass, improved glucose control, mitigated risk factors of CVD such as blood pressure, lipids and inflammation in individuals with overweight and obesity. However, most of the TRE clinical trials are pilot studies that have only examined adherence, feasibility and body weight with a limited sample size and number. Few have explored the potential mechanisms involved in restoring the circadian clocks in human tissue and blood. Hence, I conducted the first human trial with a highly controlled metabolic ward stay to examine the effects of eight weeks of 10-hour TRE (self-selected eating duration except consuming the last meal before 7:30 pm) on glucose metabolism in men with increased risk of T2DM. Fifteen men with obesity but no history of diabetes were enrolled and underwent two weeks of baseline monitoring, before they were instructed to eat their regular diets within a contiguous 10-hour time frame each day for 8-weeks. Metabolic testing was performed at baseline and week 8 during a 35-hour metabolic ward stay, during which all food intake was strictly timed and controlled. Eight weeks of TRE did not alter the primary outcome plasma glucose area under the curve (AUC) at breakfast, but increased glucose AUC at dinner. TRE reduced fasting glucose, glycated haemoglobin (HbA1c), body weight and body fat. In SAT after an overnight fast, 117 genes were upregulated and 202 genes were downregulated by TRE. Pathway analysis revealed the downregulation of genes involved in proteasome function and mitochondrial regulation. These results suggest that TRE had a net effect to reduce glycaemia and dampened energy-consuming pathways in SAT. To further explore the mechanisms of TRE on these improvements, I evaluated the impacts of TRE on markers of central clocks (dim light melatonin onset, DLMO) and SAT clocks, as well as the 24-hour profiles of blood metabolites, glucoregulatory hormones and transcriptome profiles in human SAT in the same cohort. TRE did not alter DLMO, suggesting central clocks were not altered by TRE. TRE reduced morning cortisol levels, which correlated to the changes in HbA1c, indicating an improved glycaemic control. TRE altered the 24-hour profile of insulin, non-esterified fatty acids (NEFA), triglyceride and glucose-dependent insulinotropic peptide. TRE increased CLOCK and NR1D2 and decreased PER1 and NR1D1 at 12 am. The rhythmicity of 450 genes were altered by TRE. Pathway analysis showed enrichment in transcription co-repressor activity, DNA binding transcription factor binding, regulation of chromatin organization and small GTPase binding pathways. Weighted Gene Co-expression Network Analysis of these 450 genes revealed the three module eigengenes that were strongly correlated with BMI, insulin and NEFA. This study suggests that TRE altered the rhythms of blood metabolites, insulin, and increased key clock genes, restored the expression of genes involving in chromatin regulation and vesicular translocation of glucose transporters in human SAT. In conclusion, this research showed that routine consumption of breakfast meals may optimise temporal regulation to anticipate and respond appropriately to a glucose challenge. This was partially supported by the second study showing that IF protocol initiated at breakfast time does not alter peripheral clocks in muscle or fat, which could be essential to prevent circadian misalignment during IF in humans. Finally, I provided novel evidence that TRE could be a preventative tool for individuals with obesity who are at increased risk of T2DM to improve glucose control and reset circadian rhythms, and thus may potentially be a therapeutic strategy for patients with prediabetes or T2DM.