Introduction
Obesity is a major risk factor for a spectrum of diseases including type II diabetes, nonalcoholic fatty liver disease, cardiovascular disease, and cancer. The incidence of obesity is on the increase and, although the driving causes are multifactorial, nutritional imbalance is a major contributor (
Pontzer et al., 2012
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). Murine models have been invaluable in understanding the mechanisms of nutrient homeostasis and the consequences of nutrient imbalance, and as discovery platforms for pharmacological and behavioral interventions. Ad libitum access to a high-fat diet (HFD) in mice causes obesity, insulin resistance, hepatic steatosis, hypercholesterolemia, and dyslipidemia (
). Ad libitum access to high-fructose diet (Fr diet) on the other hand does not cause marked increase in adiposity, yet leads to glucose intolerance and hepatic steatosis (
Mellor et al., 2011
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Myocardial autophagy activation and suppressed survival signaling is associated with insulin resistance in fructose-fed mice.
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Samuel, 2011
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).
Diseases like obesity, arising from nutrient imbalance or excess, are often accompanied by disruptions of multiple pathways in different organ systems. For example, the regulation of glucose, lipids, cholesterol, and amino acids (aa) homeostasis involves the liver, white adipose tissue (WAT), brown adipose tissue (BAT), and muscle. In each tissue, nutrient homeostasis is maintained by balancing energy storage and energy utilization. Pharmacological agents directed against specific targets effectively treat certain aspects of this homeostatic imbalance. However, treating one aspect of a metabolic disease sometimes worsens other symptoms (e.g., increased adiposity seen with insulin sensitizers), and beneficial effects are often short lived (e.g., sulfonylureas) (
). Furthermore, recent studies have shown that early perturbation of nutrient homeostasis can cause epigenetic changes that predispose an individual to metabolic diseases later in life (
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Metabolic imprinting, programming and epigenetics—a review of present priorities and future opportunities.
). Hence, finding interventions that impact multiple organ systems and can reverse existing disease will likely be more potent in combating the pleiotropic effect of nutrient imbalance.
Lifestyle interventions, including changes in diet, reduced caloric intake, and increased exercise, have been the first-line therapy in efforts to combat obesity and metabolic diseases. However, these lifestyle changes require constant attention to nutrient quality and quantity and physical activity. Their success has been limited to a small percentage of individuals (
Anderson et al., 2001
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). Hence, novel interventions are urgently needed. Temporal regulation of feeding offers an innovative strategy to prevent and treat obesity and associated metabolic diseases (
). Recent discoveries have shown that many metabolic pathways, including current pharmacological targets, have diurnal rhythms (
Gamble et al., 2014
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Coordinated transcription of key pathways in the mouse by the circadian clock.
). It is hypothesized that under normal healthy conditions the cyclical expression of metabolic regulators coordinates a wide range of cellular processes for more efficient metabolism. In HFD-induced obesity, such temporal regulation is blunted (
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High-fat diet disrupts behavioral and molecular circadian rhythms in mice.
). Tonic activation or inhibition of a metabolic pathway, as is the case with pharmacological therapy, cannot restore normal rhythmic activity pattern. Therefore, interventions that restore diurnal regulation in multiple pathways and tissue types might be effective in countering the pleiotropic effect of nutrient imbalance.
Gene expression and metabolomics profiling, as well as targeted assay of multiple metabolic regulators, have revealed that a defined daily period of feeding and fasting is a dominant determinant of diurnal rhythms in metabolic pathways (
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,
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Bray et al., 2010
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Vollmers et al., 2009
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Time of feeding and the intrinsic circadian clock drive rhythms in hepatic gene expression.
). Accordingly, early introduction of time-restricted feeding (TRF), where access to food is limited to 8 hr during the active phase, prevents the adverse effects of HFD-induced metabolic diseases without altering caloric intake or nutrient composition (
Hatori et al., 2012
- Hatori M.
- Vollmers C.
- Zarrinpar A.
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Time-restricted feeding without reducing caloric intake prevents metabolic diseases in mice fed a high-fat diet.
). However, it is unclear whether TRF (i) is effective against other nutritional challenges, (ii) can be used to treat existing obesity, (iii) has a legacy effect after cessation, and (iv) can be adapted to different lifestyles. In lieu of the metabolic imprinting that renders mice susceptible to disease later in life, the therapeutic effect of TRF on preexisting diet-induced obesity (DIO) remained to be explored. The effectiveness of TRF as a single 8 hr feeding duration prompts exploration of the temporal window of food access that would still be effective against nutrition challenge. This is important before any human study can commence, given the incompatibility of an 8 hr restricted diet with a modern work schedule. Additionally, a change in eating pattern between weekday and weekend even when the mice are fed a standard diet has been suggested to contribute to obesity and metabolic diseases. This intimates that occasional deviation from TRF might exacerbate the disease. Addressing these questions is fundamental to elucidate the effectiveness and limitations of TRF and will offer novel insight into the relative role of eating pattern and nutrition on metabolic homeostasis.
This comprehensive study investigates the effectiveness of TRF against different nutritional challenges including high-fat, high-fructose, and high-fat-plus-high-fructose diets, all of which have been shown to cause dysmetabolism. We varied the duration of food access to characterize the temporal boundaries within which TRF benefits persist. We also evaluated both the therapeutic and legacy effects of TRF when interrupted by periods of unlimited access to energy-dense food. Results indicate pleiotropic beneficial effects of TRF that can prevent and alleviate multiple adverse effects of nutrient imbalance, and the benefits were proportional to the duration of fasting. Finally, and most importantly from a translational perspective for obese humans, TRF reversed obesity and metabolic disease and can potentially serve as an additional therapeutic intervention in the arsenal against this pandemic.
Discussion
Obesity is associated with multiple comorbidities including diabetes, heart disease, and cancer. In the United States, more than one-third (35.7%) of adults are obese. Hence, there is an imperative to find treatments and preventative measures against obesity and metabolic disease. TRF is a potential behavioral intervention. It de-emphasizes caloric intake, hence making it an attractive and easily adoptable lifestyle modification. Restricting feeding to 8 hr of a rodent’s preferred nocturnal feeding time protects against weight gain and associated metabolic diseases (
Hatori et al., 2012
- Hatori M.
- Vollmers C.
- Zarrinpar A.
- DiTacchio L.
- Bushong E.A.
- Gill S.
- Leblanc M.
- Chaix A.
- Joens M.
- Fitzpatrick J.A.
- et al.
Time-restricted feeding without reducing caloric intake prevents metabolic diseases in mice fed a high-fat diet.
). In this study, we show that TRF exerts pleiotropic beneficial effects on multiple measures of metabolic fitness under various nutritional challenges that are faced by most modern human societies. This sets the stage for exploring TRF in treating human obesity and dysmetabolism.
Mice fed a diet in which fat represents more than 40% of the caloric content gained excessive body weight relative to mice fed NC. Yet, daily TRF (9–15 hr) protected mice fed a HFD from obesity. Weight gain was equivalent among mice on TRFs of 8, 9, or 12 hr (
Hatori et al., 2012
- Hatori M.
- Vollmers C.
- Zarrinpar A.
- DiTacchio L.
- Bushong E.A.
- Gill S.
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- Chaix A.
- Joens M.
- Fitzpatrick J.A.
- et al.
Time-restricted feeding without reducing caloric intake prevents metabolic diseases in mice fed a high-fat diet.
;
Figure 2). Only when mice were subjected to 15 hr TRF was moderate obesity observed. In other words, a daily fast of < 12 hr may not elicit a response that is sufficient to protect against obesity. The duration of feeding and fasting likely determine the overall anabolic and catabolic signals needed to maintain body weight at a steady-state value. HFD-induced obesity is associated with insulin hypersecretion and insulin resistance (
McArdle et al., 2013
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,
). Because insulin itself is an anabolic signal, reducing the feeding period likely reduces the net daily anabolic effect of insulin on fatty acid synthesis and storage. Conversely, increasing the fasting duration supports fatty acid utilization. Both reduced insulin signaling during fasting and switching energy usage from glucose to fat within a few hours of fasting likely contribute to the observed reduction in adiposity in mice fed a HFD (FTT) to levels found in mice fed NC ad libitum (NAA).
Mice fed NC did not show profound differences in body weight between ALF and TRF. Nevertheless, there were other positive consequences of TRF. In the 38-week-long TRF, mice on NC (NAA, NTT, NAT, and NTA) exhibited equivalent body weights, yet NTT mice had significantly more lean mass and less fat mass than other NC cohorts (
Figure S2D). Furthermore, NTT mice were protected from mild hepatic steatosis, which is usually observed in old mice fed NC ad libitum (
Jin et al., 2013
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Increased expression of enzymes of triglyceride synthesis is essential for the development of hepatic steatosis.
). Similarly, mice fed an Fr diet did not show any significant change in body weight between ALF and TRF cohorts, yet FrT mice had less fat mass, increased lean mass, and better glucose tolerance relative to FrA mice (
Figures S2E and
S4B).
Although chronic TRF paradigms highlight the benefits of a daily feed-fast cycle, the 5T2A paradigm, in which mice had ad libitum access to food during the weekend, was just as effective. Mice subjected to 5T2A did not “learn” a daily feed-fast behavioral rhythm. Instead, during the weekend they consumed almost equivalent calories during days and nights (
Figure S2F). Yet, surprisingly, the gene expression signature of 5T2A mice sacrificed during the weekend ALF phase resembled that of TRF mice (
Figures 5C, 5D, and
7B), suggesting that the weekday TRF imprints a gene expression signature that can resist occasional changes in the feeding pattern. However, when the mice were completely transitioned from TRF to chronic ALF (as in the 13:12 and 26:12 mice), they gradually adopted an ALF gene expression signature, which eventually blunted the previous beneficial effect of TRF. Conversely, transferring mice from ALF to TRF effectively alleviated the adverse health status of existing obesity. The outcome of the crossover on body weight regulation depended on the age at which the switch happened. When switched at 25 weeks old (13:12 study), within 12 weeks, body weights of mice maintained on TRF (FTT) or switched to TRF (FAT) were equivalent. At 38 weeks old (26:12 study), crossed-over mice (FTA and FAT) attained a body weight that was intermediate between FAA and FTT mice. Therefore, TRF is more effective in normalizing body weight when adopted early in life or when there is moderate adiposity. Nevertheless, in older mice, TRF reduced excessive body weight by 20% and prevented further weight gain. Attaining an ideal body weight equivalent to that observed in chronic TRF mice might require additional interventions. In summary, TRF for 12 hr or shorter offers metabolic benefits irrespective of diet type, so much so that even occasional ALF did not blunt the TRF benefits. In addition, age is an important factor in TRF, as young individuals may be more susceptible to its benefits than older counterparts.
These different studies revealed that the benefits of TRF seem to be directly related to adiposity. PPARγ, a transcription factor involved in lipid storage and adipocytes differentiation, appears to play an important role. The overexpression of PPARγ in WAT of TRF mice is reminiscent of the protective role it plays by promoting the development of “better-quality” fat tissue (
Sharma and Staels, 2007
Review: Peroxisome proliferator-activated receptor gamma and adipose tissue—understanding obesity-related changes in regulation of lipid and glucose metabolism.
). Conversely, PPARγ expression was much higher in BAT of ALF mice, which could explain its observed “whitening.” Elevated hepatic PPARγ is a protective measure to prevent serum hyperlipidemia, triglyceride accumulation in other tissues, and associated insulin resistance (
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). It is unclear how the feeding pattern tunes PPARγ levels in a tissue-specific manner, as observed in our study. Such differential regulation is unlikely to be an acute effect of daily feeding, as PPARγ levels of 5T2A livers during ALF days were similar to those seen with FTT or FAT livers.
In addition to lipid storage, TRF has a tremendous effect on lipid regulation itself. SREBPs are master transcription factors involved in lipid and sterol homeostasis. Although there were no differences in hepatic mRNA levels between ALF and TRF, levels of the shorter, proteolytically activated form of SREBP were higher in TRF, indicating higher activation of the pathway. An increase in active SREBP, as well as its known target enzymes Cyp7a1 and Cyp7b1, in the liver of TRF mice likely restores cholesterol homeostasis. Serum levels of cholesterol, its precursor cholestanol, and its hormonal or bile acid derivatives (corticosterone, TCA, and TCDA) were better regulated in TRF mice (
Figure 6). Importantly, protection from hypercholesterolemia was a hallmark of all mice experiencing TRF, independent of the dietary challenge or the duration of feeding (
Figure 7).
In addition to improving lipid and cholesterol homeostasis, TRF had far-reaching effects on glucose and protein metabolism. ALF mice showed constitutively higher activation of the gluconeogenesis pathway, a characteristic of insulin resistance (
Figure 5D). All mice on TRF were protected against insulin resistance (HOMA-IR;
Figure S4A). Irrespective of adiposity, when TRF mice were challenged with a glucose bolus they were able to restore normoglycemia much faster than ALF mice (
Figure 4C). In mice fed a normal diet and mice on TRF, serum levels of free aa oscillated with a daytime peak (i.e., when mice were fasting). Accordingly, phospho-S6, an indicator of protein synthesis, oscillated with a nighttime peak. In mice fed HFD ad libitum (FAA), the average levels of free aa (including BCAA) were elevated, and the peak phase was shifted by 12 hr. In addition, the phospho-S6 peak in liver and muscle was reduced and phase shifted by 12 hr. The FA condition desynchronized the diurnal profile of free aa in the serum and protein synthesis pathways in the liver and muscle. TRF restored the temporal regulation and overall levels of these pathways. This improved protein homeostasis may contribute to the remarkable endurance exhibited by TRF mice (
Figures 5E and
6D;
).
Motivated by the multifactorial improvement in diverse metabolic pathways, we used metabolomics to explore the global impact of TRF. Biomarkers for inflammation and protection against reactive oxygen species were reduced and elevated, respectively. The effect of TRF on both of these health-promoting pathways may be important for the systemic health improvements seen under TRF. For most metabolites it is unclear whether changes in the average level or the temporal pattern of abundance is important. However, because TRF affects metabolites in a number of key pathways, this aspect of our findings warrants further investigation.
Ultimately, our results highlight the great potential for TRF in counteracting human obesity and its associated metabolic disorders. Future work should explore the role of known metabolic and circadian regulators in restoring the organism’s energetics to normalcy under TRF. Furthermore, it is worth investigating whether the physiological observations found in mice apply to humans. A large-scale randomized control trial investigating the role of TRF would show whether it is applicable to humans.
Acknowledgments
This work was partially supported by NIH grants DK091618-04, EY016807, and NS066457 and AFAR grant M14322 to S.P. and funding to research cores through NIH P30 CA014195, P30 EY019005, P50 GM085764, R24 DK080506, Leona M. and Harry B. Helmsley Charitable Trust’s grant #2012-PG-MED002, and the Glenn Center for Aging. A.C. was supported by a mentor-based postdoctoral fellowship from the American Diabetes Association (7-12-MN-64) and has received support from the Philippe Foundation, New York. A.Z. received support from NIH T32 DK07202 and an AASLD Liver Scholar Award. The authors would like to thank An Qi Yao, Shih-Han Huang, Seung Mi Oh, Hiep Le, Naomi Goebel-Phipps, SAF staff, and Scott McDonnell for technical assistance and input. We thank Shubhroz Gill for careful, critical, and constructive editing of the manuscript.
Article info
Publication history
Published: December 2, 2014
Accepted: October 31, 2014
Received in revised form: September 15, 2014
Received: July 23, 2014
Copyright
© 2014 Elsevier Inc. Published by Elsevier Inc.