Life extension – healthspan – strategies for successful aging

Dietary restriction with and without caloric restriction for healthy aging

“Caloric restriction is the most effective and reproducible dietary intervention known to regulate aging and increase the healthy lifespan in various model organisms, ranging from the unicellular yeast to worms, flies, rodents, and primates.  However, caloric restriction, which in most cases entails a 20–40% reduction of food consumption relative to normal intake, is a severe intervention that results in both beneficial and detrimental effects. Specific types of chronic, intermittent, or periodic dietary restrictions without chronic caloric restriction have instead the potential to provide a significant healthspan increase while minimizing adverse effects.”

CAloric restriction (CR)  refers to a dietary intervention with an overall 20–40% reduction of total caloric intake, and dietary restriction to represent a broader scope of dietary interventions that encompass those with specific macronutrient and feeding pattern restrictions. There is mounting evidence that healthspan can be maximized and aging  can be reduced by  methods other than caloric reduction.

In 1935, Crowell and McCay demonstrated that simply reducing caloric intake without causing malnutrition nearly doubled the lifespan of rats.

Walford and Weindruch reported that “adult-initiated” caloric restriction started at 12 months of age not only increased lifespan but also reduced the incidence of spontaneous cancer by more than 50% in rats. Dietary Restriction in Mice Beginning at 1 Year of Age effect on life span and cancer  This same effect has been shown in yeast, worms, flies, and other animals, hence the genes for this are highly conserved.  Caloric restriction reduces age-related and all-cause mortality in rhesus monkeys

Caloric restriction and lifespan extension involves the down-regulation of insulin and insulin-like signalling (IIS)[ The genetics of ageing ], as well as of the amino signalling target of rapamycin (TOR)-S6 kinase pathway [Regulation of Lifespan in Drosophila by Modulation of Genes in the TOR Signaling Pathway and Regulation of Longevity and Stress Resistance by Sch9 in Yeast , and the glucose signalling Ras-protein kinase A (PKA) pathway[The Ras-Erk-ETS-Signaling Pathway Is a Drug Target for longevity]

In yeast, down-regulation of (a) the amino acid-sensing TOR and the ribosomal protein S6 kinase (S6K) ortholog Sch9 pathway6 , and (b) the Ras-AC-PKA pathway13 [Life Span Extension by Calorie Restriction Depends on Rim15 and Transcription Factors Downstream of Ras PKA, Tor, and Sch9 ] are key changes mediating part of the effects of caloric restriction on chronological lifespan, the measurement of cellular survival under non-dividing conditions. In contrast, elevated activity of sirtuin (SIR2) [ Sir2 Blocks Extreme Life-Span Extension] and [Requirement of NAD and SIR2 for Life-Span Extension by Calorie Restriction in Saccharomyces cerevisiae  has been described as a key change in the extension of replicative lifespan, measured by counting the number of buds generated by an individual mother cell In worms, the lifespan extension caused by the inactivation of IIS, or by different forms of caloric restriction, requires Forkhead FoxO transcription factor daf-16 Different dietary restriction regimens extend lifespan by both independent and overlapping genetic pathways in C. elegans  . In rodents, growth hormone (GH) and IGF-1 levels are reduced following caloric restriction Fasting vs dietary restriction in cellular protection and cancer treatment from model organisms to patients, but the link between dietary restriction, GH and aging is still being investigated, with focus on the genes and pathways regulating longevity in the simple organisms described above.

The ultimate question that lingers is the relevance of these models to human lifespan and healthspan.  Two notable studies performed by independent programs, the National Institute on Aging (NIA) Intramural Research Program and the Wisconsin National Primate Research Center (WNPRC), subjected male and female rhesus monkeys to 30% caloric restriction from levels of baseline caloric intake. The NIA reported no improvement in lifespan but observed a positive trend for the delay of age-related diseases (i.e. healthspan) Impact of caloric restriction on health and survival in rhesus monkes – the NIA study, whereas WNPRC reported significant improvement in both lifespan and healthspan Caloric restriction delays disease onset and mortality in rhesus monkeys

CALERIE (Comprehensive Assessment of the Long term Effects of Reducing Intake of Energy), recently reported that a two year 25% caloric restriction is feasible for humans and provides health benefits, such as reduced inflammatory markers and cardiometabolic risk factors. A 2-Year Randomized Controlled Trial of Human Caloric Restriction Feasibility and Effects on Predictors of Health Span and Longevity  and Energy requirements in nonobese men and women results from CALERIE and Caloric Restriction in Humans

CALERIE was conducted in three independent centers and involved 218 overweight participants, suggesting that caloric restriction can be beneficial even in a very genetically heterogeneous group. Comprehensive Assessment of Long-Term Effects of Reducing Intake of Energy Phase 2 (CALERIE Phase 2) Screening and Recruitment Methods and Results

The ability of caloric restriction to prevent the damage caused by exogenous toxins is likely to be associated with the protection, repair and replacement effects that prevent the age-dependent dysfunction caused by endogenous processes and toxic molecules. CALORIE RESTRICTION AND AGING A LIFE-HISTORY ANALYSIS At the cellular level, caloric restriction and longevity mutations allow resistance to stressors, especially oxidative stress.

Likewise, it could be that hormesis occurs, in which repeat stresses allow adaptation and survival.

Dietary options for food restriction include: short-term starvation, periodic fasting, fasting-mimetic diets, intermittent fasting, normocaloric diets with planned deficiencies (in particular macronutrients: proteins, carbohydrates, etc.), and time-restricted feeding.Fasting involves a 60% decrease in food intake. Intermittent fasting refers to practicing this intervention every other day whereas periodic fasting refers to severe restriction for two or more days periodically (every two weeks, month, etc.) Caloric restriction and fasting result in lower glucose levels and insulin levels. Fasting vs dietary restriction in cellular protection and cancer treatment from model organisms to patients  Both intermittent and periodic fasting can increase lifespan, even when there is little or no overall decrease in calorie intake. Intermittent fasting dissociates beneficial effects of dietary restriction on glucose metabolism and neuronal resistance to injury from calorie intake and Brandhorst S, Choi IY, Wei M, et al.: A Periodic Diet that Mimics Fasting Promotes Multi-System Regeneration, Enhanced Cognitive Performance, and Healthspan. Cell Metab. 2015; 22(1): 86–99. 

The restriction of specific macronutrients (or macronutrient restriction) without the restriction of calories is among the most promising interventions that have emerged to promote healthy aging in humans, with  reduced intake of proteins and amino acids is the most effective pro-longevity intervention: Protein and amino acid restriction, aging and disease from yeast to humans [ Protein or Anino Acid (AA)  restriction has been shown to be as potent as calorie restriction in extending healthspan in multiple model organisms. AA restriction affects lifespan partly through modulation of the amino acid sensing pathways TOR and GCN2. Human epidemiological studies highlight the detrimental effects of high protein diets, in particular animal-derived protein sources in contrast to plant-based sources. Epidemiological studies indicate that low protein diets are associated with lower risk of chronic and age-related diseases such as CVDs, diabetes, and cancer.]  Also, Amino acid imbalance explains extension of lifespan by dietary restriction in Drosophila : Adding essential amino acids to a DR diet increased fecundity and decreased lifespan, similar to full feeding, with other nutrients having little or no effect. However, methionine alone increased fecundity as much as full feeding, but without reducing lifespan. Reallocation of nutrients therefore does not explain the DR responses. Lifespan was reduced by amino acids, particularly essential amino acids. Hence an imbalance in dietary amino acids away from the ratio optimal for reproduction shortens lifespan during full feeding and limits fecundity during DR. Reduced activity of the insulin/Igf signaling pathway extends lifespan in diverse organisms 7, and it protected against the shortening of lifespan with full feeding. In other organisms, including mammals, it may be possible to obtain the benefits for lifespan of DR without reduced fecundity, through a suitable balance of nutrients in the diet.

A recent analysis of the National Health and Nutrition Examination Survey (NHANES) showed that low protein intake was associated with reduced overall mortality for those under 65 years of age. Low Protein Intake Is Associated with a Major Reduction in IGF-1, Cancer, and Overall Mortality in the 65 and Younger but Not Older Population : High protein intake is linked to increased cancer, diabetes, and overall mortality. High IGF-1 levels increased the relationship between mortality and high protein. Higher protein consumption may be protective for older adults. Plant-derived proteins are associated with lower mortality than animal-derived proteins. These results suggest that low protein intake during middle age followed by moderate to high protein consumption in old adults may optimize healthspan and longevity.

A high-carbohydrate, low-protein diet resulted in longer lifespan and improved cardiometabolic health, despite increased food intake and body fat: The Ratio of Macronutrients, Not Caloric Intake, Dictates Cardiometabolic Health, Aging, and Longevity in Ad Libitum-Fed Mice —  Food intake is regulated primarily by dietary protein and carbohydrate. Low-protein, high-carbohydrate diets are associated with the longest lifespans. Energy reduction from high-protein diets or dietary dilution does not extend life. Diet influences hepatic mTOR via branched-chain amino acids and glucose. Longevity and health were optimized when protein was replaced with carbohydrate to limit compensatory feeding for protein and suppress protein intake.Calorie restriction achieved by high-protein diets or dietary dilution had no beneficial effects on lifespan. 

Also: Dietary Protein to Carbohydrate Ratio and Caloric Restriction Comparing Metabolic Outcomes in Mice Abstract: Both caloric restriction (CR) and low-protein, high-carbohydrate (LPHC) ad-libitum-fed diets increase lifespan and improve metabolic parameters such as insulin, glucose, and blood lipids. Severe CR, however, is unsustainable for most people; therefore, it is important to determine whether manipulating macronutrient ratios in ad-libitum-fed conditions can generate similar health outcomes. We present the results of a short-term (8 week) dietary manipulation on metabolic outcomes in mice. We compared three diets varying in protein to carbohydrate ratio under both CR and ad libitum conditions. Ad libitum LPHC diets delivered similar benefits to CR in terms of levels of insulin, glucose, lipids, and HOMA, despite increased energy intake. CR on LPHC diets did not provide additional benefits relative to ad libitum LPHC. We show that LPHC diets under ad-libitum-fed conditions generate the metabolic benefits of CR without a 40% reduction in total caloric intake Ad libitum low-protein, high-carbohydrate diets (LPHC) improve metabolic health. Caloric restriction combined with LPHC diet does not provide added health benefits. Energy intake and energy expenditure are increased on LPHC diets.

The restriction of a single essential amino acid in a normal diet increased lifespan and stress resistance: Zimmerman JA, Malloy V, Krajcik R, et al.: Nutritional control of aging. Exp Gerontol. 2003; 38(1–2): 47–52.  In this paper, reducing specific essential amino acids in rats demonstrated longevity effects. Reduced tryptophan content in the diet extended maximum lifespan. In fact, decreasing sulfhydryl-containing amino acids in the diet by removing cysteine and methionine extended survival. Calorie restriction (CR) delays cancer, reduces the diminution of the immune system in aging, and improves insulin sensitivity.  Essential amino acid restriction in this study, i.e. methionine restriction (MR) , resulted in decreased growth in rats, but produced life extension in the same was as CR would. MR produced 42% increase in longevity. The animals grew less, but they lived longer. As for tryptophan- restricted mice who were fed ad libitum, they too had less growth but lived 10% longer. CR, on the other hand, results in a 65% life extension, but off course the animals had to eat much less. It is felt that oxidation results in age-related physiological defects and that CR attenuates the generation of oxidative end products in aged animals. Glutathions (GSH) is one of the anti-oxidants and detoxifying agents that decreases in aging. This study demonstrated that cysteine-restriction did not decrease levels of GSH, but rather increased GSH levels in mice. Considering that cyteine is a precursor of GSh, this is counter intuitive.

The Abstract:  For more than 60 years the only dietary manipulation known to retard aging was caloric restriction, in which a variety of species respond to a reduction in energy intake by demonstrating extended median and maximum life span. More recently, two alternative dietary manipulations have been reported to also extend survival in rodents. Reducing the tryptophan content of the diet extends maximum life span, while lowering the content of sulfhydryl-containing amino acids in the diet by removing cysteine and restricting the concentration of methionine has been shown to extend all parameters of survival, and to maintain blood levels of the important anti-oxidant glutathione. To control for the possible reduction in energy intake in methionine-restricted rats, animals were offered the control diet in the quantity consumed by rats fed the low methionine diet. Such pair-fed animals experienced life span extension, indicating that methionine restriction-related life span extension is not a consequence of reduced energy intake. By feeding the methionine restricted diet to a variety of rat strains we determined that lowered methionine in the diet prolonged life in strains that have differing pathological profiles in aging, indicating that this intervention acts by altering the rate of aging, not by correcting some single defect in a single strain.

Also: Methionine restriction increases blood glutathione and longevity in F344 rats

Low-protein, high-carbohydrate diet increases glucose uptake and fatty acid synthesis in brown adipose tissue of rats.

De Marte ML, Enesco HE: Influence of low tryptophan diet on survival and organ growth in mice. Mech Ageing Dev. 1986; 36(2): 161–71. Abstract: Greater survival and reduced growth were found to characterize mice on a tryptophan deficient diet as compared to fully fed control mice. The 50% survival point was reached by the tryptophan restricted group at 683 days, and by the control group at 616 days. Measurements of body weight, organ weight, and DNA level were made at 8, 12, 24, 36, 52 and 78 weeks of age. Both whole body weight and organ weight of liver, kidney, heart and spleen were about 30% lower in the tryptophan restricted group as compared to the controls, so that the ratio of organ weight to body weight remained at a constant value for both groups. There was no significant change in cell number as determined by DNA measurements, as a result of the tryptophan restriction.

Ooka H, Segall PE, Timiras PS: Histology and survival in age-delayed low-tryptophan-fed rats. Mech Ageing Dev. 1988; 43(1): 79–98. Abstract:  Diets containing tryptophan in concentrations 30 and 40 percent of those fed to controls from weaning to 24-30 months or more, can delay aging in Long-Evans female rats. Mortality among low-tryptophan-fed rats was greater in the juvenile period, but substantially less than controls at late ages. Histological biomarkers of aging were also delayed after tryptophan restriction in some organs (liver, heart, uterus, ovary, adrenal and spleen) but not in others (kidney, lung, aorta). Brain serotonin levels were low in tryptophan-deficient rats but showed remarkable capacity for rehabilitation. Effects on early and late mortality and brain levels of serotonin were proportional to the severity of the restriction.

Laboratory rodents fed a methionine-restricted diet displayed an extended lifespan with decreased age-dependent diseases and increased resistance to oxidative stress, in part due to increased antioxidant capacity : Methionine restriction increases blood glutathione and longevity in F344 rats. Abstract as follows: Little is known about the biochemical mechanisms responsible for the biological aging process. Our previous results and those of others suggest that one possible mechanism is based on the loss of glutathione (GSH), a multifunctional tripeptide present in high concentrations in nearly all living cells. The recent finding that life-long dietary restriction of the GSH precursor methionine (Met) resulted in increased longevity in rats led us to hypothesize that adaptive changes in Met and GSH metabolism had occurred, leading to enhanced GSH status. To test this, blood and tissue GSH levels were measured at different ages throughout the life span in F344 rats on control or Met-restricted diets. Met restriction resulted in a 42% increase in mean and 44% increase in maximum life span, and in 43% lower body weight compared to controls (P < 0.001). Increases in blood GSH levels of 81% and 164% were observed in mature and old Met-restricted animals, respectively (P < 0.001). Liver was apparently the source for this increase as hepatic GSH levels decreased to 40% of controls. Except for a 25% decrease in kidney, GSH was unchanged in other tissues. All changes in GSH occurred as early as 2 months after the start of the diet. Altogether, these results suggest that dramatic adaptations in sulfur amino acid metabolism occur as a result of chronic Met restriction, leading to increases in blood GSH levels and conservation of tissue GSH during aging.

Methionine-deficient diet extends mouse lifespan, slows immune and lens aging, alters glucose, T4, IGF-I and insulin levels, and increases hepatocyte MIF levels and stress resistance  : A diet deficient in the amino acid methionine has previously been shown to extend lifespan in several stocks of inbred rats. We report here that a methionine-deficient (Meth-R) diet also increases maximal lifespan in (BALB/ cJ × C57BL/6 J)F1 mice. Compared with controls, Meth-R mice have significantly lower levels of serum IGF-I, insulin, glucose and thyroid hormone. Meth-R mice also have higher levels of liver mRNA for MIF (macrophage migration inhibition factor), known to be higher in several other mouse models of extended longevity. Meth-R mice are significantly slower to develop lens turbidity and to show age-related changes in T-cell subsets. They are also dramatically more resistant to oxidative liver cell injury induced by injection of toxic doses of acetaminophen. The spectrum of terminal illnesses in the Meth-R group is similar to that seen in control mice. Studies of the cellular and molecular biology of methionine-deprived mice may, in parallel to studies of calorie-restricted mice, provide insights into the way in which nutritional factors modulate longevity and late-life illnesses.

Life-Span Extension in Mice by Preweaning Food Restriction and by Methionine Restriction in Middle Age  Abstract:   Life span can be extended in rodents by restricting food availability (caloric restriction [CR]) or by providing food low in methionine (Meth-R). Here, we show that a period of food restriction limited to the fi rst 20 days of life, via a 50% enlargement of litter size, shows extended median and maximal life span relative to mice from normal sized litters and that a Meth-R diet initiated at 12 months of age also signifi cantly increases longevity. Furthermore, mice exposed to a CR diet show changes in liver messenger RNA patterns, in phosphorylation of Erk, Jnk2, and p38 kinases, and in phosphorylation of mammalian target of rapamycin and its substrate 4EBP1, HE-binding protein 1 that are not observed in liver from agematched Meth-R mice. These results introduce new protocols that can increase maximal life span and suggest that the spectrum of metabolic changes induced by low-calorie and low-methionine diets may differ in instructive ways.

Methionine restriction decreases visceral fat mass and preserves insulin action in aging male Fischer 344 rats independent of energy restriction Abstract: Reduced dietary methionine intake (0.17% methionine, MR) and calorie restriction (CR) prolong lifespan in male Fischer 344 rats. Although the mechanisms are unclear, both regimens feature lower body weight and reductions in adiposity. Reduced fat deposition in CR is linked to preservation of insulin responsiveness in older animals. These studies examine the relationship between insulin responsiveness and visceral fat in MR and test whether, despite lower food intake observed in MR animals, decreased visceral fat accretion and preservation of insulin sensitivity is not secondary to CR. Accordingly, rats pair fed (pf) control diet (0.86% methinone, CF) to match the food intake of MR for 80 weeks exhibit insulin, glucose, and leptin levels similar to control-fed animals and comparable amounts of visceral fat. Conversely, MR rats show significantly reduced visceral fat compared to CF and PF with concomitant decreases in basal insulin, glucose, and leptin, and increased adiponectin and triiodothyronine. Daily energy expenditure in MR animals significantly exceeds that of both PF and CF. In a separate cohort, insulin responses of older MR animals as measured by oral glucose challenge are similar to young animals. Longitudinal assessments of MR and CF through 112 weeks of age reveal that MR prevents age-associated increases in serum lipids. By 16 weeks, MR animals show a 40% reduction in insulin-like growth factor-1 (IGF-1) that is sustained throughout life; CF IGF-1 levels decline much later, beginning at 112 weeks. Collectively, the results indicate that MR reduces visceral fat and preserves insulin activity in aging rats independent of energy restriction.

A fasting-mimicking diet, consisting of very low calorie and protein that leads to similar physiological response to fasting, including reduced levels of glucose and IGF-1 and increased levels of ketone bodies IGFBP-1, enhanced healthspan and rejuvenated the hematopoietic system while promoting adult neurogenesis :  Brandhorst S, Choi IY, Wei M, et al.: A Periodic Diet that Mimics Fasting Promotes Multi-System Regeneration, Enhanced Cognitive Performance, and Healthspan. Cell Metab. 2015; 22(1): 86–99. 

Feeding schedule has also been shown to have a significant impact on health and survival. In flies, time-restricted feeding (limited to 12 daytime hours every day) had profound effects on neural, peripheral, and cardiovascular physiology and improved sleep, body weight maintenance, and delayed signs of cardiac aging, under unchanged caloric intake and activity.  Time-restricted feeding attenuates age-related cardiac decline in Drosophila  Abstract: Circadian clocks orchestrate periods of rest or activity and feeding or fasting over the course of a 24-hour day and maintain homeostasis. To assess whether a consolidated 24-hour cycle of feeding and fasting can sustain health, we explored the effect of time-restricted feeding (TRF; food access limited to daytime 12 hours every day) on neural, peripheral, and cardiovascular physiology in Drosophila melanogaster. We detected improved sleep, prevention of body weight gain, and deceleration of cardiac aging under TRF, even when caloric intake and activity were unchanged. We used temporal gene expression profiling and validation through classical genetics to identify the TCP-1 ring complex (TRiC) chaperonin, the mitochondrial electron transport chain complexes, and the circadian clock as pathways mediating the benefits of TRF.

When mice were given access to food for only 8–9 hours during the active phase of the day, metabolic diseases induced by a high-fat, high-fructose, and high-sucrose diet, were reduced without lowering caloric intake : Time restricted feeding without reducing caloric intake prevents metabolic diseases in mice fed a high fat diet  Abstract: While diet-induced obesity has been exclusively attributed to increased caloric intake from fat, animals fed a high-fat diet (HFD) ad libitum (ad lib) eat frequently throughout day and night, disrupting the normal feeding cycle. To test whether obesity and metabolic diseases result from HFD or disruption of metabolic cycles, we subjected mice to either ad lib or time-restricted feeding (tRF) of a HFD for 8 hr per day. Mice under tRF consume equivalent calories from HFD as those with ad lib access yet are protected against obesity, hyperinsulinemia, hepatic steatosis, and inflammation and have improved motor coordination. The tRF regimen improved CREB, mTOR, and AMPK pathway function and oscillations of the circadian clock and their target genes’ expression. These changes in catabolic and anabolic pathways altered liver metabolome and improved nutrient utilization and energy expenditure. We demonstrate in mice that tRF regimen is a nonpharmacological strategy against obesity and associated diseases.

Ad lib feeding during the weekend did not interfere with the protective effects of time-restricted feeding: Time-Restricted Feeding Is a Preventative and Therapeutic Intervention against Diverse Nutritional Challenges Abstract:  Time-restricted feeding (TRF) confines food access to 9–12 hr during the active phase. TRF is a therapeutic intervention against obesity without calorie restriction.TRF protects against metabolic diseases even when briefly interrupted on weekends. TRF is effective against high-fat, high-fructose, and high-sucrose diets. Because current therapeutics for obesity are limited and only offer modest improvements, novel interventions are needed. Preventing obesity with time-restricted feeding (TRF; 8–9 hr food access in the active phase) is promising, yet its therapeutic applicability against preexisting obesity, diverse dietary conditions, and less stringent eating patterns is unknown. Here we tested TRF in mice under diverse nutritional challenges. We show that TRF attenuated metabolic diseases arising from a variety of obesogenic diets, and that benefits were proportional to the fasting duration. Furthermore, protective effects were maintained even when TRF was temporarily interrupted by ad libitum access to food during weekends, a regimen particularly relevant to human lifestyle. Finally, TRF stabilized and reversed the progression of metabolic diseases in mice with preexisting obesity and type II diabetes. We establish clinically relevant parameters of TRF for preventing and treating obesity and metabolic disorders, including type II diabetes, hepatic steatosis, and hypercholesterolemia.

A restricted feeding pattern reversed the progression of pre-existing obesity and type II diabetes, suggesting it has the potential to be a clinically relevant and feasible dietary intervention, useful to prevent and treat obesity and metabolic disorders : Time-Restricted Feeding Is a Preventative and Therapeutic Intervention against Diverse Nutritional Challenges

. Considering that key metabolic factors, such as 5’ AMP-activated protein kinase (AMPK), sirtuins, and protein kinase B (AKT), are regulated by an interplay of circadian rhythm and feeding time54,55, dietary schedules should be more carefully studied in the context of dietary restriction. Metabolism and the Circadian Clock Converge : Abstract: Circadian rhythms occur in almost all species and control vital aspects of our physiology, from sleeping and waking to neurotransmitter secretion and cellular metabolism. Epidemiological studies from recent decades have supported a unique role for circadian rhythm in metabolism. As evidenced by individuals working night or rotating shifts, but also by rodent models of circadian arrhythmia, disruption of the circadian cycle is strongly associated with metabolic imbalance. Some genetically engineered mouse models of circadian rhythmicity are obese and show hallmark signs of the metabolic syndrome. Whether these phenotypes are due to the loss of distinct circadian clock genes within a specific tissue versus the disruption of rhythmic physiological activities (such as eating and sleeping) remains a cynosure within the fields of chronobiology and metabolism. Becoming more apparent is that from metabolites to transcription factors, the circadian clock interfaces with metabolism in numerous ways that are essential for maintaining metabolic homeostasis. 

Time of feeding and the intrinsic circadian clock drive rhythms in hepatic gene expression Abstract: In mammals, the circadian oscillator generates approximately 24-h rhythms in feeding behavior, even under constant environmental conditions. Livers of mice held under constant darkness exhibit circadian rhythm in abundance in up to 15% of expressed transcripts. Therefore, oscillations in hepatic transcripts could be driven by rhythmic food intake or sustained by the hepatic circadian oscillator, or a combination of both. To address this question, we used distinct feeding and fasting paradigms on wild-type (WT) and circadian clock-deficient mice. We monitored temporal patterns of feeding and hepatic transcription. Both food availability and the temporal pattern of feeding determined the repertoire, phase, and amplitude of the circadian transcriptome in WT liver. In the absence of feeding, only a small subset of transcripts continued to express circadian patterns. Conversely, temporally restricted feeding restored rhythmic transcription of hundreds of genes in oscillator deficient mouse liver. Our findings show that both temporal pattern of food intake and the circadian clock drive rhythmic transcription, thereby highlighting temporal regulation of hepatic transcription as an emergent property of the circadian system. 








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