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Cell Metab. Author manuscript; available in PMC 2015 February 04.

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Cell Metab. 2014 February 4; 19(2): 181–192. doi:10.1016/j.cmet.2013.12.008.

Fasting: Molecular Mechanisms and Clinical Applications

Valter D. Longo1 and Mark P. Mattson2,3

1Longevity Institute, Davis School of Gerontology and Department of Biological Sciences,University of Southern California, Los Angeles, CA 90089-2520, USA

2National Institute on Aging Intramural Research Program, National Institutes of Health,Baltimore, Maryland 21224, USA

3Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland21205, USA

Abstract

Fasting has been practiced for millennia, but only recently studies have shed light on its role inadaptive cellular responses that reduce oxidative damage and inflammation, optimize energymetabolism and bolster cellular protection. In lower eukaryotes, chronic fasting extends longevityin part by reprogramming metabolic and stress resistance pathways. In rodents intermittent orperiodic fasting protects against diabetes, cancers, heart disease and neurodegeneration, while inhumans it helps reduce obesity, hypertension, asthma and rheumatoid arthritis. Thus, fasting hasthe potential to delay aging and help prevent and treat diseases while minimizing the side effectscaused by chronic dietary interventions.

Introduction

In humans, fasting is achieved by ingesting no or minimal amounts of food and caloricbeverages for periods that typically range from 12 hours to three weeks. Many religiousgroups incorporate periods of fasting into their rituals including Muslims who fast fromdawn until dusk during the month of Ramadan, and Christians, Jews, Buddhists and Hinduswho traditionally fast on designated days of the week or calendar year. In many clinics,patients are now monitored by physicians while undergoing water only or very low calorie(less than 200 kcal/day) fasting periods lasting from 1 week or longer for weightmanagement, and for disease prevention and treatment. Fasting is distinct from caloricrestriction (CR) in which the daily caloric intake is reduced chronically by 20–40%, butmeal frequency is maintained. Starvation is instead a chronic nutritional insufficiency that iscommonly used as a substitute for the word fasting, particularly in lower eukaryotes, but thatis also used to define extreme forms of fasting, which can result in degeneration and death.We now know that fasting results in ketogenesis, promotes potent changes in metabolicpathways and cellular processes such as stress resistance, lipolysis and autophagy, and canhave medical applications that in some cases are as effective as those of approved drugssuch as the dampening of seizures and seizure-associated brain damage and the ameliorationof rheumatoid arthritis (Bruce-Keller et al., 1999; Hartman et al., 2012; Muller et al., 2001).As detailed in the remainder of this article, findings from well-controlled investigations inexperimental animals, and emerging findings from human studies, indicate that different forms of fasting may provide effective strategies to reduce weight, delay aging, and optimizehealth. Here we review the fascinating and potent effects of different forms of fastingincluding intermittent fasting (IF, including alternate day fasting, or twice weekly fasting,for example) and periodic fasting (PF) lasting several days or longer every 2 or more weeks.We focus on fasting and minimize the discussion of CR, a topic reviewed elsewhere(Fontana et al., 2010; Masoro, 2005).

Lessons from simple organisms

The remarkable effects of the typical 20–40% CR on aging and diseases in mice and rats areoften viewed as responses evolved in mammals to adapt to periods of limited availability offood (Fontana and Klein, 2007; Fontana et al., 2010; Masoro, 2005; Weindruch andWalford, 1988). However, the cellular and molecular mechanisms responsible for theprotective effects of CR have likely evolved billions of years earlier in prokaryotesattempting to survive in an environment largely or completely devoid of energy sourceswhile avoiding age-dependent damage that could compromise fitness. In fact, E. coliswitched from a nutrient rich broth to a calorie-free medium survive 4 times longer, aneffect reversed by the addition of various nutrients but not acetate, a carbon sourceassociated with starvation conditions (Figure 1A) (Gonidakis et al., 2010). The effect of richmedium but not acetate in reducing longevity raises the possibility that a ketone body-likecarbon source such as acetate may be part of an “alternate metabolic program” that evolvedbillions of years ago in microorganisms and that now allows mammals to survive duringperiods of food deprivation by obtaining much of the energy by catabolizing fatty acids andketone bodies including acetoacetate and β-hydroxybutyrate (Cahill, 2006).

In the yeast S. cerevisiae, switching cells from standard growth medium to water also causesa consistent 2-fold chronological lifespan extension as well as a major increase in theresistance to multiple stresses (Figure 1B) (Longo et al., 1997; Longo et al., 2012). Themechanisms of food deprivation-dependent lifespan extension involve the down-regulationof the amino acid response Tor-S6K (Sch9) pathway as well as of the glucose responsiveRas-adenylate cyclase-PKA pathway resulting in the activation of the serine/threoninekinase Rim15, a key enzyme coordinating the protective responses (Fontana et al., 2010).The inactivation of Tor-S6K, Ras-AC-PKA and activation of Rim15 result in increasedtranscription of genes including superoxide dismutases and heat shock proteins controlled bystress responsive transcription factors Msn2, Msn4 and Gis1, required for the majority of theprotective effects caused by food deprivation (Wei et al., 2008). Notably, when switched tofood deprivation conditions, both bacteria and yeast enter a hypometabolic mode that allowsthem to minimize the use of reserve carbon sources and can also accumulate high levels ofthe ketone body-like acetic acid, analogously to mammals.

Another major model organism in which fasting extends lifespan is the nematode C.elegans. Food deprivation conditions achieved by feeding worms little or no bacteria, lead toa major increase in lifespan (Figure 1C) (Kaeberlein et al., 2006; Lee et al., 2006), whichrequires AMPK as well as the stress resistance transcription factor DAF-16, similarly to therole of transcription factors Msn2/4 and Gis1 in yeast and FOXOs in flies and mammals(Greer et al., 2007). Intermittent food deprivation also extends lifespan in C. elegans by amechanism involving the small GTPase RHEB-1 (Honjoh et al., 2009).

In flies, most studies indicate that intermittent food deprivation does not affect lifespan(Grandison et al., 2009). However, food reduction or food dilution have been consistentlyshown to extend Drosophila longevity (Piper and Partridge, 2007) suggesting that flies canbenefit from dietary restriction but may be sensitive to even short starvation periods.

Together these results indicate that food deprivation can result in pro-longevity effects in awide variety of organisms, but also underline that different organisms have differentresponses to fasting.

Adaptive responses to fasting in mammals

In most mammals, the liver serves as the main reservoir of glucose, which is stored in theform of glycogen. In humans, depending upon their level of physical activity, 12 to 24 hoursof fasting typically results in a 20% or greater decrease in serum glucose and depletion ofthe hepatic glycogen, accompanied by a switch to a metabolic mode in which non-hepaticglucose, fat-derived ketone bodies and free fatty acids are used as energy sources (Figures 2and 3). Whereas most tissues can utilize fatty acids for energy, during prolonged periods offasting, the brain relies on the ketone bodies β-hydroxybutyrate and acetoacetate in additionto glucose for energy consumption (Figure 3B). Ketone bodies are produced in hepatocytesfrom the acetyl-CoA generated from β oxidation of fatty acids released into the bloodstreamby adipocytes, and also by the conversion of ketogenic amino acids. After hepatic glycogendepletion, ketone bodies, fat-derived glycerol, and amino acids account for thegluconeogenesis-dependent generation of approximately 80 grams/day of glucose, which ismostly utilized by the brain. Depending on body weight and composition, the ketone bodies,free fatty acids and gluconeogenesis allow the majority of human beings to survive 30 ormore days in the absence of any food and allow certain species, such as king penguins, tosurvive for over 5 months without food (Eichhorn et al., 2011) (Figure 3C). In humans,during prolonged fasting, the plasma levels of 3-β-hydroxybutyrate are about 5 times thoseof free fatty acids and acetoacetic acid (Figure 3A and 3B). The brain and other organsutilize ketone bodies in a process termed ketolysis, in which acetoacetic acid and 3-β-hydroxybutyrate are converted into acetoacetyl-CoA and then acetyl-CoA. These metabolicadaptations to fasting in mammals are reminiscent of those described earlier for E. coli andyeast, in which acetic acid accumulates in response to food deprivation (Gonidakis et al.,2010; Longo et al., 2012). In yeast, glucose, acetic acid and ethanol, but not glycerol whichis also generated during fasting from the breakdown of fats, accelerate aging (Fabrizio et al.,2005; Wei et al., 2009). Thus, glycerol functions as a carbon source that does not activatethe pro-aging nutrient signaling pathways but can be catabolized by cells. It will beimportant to understand how the different carbon sources generated during fasting affectcellular protection and aging. and to determine whether glycerol, specific ketone bodies orfatty acids can provide nourishment while reducing cellular aging in mammals, a possibilitysuggested by beneficial effects of a dietary ketone precursor in a mouse model ofAlzheimer’s disease (Kashiwaya et al., 2012). It will also be important to study, in variousmodel organisms and humans, how high intake of specific types of fats (medium- vs. long-chain fatty acids, etc.) in substitution of carbohydrates and proteins influencesgluconeogenesis and glucose levels as well as aging and diseases.

Fasting and the brain

In mammals, severe CR/food deprivation results in a decrease in the size of most organsexcept the brain, and the testicles in male mice (Weindruch and Sohal, 1997). From anevolutionary perspective this implies that maintenance of a high level of cognitive functionunder conditions of food scarcity is of preeminent importance. Indeed, a highly conservedbehavioral trait of all mammals is to be active when hungry and sedentary when satiated. Inrodents, alternating days of normal feeding and fasting (IF) can enhance brain function asindicated by improvements in performance on behavioral tests of sensory and motorfunction (Singh et al., 2012) and learning and memory (Fontan-Lozano et al., 2007). Thebehavioral responses to IF are associated with increased synaptic plasticity and increasedproduction of new neurons from neural stem cells (Lee et al., 2002).

Particularly interesting with regards to adaptive responses of the brain to limited foodavailability during human evolution is brain-derived neurotrophic factor (BDNF). The genesencoding BDNF and its receptor TrkB appeared in genomes relatively recently as they arepresent in vertebrates, but absent from worms, flies and lower species (Chao, 2000). Theprominent roles of BDNF in the regulation of energy intake and expenditure in mammals ishighlighted by the fact that the receptors for both BDNF and insulin are coupled to thehighly conserved PI3 kinase – Akt, and MAP kinase signaling pathways (Figure 4). Studiesof rats and mice have shown that running wheel exercise and IF increase BDNF expressionin several regions of the brain, and that BDNF in part mediates exercise- and IF-inducedenhancement of synaptic plasticity, neurogenesis and neuronal resistance to injury anddisease (see sections on fasting and neurodegeneration below). BDNF signaling in the brainmay also mediate behavioral and metabolic responses to fasting and exercise includingregulation of appetite, activity levels, peripheral glucose metabolism and autonomic controlof the cardiovascular and gastrointestinal systems (Mattson, 2012a, b; Rothman et al., 2012).

Hunger is an adaptive response to food deprivation that involves sensory, cognitive andneuroendocrine changes which motivate and enable food seeking behaviors. It has beenproposed that hunger-related neuronal networks, neuropeptides and hormones play pivotalroles in the beneficial effects of energy restriction on aging and disease susceptibility. Asevidence, when mice in which the hypothalamic ‘hunger peptide’ NPY is selectively ablatedare maintained on a CR diet, the ability of CR to suppress tumor growth is abolished (Shi etal., 2012). The latter study further showed that the ability of CR to elevate circulatingadiponectin levels was also compromised in NPY-deficient mice, suggesting a key role forthe central hunger response in peripheral endocrine adaptations to energy restriction.Adiponectin levels increase dramatically in response to fasting; and data suggest roles foradiponectin in the beneficial effects of IF on the cardiovascular system (Wan et al., 2010).The hunger response may also improve immune function during aging as ghrelin-deficientmice exhibit accelerated thymic involution during aging, and treatment of middle age micewith ghrelin increases thymocyte numbers and improves the functional diversity ofperipheral T cell subsets (Peng et al., 2012). In addition to its actions on the hypothalamusand peripheral endocrine cells, fasting may increase neuronal network activity in brainregions involved in cognition, resulting in the production of BDNF, enhanced synapticplasticity and improved stress tolerance (Rothman et al., 2012). Thus, hunger may be acritical factor involved in widespread central and peripheral adaptive responses to thechallenge of food deprivation for extended time periods.

Fasting, aging, and disease in rodent models

Different fasting methods and aging

The major differences between IF and PF in mice are the length and the frequency of the fastcycles. IF cycles usually last 24 hours and are one to a few days apart, whereas PF cycleslast 2 or more days and are at least 1 week apart, which is necessary for mice to regain theirnormal weight. One difference in the molecular changes caused by different fasting regimesis the effect on a variety of growth factors and metabolic markers, with IF causing morefrequent but less pronounced changes than PF. It will be important to determine how thefrequency of specific changes such as the lowering of IGF-1 and glucose affect cellularprotection, diseases and longevity. The most extensively investigated IF method in animalstudies of aging has been alternate day fasting (food is withdrawn for 24 hours on alternatedays, with water provided ad libitum) (Varady and Hellerstein, 2007). The magnitude of theeffects of alternate day fasting on longevity in rodents depends upon the species and age atregimen initiation, and can range from a negative effect to as much as an 80% lifespanextension (Arum et al., 2009; Goodrick et al., 1990). IF every other day extended thelifespan of rats more than fasting every 3rd or 4th day (Carlson and Hoelzel, 1946). Fasting for 24 hours twice weekly throughout adult life resulted in a significant increase in lifespanof black-hooded rats (Kendrick, 1973). In rats, the combination of alternate day fasting andtreadmill exercise resulted in greater maintenance of muscle mass than did IF or exercisealone (Sakamoto and Grunewald, 1987). Interestingly, when rats were maintained for 10weeks on a PF diet in which they fasted 3 consecutive days each week, they were less proneto hypoglycemia during 2 hours of strenuous swimming exercise as a result of theiraccumulation of larger intramuscular stores of glycogen and triglycerides (Favier and Koubi,1988). Several major physiological responses to fasting are similar to those caused byregular aerobic exercise including increased insulin sensitivity and cellular stress resistance,reduced resting blood pressure and heart rate, and increased heart rate variability as a resultof increased parasympathetic tone (Figure 2) (Anson et al., 2003; Mager et al., 2006; Wan etal., 2003). Emerging findings suggest that exercise and IF retard aging and some age-relateddiseases by shared mechanisms involving improved cellular stress adaptation (Stranahan andMattson, 2012). However, in two different mouse genetic backgrounds, IF did not extendmean lifespan and even reduced lifespan when initiated at 10 months (Goodrick et al.,1990). When initiated at 1.5 months, IF either increased longevity or had no effect (Figure1D) (Goodrick et al., 1990). These results in rodents point to conserved effects of fasting onlifespan, but also to the need for a much better understanding of the type of fasting that canmaximize its longevity effects and the mechanisms responsible for the detrimental effectsthat may be counterbalancing its anti-aging effects. For example, one possibility is thatfasting may be consistently protective in young and middle aged laboratory rodents that areeither gaining or maintaining a body weight, but may be detrimental in older animals that,similarly to humans, begin to lose weight prior to their death. Notably, whereas bacteria,yeast and humans can survive for several weeks or more without nutrients, most strains ofmice are unable to survive more than 3 days without food. The age-dependent weight lossmay make this sensitivity to long periods of fasting worse.

Fasting and cancer

Fasting can have positive effects in cancer prevention and treatment. In mice, alternate dayfasting caused a major reduction in the incidence of lymphomas (Descamps et al., 2005) andfasting for 1 day per week delayed spontaneous tumorigenesis in p53-deficient mice(Berrigan et al., 2002). However, the major decrease in glucose, insulin and IGF-1 caused byfasting, which is accompanied by cell death and/or atrophy in a wide range of tissues andorgans including the liver and kidneys, is followed by a period of abnormally high cellularproliferation in these tissues driven in part by the replenishment of growth factors duringrefeeding. When combined with carcinogens during refeeding, this increased proliferativeactivity can actually increase carcinogenesis and/or pre-cancerous lesions in tissuesincluding liver and colon (Tessitore et al., 1996). Although these studies underline the needfor an in depth understanding of its mechanisms of action, fasting is expected to have cancerpreventive effects as indicated by the studies above and by the findings that multiple cyclesof periodic fasting can be as effective as toxic chemotherapy in the treatment of somecancers in mice (Lee et al., 2012).

In the treatment of cancer, fasting has been shown to have more consistent and positiveeffects. PF for 2–3 days was shown to protect mice from a variety of chemotherapy drugs,an effect called differential stress resistance (DSR) to reflect the inability of cancer cells tobecome protected based on the role of oncogenes in negatively regulating stress resistance,thus rendering cancer cells, by definition, unable to become protected in response to fastingconditions (Figure 5) (Raffaghello et al., 2008). PF also causes a major sensitization ofvarious cancer cells to chemo-treatment, since it fosters an extreme environment incombination with the stress conditions caused by chemotherapy. In contrast to the protectedstate entered by normal cells during fasting, cancer cells are unable to adapt, a phenomenon called differential stress sensitization (DSS), based on the notion that most mutations aredeleterious and that the many mutations accumulated in cancer cells promote growth understandard conditions but render them much less effective in adapting to extremeenvironments (Lee et al., 2012). In mouse models of metastatic tumors, combinations offasting and chemotherapy that cause DSR and DSS, result in 20 to 60% cancer-free survivalcompared to the same levels of chemotherapy or fasting alone, which are not sufficient tocause any cancer-free survival (Lee et al., 2012; Shi et al., 2012). Thus, the idea that cancercould be treated with weeks of fasting alone, made popular decades ago, may be onlypartially true, at least for some type of cancers, but is expected to be ineffective for othertypes of cancers. The efficacy of long-term fasting alone (2 weeks or longer) in cancertreatment will need to be tested in carefully designed clinical trials in which side effectsincluding malnourishment and possibly a weakened immune system and increasedsusceptibility to certain infections are carefully monitored. By contrast, animal data frommultiple laboratories indicate that the combination of fasting cycles with chemotherapy ishighly and consistently effective in enhancing chemotherapeutic index and has hightranslation potential. A number of ongoing trials should soon begin to determine the efficacyof fasting in enhancing cancer treatment in the clinic.

Fasting and neurodegeneration

Compared to ad libitum-fed controls, rats and mice maintained on an IF diet exhibit lessneuronal dysfunction and degeneration, and fewer clinical symptoms in models ofAlzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease (HD). Thesemodels include transgenic mice expressing mutant human genes that cause dominantlyinherited AD (amyloid precursor protein and presenilin-1) and frontotemporal lobe dementia(Tau) (Halagappa et al., 2007), PD (α-synuclein) (Griffioen et al., 2012) and HD(huntingtin) (Duan et al., 2003), as well as neurotoxin-based models pertinent to AD, PDand HD (Bruce-Keller et al., 1999; Duan and Mattson, 1999). Animals on an IF diet alsofare better than ad libitum-fed controls after acute injury including severe epileptic seizures,stroke, and traumatic brain and spinal cord injuries (Arumugam et al., 2010; Bruce-Keller etal., 1999; Plunet et al., 2008).

Several interrelated cellular mechanisms contribute to the beneficial effects of IF on thenervous system including reduced accumulation of oxidatively damaged molecules,improved cellular bioenergetics, enhanced neurotrophic factor signaling, and reducedinflammation (Mattson, 2012a). The latter neuroprotective mechanisms are supported bystudies showing that IF diets boost levels of antioxidant defenses, neurotrophic factors(BDNF and FGF2) and protein chaperones (HSP-70 and GRP-78), and reduce levels of pro-inflammatory cytokines (TNFα, IL-1β and IL-6) (Figure 4) (Arumugam et al., 2010). IF mayalso promote restoration of damaged nerve cell circuits by stimulating synapse formationand the production of new neurons from neural stem cells (neurogenesis) (Lee et al., 2002).Interestingly, while beneficial in models of most neurodegenerative conditions, there isevidence that fasting can hasten neurodegeneration in some models of inherited amyotrophiclateral sclerosis, perhaps because the motor neurons affected in those models are unable torespond adaptively to the moderate stress imposed by fasting (Mattson et al., 2007; Pedersenand Mattson, 1999).

Fasting and the metabolic syndrome

Metabolic syndrome (MS), defined as abdominal adiposity, combined with insulinresistance, elevated triglycerides and/or hypertension, greatly increases the risk ofcardiovascular disease, diabetes, stroke and AD. Rats and mice maintained under the usualad libitum feeding condition develop an MS-like phenotype as they age. MS can also beinduced in younger animals by feeding them a diet high in fat and simple sugars (Martin etal., 2010). IF can prevent and reverse all aspects of the MS in rodents: abdominal fat,inflammation and blood pressure are reduced, insulin sensitivity is increased, and thefunctional capacities of the nervous, neuromuscular and cardiovascular systems areimproved (Castello et al., 2010; Wan et al., 2003). Hyperglycemia is ameliorated by IF inrodent models of diabetes (Pedersen et al., 1999) and the heart is protected against ischemicinjury in myocardial infarction models (Ahmet et al., 2005). A protective effect of fastingagainst ischemic renal and liver injury occurs rapidly, with 1 – 3 days of fasting improvingfunctional outcome and reducing tissue injury and mortality (Mitchell et al., 2010). Six dayson a diet missing just a single essential amino acid such as tryptophan can also elicit changesin metabolism and stress resistance, similar to those caused by fasting, which are dependenton the amino acid sensing kinase Gcn2 (Peng et al., 2012).

Multiple hormonal changes that typify MS in humans a re observed in rodents maintainedon high fat and sugar diets including elevated levels of insulin and leptin and reduced levelsof adiponectin and ghrelin. Elevated leptin levels are typically reflective of a pro-inflammatory state, whereas adiponectin and ghrelin can suppress inflammation and increaseinsulin sensitivity (Baatar et al., 2011; Yamauchi et al., 2001). Local inflammation inhypothalamic nuclei that control energy intake and expenditure may contribute to asustained positive energy balance in MS (Milanski et al., 2012). Fasting results in a loweringof insulin and leptin levels and an elevation of adiponectin and ghrelin levels. By increasinginsulin and leptin sensitivity, suppressing inflammation and stimulating autophagy, fastingreverses all the major abnormalities of the MS in rodents (Singh et al., 2009; Wan et al.,2010). Finally, in addition to its many effects on cells throughout the body and brain, IF mayelicit changes in the gut microbiota that protect against MS (Tremaroli and Backhed, 2012).Naturally, the challenge of applying fasting-based interventions to treat MS in humans is amajor one, as some obese individuals may have difficulties in following IF for long periods.

Fasting, aging, and disease in humans

Fasting and factors implicated in aging

Clinical and epidemiological data are consistent wit h an ability of fasting to retard the agingprocess and associated diseases. Major factors implicated in aging whose generation areaccelerated by gluttonous lifestyles and slowed by energy restriction in humans include: 1)oxidative damage to proteins, DNA and lipids; 2) inflammation; 3) accumulation ofdysfunctional proteins and organelles; and 4) elevated glucose, insulin and IGF-I, althoughIGF-1decreases with aging and its severe deficiency can be associated with certainpathologies (Bishop et al., 2010; Fontana and Klein, 2007). Serum markers of oxidativedamage and inflammation as well as clinical symptoms are reduced over a period of 2–4weeks in asthma patients maintained on an alternate day fasting diet (Johnson et al., 2007).Similarly, when on a 2 days/week fasting diet overweight women at risk for breast cancerexhibited reduced oxidative stress and inflammation (Harvie et al., 2011) and elderly menexhibited reductions in body weight and body fat, and improved mood (Teng et al., 2011).Additional effects of fasting in human cells that can be considered as potentially ‘anti-aging’are inhibition the mTOR pathway, stimulation of autophagy and ketogenesis (Harvie et al.,2011; Sengupta et al., 2010).

Among the major effects of fasting relevant to aging and diseases are changes in the levelsof IGF-1, IGFBP1, glucose, and insulin. Fasting for 3 or more days causes a 30% or moredecrease in circulating insulin and glucose, as well as rapid decline in the levels of insulin-like growth factor 1 (IGF-1), the major growth factor in mammals, which together withinsulin is associated with accelerated aging and cancer (Fontana et al., 2010). In humans,five days of fasting causes an over 60% decrease in IGF-1and a 5-fold or higher increase inone of the principal IGF-1-inhibiting proteins: IGFBP1 (Thissen et al., 1994a). This effect of fasting on IGF-1is mostly due to protein restriction, and particularly to the restriction ofessential amino acids, but is also supported by calorie restriction since the decrease ininsulin levels during fasting promotes reduction in IGF-1(Thissen et al., 1994a). Notably, inhumans, chronic calorie restriction does not lead to a decrease in IGF-1unless combinedwith protein restriction (Fontana et al., 2008).

IF can be achieved in with a minimal decrease in overall calorie intake if the refeedingperiod in which subjects overeat is considered. Thus, fasting cycles provide a much morefeasible strategy to achieve the beneficial effects of CR, and possibly stronger effects,without the burden of chronic underfeeding and some of the potentially adverse effectsassociated with weight loss or very low BMIs. In fact, subjects who are moderatelyoverweight (BMI of 25–30) in later life can have reduced overall mortality risk compared tosubjects of normal weight (Flegal et al., 2013). Although these results may be affected bythe presence of many existing or developing pathologies in the low weight control group,they underline the necessity to differentiate between young individuals and elderlyindividuals who may use CR or fasting to reduce weight or delay aging. Although extremedietary interventions during old age may continue to protect from age-related diseases, theycould have detrimental effects on the immune system and the ability to respond to certaininfectious diseases, wounds and other challenges (Kristan, 2008; Reed et al., 1996).However, IF or PF designed to avoid weight loss and maximize nourishment have thepotential to have beneficial effects on infectious diseases, wounds and other insults even inthe very old. Nourishment of subjects can be achieved by complementing IF or PF withmicro- and macro Studies to test the effect of IF or PF regimens on markers of aging, cancer,cognition and obesity are in progress (V. Longo and M. Mattson).

Fasting and cancer

Fasting has the potential for applications in both cancer prevention and treatment. Althoughno human data are available on the effect of IF or PF in cancer prevention, their effect onreducing IGF-1, insulin and glucose levels, and increasing IGFBP1 and ketone body levelscould generate a protective environment that reduces DNA damage and carcinogenesis,while at the same time creating hostile conditions for tumor and pre-cancerous cells (Figure5). In fact, elevated circulating IGF-1 is associated with increased risk of developing certaincancers (Chan et al., 2000; Giovannucci et al., 2000) and individuals with severeIGF-1deficiency caused by growth hormone receptor deficiency, rarely develop cancer(Guevara-Aguirre et al., 2011; Shevah and Laron, 2007; Steuerman et al., 2011).Furthermore, the serum from these IGF-1deficient subjects protected human epithelial cellsfrom oxidative stress-induced DNA damage. Furthermore, once their DNA becamedamaged, cells were more likely to undergo programmed cell death (Guevara-Aguirre et al.,2011). Thus, fasting may protect from cancer by reducing cellular and DNA damage butalso by enhancing the death of pre-cancerous cells.

fasting on IGF-1is mostly due to protein restriction, and particularly to the restriction ofessential amino acids, but is also supported by calorie restriction since the decrease ininsulin levels during fasting promotes reduction in IGF-1(Thissen et al., 1994a). Notably, inhumans, chronic calorie restriction does not lead to a decrease in IGF-1unless combinedwith protein restriction (Fontana et al., 2008).IF can be achieved in with a minimal decrease in overall calorie intake if the refeedingperiod in which subjects overeat is considered. Thus, fasting cycles provide a much morefeasible strategy to achieve the beneficial effects of CR, and possibly stronger effects,without the burden of chronic underfeeding and some of the potentially adverse effectsassociated with weight loss or very low BMIs. In fact, subjects who are moderatelyoverweight (BMI of 25–30) in later life can have reduced overall mortality risk compared tosubjects of normal weight (Flegal et al., 2013). Although these results may be affected bythe presence of many existing or developing pathologies in the low weight control group,they underline the necessity to differentiate between young individuals and elderlyindividuals who may use CR or fasting to reduce weight or delay aging. Although extremedietary interventions during old age may continue to protect from age-related diseases, theycould have detrimental effects on the immune system and the ability to respond to certaininfectious diseases, wounds and other challenges (Kristan, 2008; Reed et al., 1996).However, IF or PF designed to avoid weight loss and maximize nourishment have thepotential to have beneficial effects on infectious diseases, wounds and other insults even inthe very old. Nourishment of subjects can be achieved by complementing IF or PF withmicro- and macro Studies to test the effect of IF or PF regimens on markers of aging, cancer,cognition and obesity are in progress (V. Longo and M. Mattson).Fasting and cancerFasting has the potential for applications in both cancer prevention and treatment. Althoughno human data are available on the effect of IF or PF in cancer prevention, their effect onreducing IGF-1, insulin and glucose levels, and increasing IGFBP1 and ketone body levelscould generate a protective environment that reduces DNA damage and carcinogenesis,while at the same time creating hostile conditions for tumor and pre-cancerous cells (Figure5). In fact, elevated circulating IGF-1 is associated with increased risk of developing certaincancers (Chan et al., 2000; Giovannucci et al., 2000) and individuals with severeIGF-1deficiency caused by growth hormone receptor deficiency, rarely develop cancer(Guevara-Aguirre et al., 2011; Shevah and Laron, 2007; Steuerman et al., 2011).Furthermore, the serum from these IGF-1deficient subjects protected human epithelial cellsfrom oxidative stress-induced DNA damage. Furthermore, once their DNA becamedamaged, cells were more likely to undergo programmed cell death (Guevara-Aguirre et al.,2011). Thus, fasting may protect from cancer by reducing cellular and DNA damage butalso by enhancing the death of pre-cancerous cells.In a preliminary study of 10 subjects with a variety of malignancies, the combination ofchemotherapy with fasting resulted in a decrease in a range of self-reported common sideeffects caused by chemotherapy compared to the same subjects receiving chemotherapywhile on a standard diet (Safdie et al., 2009). The effect of fasting on chemotherapy toxicityand cancer progression is now being tested in clinical trials in both Europe and the US(0S-08-9, 0S-10-3).

Fasting and neurodegeneration

Our current understanding of the impact of IF on the nervous system and cognitive functionsis largely inferred from animal studies (see above). Interventional studies to determine theimpact of fasting on brain function and neurodegenerative disease processes are lacking After 3–4 month, CR improved cognitive function (verbal memory) in overweight women(Kretsch et al., 1997) and in elderly subjects (Witte et al., 2009). Similarly, when subjectswith mild cognitive impairment were maintained for 1 month on a low glycemic diet, theyexhibited improved delayed visual memory, cerebrospinal fluid biomarkers of Aβmetabolism and brain bioenergetics (Bayer-Carter et al., 2011). Studies in which cognitivefunction, regional brain volumes, neural network activity, and biochemical analyses ofcerebrospinal fluid are measured in human subjects before and during an extended period ofIF should clarify the impact of IF on human brain structure and function.

Fasting, inflammation and hypertension

In humans, one of the best demonstrations of the beneficial effects of long-term fastinglasting one to 3 weeks is in the treatment of rheumatoid arthritis (RA). In agreement with theresults in rodents, there is little doubt that during the period of fasting both inflammation andpain are reduced in RA patients (Muller et al., 2001). However, after the normal diet isresumed, inflammation returns unless the fasting period is followed by a vegetarian diet(Kjeldsen-Kragh et al., 1991), a combination therapy that has beneficial effects lasting fortwo years or longer (Kjeldsen-Kragh et al., 1994). The validity of this approach is supportedby four differently controlled studies, including two randomized trials (Muller et al., 2001).Therefore, fasting combined with a vegetarian diet and possibly with other modified dietsprovides beneficial effects in the treatment of RA. Alternate day IF also resulted insignificant reductions in serum TNFα and ceramides in asthma patients during a 2 monthperiod (Johnson et al., 2007). The latter study further showed that markers of oxidativestress often associated with inflammation (protein and lipid oxidation) were significantlyreduced in response to IF. Thus, for many patients able and willing to endure long-termfasting and to permanently modify their diet, fasting cycles would have the potential to notonly augment but also replace existing medical treatments.

Water only and other forms of long-term fasting have also been documented to have potenteffects on hypertension. An average of 13 days of water only fasting resulted in theachievement of a systolic blood pressure (BP) below 120 in 82% of subjects with borderlinehypertension with a mean 20 mm Hg reduction in BP (Goldhamer et al., 2002). BP remainedsignificantly lower compared to baseline even after subjects resumed the normal diet for anaverage of 6 days (Goldhamer et al., 2002). A small pilot study of patients with hypertension(140 mm and above systolic BP) also showed that 10–11 days of fasting caused a 37–60 mmdecrease in systolic BP (Goldhamer et al., 2001). These preliminary studies are promisingbut underscore the need for larger controlled and randomized clinical studies that focus onperiodic fasting strategies that are feasible for a larger portion of the population.

For both hypertension and RA it will be important to develop PF mimicking diets that are aseffective as the fasting regimens described above but that are also tolerable by the greatmajority of patients.

Fasting and the metabolic syndrome

Periodic fasting can reverse multiple features of the metabolic syndrome in humans: itenhances insulin sensitivity, stimulates lipolysis and reduces blood pressure. Body fat andblood pressure were reduced and glucose metabolism improved in obese subjects inresponse to an alternate day modified fast (Klempel et al., 2013; Varady et al., 2009).Overweight subjects maintained for 6 months on a twice weekly IF diet in which theyconsumed only 500–600 calories on the fasting days, lost abdominal fat, displayed improvedinsulin sensitivity and reduced blood pressure (Harvie et al., 2011). Three weeks of alternateday fasting resulted in reductions in body fat and insulin levels in normal weight men andwomen (Heilbronn et al., 2005) and Ramadan fasting (2 meals/day separated by approximately 12 hours) in subjects with MS resulted in decreased daily energy intake,decreased plasma glucose levels and increased insulin sensitivity (Shariatpanahi et al.,2008). Subjects undergoing coronary angiography who reported that they fasted regularlyexhibited a lower prevalence of diabetes compared to non-fasters (Horne et al., 2012). Anti-metabolic syndrome effects of IF were also observed in healthy young men (BMI of 25)after 15 days of alternate day fasting: their whole-body glucose uptake rates increasedsignificantly, levels of plasma ketone bodies and adiponectin were elevated, all of whichoccurred without a significant decrease in body weight (Halberg et al., 2005). The latterfindings are similar to data from animal studies showing that IF can improve glucosemetabolism even with little or no weight change (Anson et al., 2003). It will be important todetermine if longer fasting periods which promote a robust switch to a fat breakdown andketone body-based metabolism, can cause longer lasting and more potent effects.

Conclusions and Recommendations

Based on the existing evidence from animal and human studies described, we conclude thatthere is great potential for lifestyles that incorporate periodic fasting during adult life topromote optimal health and reduce the risk of many chronic diseases, particularly for thosewho are overweight and sedentary. Animal studies have documented robust and replicableeffects of fasting on health indicators including greater insulin sensitivity, and reduced levelsof blood pressure, body fat, IGF-I, insulin, glucose, atherogenic lipids and inflammation.Fasting regimens can ameliorate disease processes and improve functional outcome inanimal models of disorders that include myocardial infarction, diabetes, stroke, AD and PD.One general mechanism of action of fasting is that it triggers adaptive cellular stressresponses, which result in an enhanced ability to cope with more severe stress andcounteract disease processes. In addition, by protecting cells from DNA damage,suppressing cell growth and enhancing apoptosis of damaged cells, fasting could retard and/or prevent the formation and growth of cancers.

However, studies of fasting regimens have not been performed in children, the very old andunderweight individuals, and it is possible that IF and PF would be harmful to thesepopulations. Fasting periods lasting longer than 24 hours and particularly those lasting 3 ormore days should be done under the supervision of a physician and preferably in a clinic. IF-and PF-based approaches towards combating the current epidemics of overweight, diabetesand related diseases should be pursued in human research studies and medical treatmentplans. Several variations of potential ‘fasting prescriptions’ that have been adopted foroverweight subjects revolve around the common theme of abstaining from food and caloricbeverages for at least 12 – 24 hours on one or more days each week or month, depending onthe length, combined with regular exercise. For those who are overweight, physicians couldask their patients to choose a fasting-based intervention that they believe they could complywith based upon their daily and weekly schedules. Examples include the ‘5:2’ IF diet(Harvie et al., 2011), the alternate day modified fasting diet (Johnson et al., 2007; Varady etal., 2009), a 4–5 day fast or low calorie but high nourishment fasting mimicking diets onceevery 1–3 months followed by the skipping of one major meal every day if needed (V.Longo, clinical trial in progress). One of the concerns with unbalanced alternating diets suchas those in which low calorie intake is only observed for 2 days a week are the potentialeffects on circadian rhythm and the endocrine and gastrointestinal systems, which are knownto be influenced by eating habits. During the first 4 – 6 weeks of implementation of thefasting regimen, a physician or registered dietitian should be in regular contact with thepatient to monitor their progress and to provide advice and supervision.

Fasting regimens could also be tailored for specific diseases as stand-alone or adjuncttherapies. Results of initial trials of IF (fasting 2 days per week or every other day) in humansubjects suggest that there is a critical transition period of 3 – 6 weeks during which time the brain and body adapt to the new eating pattern and mood is enhanced (Harvie et al., 2011;Johnson et al., 2007). Though speculative, it is likely that during the latter transition periodbrain neurochemistry changes so that the ‘addiction’ to regular consumption of foodthroughout the day is overcome. Notably, the various fasting approaches are likely to havelimited efficacy particularly on aging and conditions other than obesity unless combinedwith diets such as the moderate calorie intake and mostly plant-based Mediterranean orOkinawa low protein diets (0.8 g protein/Kg of body weight), consistently associated withhealth and longevity.

In the future, it will be important to combine epidemiological data, studies of long-livedpopulations and their diets, results from model organisms connecting specific dietarycomponents to pro-aging and pro-disease factors, with data from studies on fasting regimensin humans, to design large clinical studies that integrate fasting with diets recognized asprotective and enjoyable. A better understanding of the molecular mechanisms by whichfasting affects various cell types and organ systems should lead to the development of novelprophylactic and therapeutic interventions for a wide range of disorders.

Acknowledgments

We thank Min Wei for all the assistance with the preparation of the manuscript. We thank Yvon Le Maho forproviding valuable information about fasting and a picture of penguins. We thank Matt Kaeberlein and MatthewPiper for panels for Figure 1. We thank William Mair for helpful discussions on fasting in Drosophila. This workwas supported, in part, by the Intramural Research Program of the National Institute on Aging, by the GlennFoundation for Medical Research, and by NIH/NIA grants AG20642, AG025135, and AG034906 to VDL.

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Figure 1. Fasting extends lifespans of yeast, worms and mice

A) lifespan of E. coli incubated in either LB medium or nutrient-free buffer (Gonidakis etal., 2010); B) lifespan of S. cerevisiae incubated in either medium or water (Wei et al.,2008); C) Lifespan of C. elegans in standard medium or in medium with a 90% reduction orcomplete removal of bacterial food (Kaeberlein et al., 2006); D) Lifespan of mal C57BL/6Jmice on alternating day fasting initiated at 1–2 month of age (Goodrick et al., 1990).

Figure 2. Pivotal roles of the nervous and endocrine systems as mediators of adaptive responsesof major organ systems to intermittent fasting

Figure 3. Fasting in mammals

A) Concentrations of ketone bodies (acetone, β-hydroxybutyric acid, acetoacetic acid) andplasma free fatty acids (FFA) during 40 days of fasting in humans. Note the more than threeorders of magnitude change in β-hydroxybutyrate and the doubling of FFA; B) Brainsubstrate utilization in three fasting obese volunteers after several weeks of food deprivation.Many studies suggest that human brain cells can survive with little to no glucose, but thishas not been clearly demonstrated (Redrawn from: (Cahill, 2006)). C) Emperor penguinscan fast for periods lasting for over 5 months. The picture shows Emperor penguins andtheir chicks a few weeks before fledging (courtesy of Yvone le Maho). The parents go backand forth between the open sea and their colony on sea ice, next to a glacier, which offersprotection against wind, to regurgitate food conserved in their stomach to feed their chickswhile they are themselves fasting. Fasting penguins undergo 3 phases (Le Maho et al., 1976;Le Maho et al., 1981; Robin et al., 1987). The first phase (phase I) represents a transitionbetween the fed state and starvation, during which the penguin stops utilizing diet-derivedenergy. This phase, which lasts between several hours and several days, is characterized by arapid decrease in protein loss. The following phase (phase II), is a ketotic phase associatedwith protein sparing which can last for several days in rats to several months in obese geese,king penguin chicks, bears, and seals (Adams and Costa, 1993; Atkinson and Ramsay, 1995;Castellini and Rea, 1992; Cherel et al., 1991; Cherel and Groscolas, 1999; Cherel and LeMaho, 1985; Cherel et al., 1988a; Cherel et al., 1988b; Fond et al., 2013; Reilly, 1991;Robin et al., 1987; Robin et al., 1988). Phase III is brief, since the high protein loss leads todeath. During phase III glucose and total plasma protein levels are reduced, and uric acidincreases while ketone bodies values remain low. Wild animals that fast for long periods areefficient at sparing proteins during long periods of fasting, with only 2–10% of total energycoming from proteins versus the 20–40% in species less adapted to fasting.

Figure 4. Neural circuits and cellular signaling pathways the mediate adaptive responses of thebrain to fasting

A) Neurons in the hippocampus play critical roles in learning and memory, and arevulnerable to dysfunction and degeneration in Alzheimer’s disease, stroke, traumatic braininjury and epilepsy. The dentate gyrus (yellow) contains neurons that receive inputs fromneurons in the entorhinal cortex (EC), with the latter brain region serving as a conduit forsensory information from higher cerebral cortical regions involved in responding to sensoryinputs and internally-generated cognitive processes. Increased activity in these neuronsoccurs in response to fasting resulting in the production of brain-derived neurotrophic factor(BDNF). BDNF promotes the growth and maintenance of dendrites and synapses, and alsoenhances the production and survival of new neurons from neural stem cells; the newly-generated neurons then integrate into the existing neural circuits; B) Signaling pathways bywhich glutamate, BDNF, insulin and glucagon-like peptide 1 (GLP-1) improve neuronalbioenergetics and protect the neurons against neurodegenerative disease and traumaticinjury. Glutamate activates AMPA and N-methyl-D-aspartate (NMDA) receptors resultingin Ca2+ influx and the activation of Ca2+/calmodulin-sensitive (CaM) kinases which, in turn,activate the transcription factors cyclic AMP response element-binding protein (CREB) andnuclear factor κB (NF-κB). Genes induced by the latter transcription factor include thoseencoding BDNF, the DNA repair enzyme APE1, the master regulator of mitochondrialbiogenesis PGC-1α, and the antioxidant enzyme manganese superoxide dismutase(MnSOD). BDNF and insulin bind their respective receptor tyrosine kinases (trkB and theinsulin receptor) resulting in the activation of the PI3 kinase and Akt kinase. BDNF alsostimulates mitogen-activated protein kinases (MAPK). Some of the gene targets of BDNFinclude PGC-1α, APE1, and the anti-apoptotic protein Bcl-2. Insulin activates themammalian target of rapamycin (mTOR) pathway to promote protein synthesis and cellgrowth. Finally, GLP-1 activates receptors (GLP-1R) coupled to cyclic AMP production,CREB activation and BDNF production.

Figure 5. Differential stress resistance and sensitization in aging, disease prevention and cancertreatment

A) In both mice and humans, fasting for 2 or 5 days, respectively causes an over 60%decrease in IGF-I, a 30% or more decrease in glucose and a 5–10 fold increase in theIGF-1binding protein and inhibitor IGFBP1 (Cahill, 2006; Lee et al., 2012; Raffaghello etal., 2008; Thissen et al., 1994a; Thissen et al., 1994b). These and other endocrinologicalalterations affect the expression of hundreds of genes in many cell types and the consequentreduction or halting of growth and elevation in stress resistance, which may be dependent inpart on FOXO and other stress resistance transcription factors. These periodically extremeconditions can promote changes, which are long-lasting and delay aging and diseaseindependently of calorie restriction, although the cellular mechanisms responsible for theseeffects remain poorly understood. In the presence of chemotherapy drugs, fasting canpromote the protection of normal but not cancer cells (differential stress resistance, DSR)since oncogenic pathways play central roles in inhibiting stress resistance and thereforecancer cells are unable to switch to the stress response mode; B) The extreme changescaused by fasting, and particularly the very low IGF-1and glucose levels and high IGFBP1also generate a tumor prevention environment which promotes cancer cell death sincetransformed cells have acquired a number of mutations which progressively decrease theirability to adapt to extreme environments (differential stress sensitization, DSS) (Guevara-Aguirre et al., 2011; Lee et al., 2012; Lee et al., 2010).