- Email: [email protected]
Alternate-Day Fasting Gets a Safe Bill of Health
Cell Metabolism
Previews availability during infections can mediate anti-virulence defenses, curbing pathogen virulence (Sanchez et al., 2018). In the CLP and influenza infection models, it will be interesting to determine how increased triglyceride availability affects bacterial and viral virulence. The important question of how triglyceride availability supports cardiac function during sepsis remains. Does increased triglyceride availability promote disease tolerance by supporting cardiac metabolism or by some other function? It remains to be determined if GDF15 mediates disease tolerance via activation of its receptor in the area postrema or some other neurons or cell types in the body. It will also be important to understand why lipid administration is only protective in settings where GDF15 is depleted and not sepsis in general.
motes host survival and transmission. Cell 168, 503–516.e12.
REFERENCES Hsu, J.Y., Crawley, S., Chen, M., Ayupova, D.A., Lindhout, D.A., Higbee, J., Kutach, A., Joo, W., Gao, Z., Fu, D., et al. (2017). Non-homeostatic body weight regulation through a brainstemrestricted receptor for GDF15. Nature 550, 255–259. Luan, H., Wang, A., Hilliard, B., Carvalho, F., Rosen, C., Ahasic, A., Herzog, E., Kang, I., Pisani, M., Yu, S., et al. (2019). GDF15 is an inflammation-induced central mediator of tissue tolerance. Cell 178, 1231–1244. Medzhitov, R., Schneider, D.S., and Soares, M.P. (2012). Disease tolerance as a defense strategy. Science 335, 936–941. Nestel, P.J. (1964). Plasma triglyceride concentration and plasma free fatty acid changes in response to norepinephrine in man. J. Clin. Invest. 43, 77–82. Rao, S., Schieber, A.M.P., O’Connor, C.P., Leblanc, M., Michel, D., and Ayres, J.S. (2017). Pathogen-mediated inhibition of anorexia pro-
Sanchez, K.K., Chen, G.Y., Schieber, A.M.P., Redford, S.E., Shokhirev, M.N., Leblanc, M., Lee, Y.M., and Ayres, J.S. (2018). Cooperative metabolic adaptations in the host can favor asymptomatic infection and select for attenuated virulence in an enteric pathogen. Cell 175, 146– 158.e15. Schieber, A.M., Lee, Y.M., Chang, M.W., Leblanc, M., Collins, B., Downes, M., Evans, R.M., and Ayres, J.S. (2015). Disease tolerance mediated by microbiome E. coli involves inflammasome and IGF-1 signaling. Science 350, 558–563. Schneider, D.S., and Ayres, J.S. (2008). Two ways to survive infection: what resistance and tolerance can teach us about treating infectious diseases. Nat. Rev. Immunol. 8, 889–895. Wang, A., Huen, S.C., Luan, H.H., Yu, S., Zhang, C., Gallezot, J.D., Booth, C.J., and Medzhitov, R. (2016). Opposing effects of fasting metabolism on tissue tolerance in bacterial and viral inflammation. Cell 166, 1512–1525.e12.
Alternate-Day Fasting Gets a Safe Bill of Health Leonie K. Heilbronn1,2,* and Satchidananda Panda3,* 1Faculty
of Health Sciences, The University of Adelaide, Adelaide, SA, Australia Health Theme, South Australian Health and Medical Research Institute, Adelaide, SA, Australia 3The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA *Correspondence: [email protected] (L.K.H.), [email protected] (S.P.) https://doi.org/10.1016/j.cmet.2019.08.006 2Lifelong
Various forms of fasting improve health and longevity in preclinical models. However, safety, outcomes, and the molecular changes underpinning human fasting are unclear. Stekovic et al. (2019) report improved markers of health for up to 6 months and associated metabolic changes among healthy adults who followed alternate-day fasting. Reducing daily caloric intake by 20%– 40% (a.k.a. caloric restriction or CR) in animals increases total and healthy lifespan—a feat rarely achieved by any drug. However, counting calories for CR in humans can be challenging, and chronic CR in rodents can dampen immune function and bone metabolism. Intermittent fasting (IF) is an alternative strategy involving 2–4 days per week when food intake is severely reduced or completely withheld, interspersed with ad libitum eating the remainder of the week. Mice subjected to alternate-day fasting (ADF)—a form of IF—double their caloric
intake on feast days, maintaining body weight close to their ad lib cohorts. Yet ADF mice enjoy pleiotropic health benefits found among CR mice who weigh much less (Mattson et al., 2014). This observation raised the possibility that prolonged fasting elicits a pro-longevity metabolic state. Thus, testing the feasibility of IF in humans and its health impacts, and assessing accompanying molecular changes are crucial to devise optimum nutrition to support long healthspan. Few human trials on IF have been conducted: they are mostly in obese individuals and are modified to allow consump-
tion of 500 kcals during the fasting day (Harvie et al., 2011, 2013; Trepanowski et al., 2017). Whether ADF imparts any benefit to healthy people and unbiased omics-based molecular changes in ADF have not been addressed. As ADF can involve fasting for up to 36 h, there was also concern that it might trigger excessive muscle loss among healthy cohorts. In a study published in this issue of Cell Metabolism (Stekovic et al., 2019), healthy individuals who had been under a selfimposed complete ADF for >6 months (long-term ADF) were matched to a comparator group who were naive to IF. This comparator group was then
Cell Metabolism 30, September 3, 2019 ª 2019 Elsevier Inc. 411
Cell Metabolism
Previews
Figure 1. Alternate-Day Fasting Is Safe for Normal Weight Healthy Adults Ad libitum eating every other day leads to both a prolonged period of fasting and an overall reduction in caloric intake. This results in several metabolic changes that correlate with healthy lifespan. ADF does not change the immune cell composition or bone mineral density.
randomized to 4 weeks of ADF (shortterm ADF) or ad lib control. The ADF group was in marked CR (37% in short-term ADF and 28% in long-term ADF) versus control (8%); i.e., the ADF group did not double their caloric intake, but modestly increased calorie intake to >125% on feast days. This inability to consume enough calories to maintain weight is observed in other studies of ADF/IF (Hutchison et al., 2019). Significant weight loss was observed in ADF, which was due to reductions in both lean (2.7%) and fat mass (8.9%). The reduction in fat mass occurred from the trunk area and particularly the android area, which is considered lipotoxic (Figure 1). The expected fasting-induced surge in ketone bodies (specifically beta hydroxybutyrate or BHBA), which are products of fatty acid oxidation, was observed. Keeping in mind that the ADF group ate >125% of caloric intake, the increase in ketones following feast days was unexpected. There was also no reduction in resting metabolic rate, despite weight loss. These findings raise important questions about whether postprandial metabolic rate, relative fuel utilization, or substrate interconversion is altered by ADF in healthy adults. In mice that undergo 16 h of fast every day, post-prandial energy expenditure was increased without any change in energy expenditure during the rest or fast period (Hatori et al., 2012). Among hormonal changes, the chronic ADF group showed relatively less circulating fT3, although the TSH and free thyroxine did not change. This reflects 412 Cell Metabolism 30, September 3, 2019
normal thyroid function even after 6 months of ADF. However, there was a marked increase in parathyroid hormone, which may partly explain the preservation of bone mineral density (BMD) following ADF. This is an important finding as a slight reduction in BMD was reported in a relatively younger cohort following chronic CR, in which participants reduced calories by 12% for more than 12 months (Villareal et al., 2016). Did ADF improve metabolic health? Since the participants were healthy to begin with, the authors assessed metabolic changes that are predictive of cardiometabolic health. Long-term ADF induced multiple beneficial health effects including reduced cholesterol and inflammatory markers and reduced risk for cardiovascular disease versus the comparator group. How comparable are these metabolic benefits with those observed in other ADF/IF studies? While there is general agreement that ADF/IF imparts metabolic health benefits, protocol differences often hinder direct comparison. For example, many IF studies prescribe a reduced caloric intake on ‘‘fast’’ day, so participants don’t experience 24 h of true fast. Other groups prescribed 24 h fasts initiated immediately after breakfast (Hutchison et al., 2019) or dinner (Halberg et al., 2005), or advocate for ‘‘breaking’’ the fast in the middle of the day (Trepanowski et al., 2017). There is accumulating evidence that timing of nutrition impacts peripheral clocks, which modulate several aspects of metabolism and physiology (Skene et al., 2018).
Uniquely, the authors used omics approaches to assess molecular changes after 12 or 36 h fast among the ADF group. Approximately 20% of the plasma metabolome was transiently elevated after 36 h fast, with lipids contributing a major portion. Another 20% of the metabolites were lowered by the extended fast, and amino acids were the major contributor. These plasma metabolite changes may reflect changes in adipose tissue and liver metabolism. The liver is a key player in extended fasting, converting fatty acids released from adipose tissue to ketones, and amino acids as substrates for gluconeogenesis. ADF also reduced the pro-aging metabolite methionine, which may have prolongevity effects (Richie et al., 1994). The proteome, measured from peripheral blood mononuclear cells (PBMCs) in fasted state, showed inactivation of pathways involved in lipid metabolism, mitochondrial biogenesis, and stress response. Although there was no significant compositional change in blood cells, the proteomic changes suggest a distinct molecular state of PBMCs. So it remains to be tested whether people under ADF mount a different immune response upon challenge. The similarity of the metabolome changes with those seen in chronic CR (Mitchell et al., 2016) either implies a common mechanism or was due to unintended CR in the current study. Whether the reduced caloric intake accompanied disproportionate changes in the source of macro- and micro-nutrient (e.g., processed or raw) cannot be ruled out. Further, a disproportionate reduction in carbohydrate relative to fat intake might also explain the observed increase in BHBA after an overnight fast. Similarly, there was some indication of slight, albeit clinically insignificant, reduction in iron, RBC, and hematocrit. Altogether, this study gives a safe bill of health to relatively long-term ADF in healthy adults and highlights that a combination of fasting with optimum nutrition may ultimately increase healthy human lifespan. ACKNOWLEDGMENTS Research in the Panda Lab is supported by the NIH (DK115214 and EY 016807), the Department of Homeland Security (EMW-2016-FP-00788), and the Department of Defense (W81XWH1810645). Research in the Heilbronn Lab is supported by
Cell Metabolism
Previews the National Heath and Medical Research Council (APP1143092). DECLARATION OF INTERESTS S.P. authored The Circadian Code, for which he collects author royalty. L.K.H. declares no competing interest. REFERENCES Halberg, N., Henriksen, M., So¨derhamn, N., Stallknecht, B., Ploug, T., Schjerling, P., and Dela, F. (2005). Effect of intermittent fasting and refeeding on insulin action in healthy men. J. Appl. Physiol. 99, 2128–2136. Harvie, M.N., Pegington, M., Mattson, M.P., Frystyk, J., Dillon, B., Evans, G., Cuzick, J., Jebb, S.A., Martin, B., Cutler, R.G., et al. (2011). The effects of intermittent or continuous energy restriction on weight loss and metabolic disease risk markers: a randomized trial in young overweight women. Int. J. Obes. 35, 714–727. Harvie, M., Wright, C., Pegington, M., McMullan, D., Mitchell, E., Martin, B., Cutler, R.G., Evans, G., Whiteside, S., Maudsley, S., et al. (2013). The effect of intermittent energy and carbohydrate restriction v. daily energy restriction on weight loss and metabolic disease risk markers in overweight women. Br. J. Nutr. 110, 1534–1547.
Hatori, M., Vollmers, C., Zarrinpar, A., DiTacchio, L., Bushong, E.A., Gill, S., Leblanc, M., Chaix, A., Joens, M., Fitzpatrick, J.A., et al. (2012). Timerestricted feeding without reducing caloric intake prevents metabolic diseases in mice fed a highfat diet. Cell Metab. 15, 848–860. Hutchison, A.T., Liu, B., Wood, R.E., Vincent, A.D., Thompson, C.H., O’Callaghan, N.J., Wittert, G.A., and Heilbronn, L.K. (2019). Effects of intermittent versus continuous energy intakes on insulin sensitivity and metabolic risk in women with overweight. Obesity (Silver Spring) 27, 50–58. Mattson, M.P., Allison, D.B., Fontana, L., Harvie, M., Longo, V.D., Malaisse, W.J., Mosley, M., Notterpek, L., Ravussin, E., Scheer, F.A., et al. (2014). Meal frequency and timing in health and disease. Proc. Natl. Acad. Sci. USA 111, 16647–16653. Mitchell, S.J., Madrigal-Matute, J., ScheibyeKnudsen, M., Fang, E., Aon, M., Gonza´lez-Reyes, J.A., Cortassa, S., Kaushik, S., Gonzalez-Freire, M., Patel, B., et al. (2016). Effects of sex, strain, and energy intake on hallmarks of aging in mice. Cell Metab. 23, 1093–1112. Richie, J.P., Jr., Leutzinger, Y., Parthasarathy, S., Malloy, V., Orentreich, N., and Zimmerman, J.A. (1994). Methionine restriction increases blood glutathione and longevity in F344 rats. FASEB J. 8, 1302–1307.
Skene, D.J., Skornyakov, E., Chowdhury, N.R., Gajula, R.P., Middleton, B., Satterfield, B.C., Porter, K.I., Van Dongen, H.P.A., and Gaddameedhi, S. (2018). Separation of circadianand behavior-driven metabolite rhythms in humans provides a window on peripheral oscillators and metabolism. Proc. Natl. Acad. Sci. USA 115, 7825–7830. Stekovic, S., Hofer, S.J., Tripolt, N., Aon, M.A., Royer, P., Pein, L., Stadler, J.T., Pendl, T., Prietl, B., Url, J., et al. (2019). Alternate day fasting improves physiological and molecular markers of aging in healthy, non-obese humans. Cell Metab. 30, this issue, 462–476. Trepanowski, J.F., Kroeger, C.M., Barnosky, A., Klempel, M.C., Bhutani, S., Hoddy, K.K., Gabel, K., Freels, S., Rigdon, J., Rood, J., et al. (2017). Effect of alternate-day fasting on weight loss, weight maintenance, and cardioprotection among metabolically healthy obese adults: a randomized clinical trial. JAMA Intern. Med. 177, 930–938. Villareal, D.T., Fontana, L., Das, S.K., Redman, L., Smith, S.R., Saltzman, E., Bales, C., Rochon, J., Pieper, C., Huang, M., et al.; CALERIE Study Group (2016). Effect of twoyear caloric restriction on bone metabolism and bone mineral density in non-obese younger adults: a randomized clinical trial. J. Bone Miner. Res. 31, 40–51.
Cell Metabolism 30, September 3, 2019 413
Previews availability during infections can mediate anti-virulence defenses, curbing pathogen virulence (Sanchez et al., 2018). In the CLP and influenza infection models, it will be interesting to determine how increased triglyceride availability affects bacterial and viral virulence. The important question of how triglyceride availability supports cardiac function during sepsis remains. Does increased triglyceride availability promote disease tolerance by supporting cardiac metabolism or by some other function? It remains to be determined if GDF15 mediates disease tolerance via activation of its receptor in the area postrema or some other neurons or cell types in the body. It will also be important to understand why lipid administration is only protective in settings where GDF15 is depleted and not sepsis in general.
motes host survival and transmission. Cell 168, 503–516.e12.
REFERENCES Hsu, J.Y., Crawley, S., Chen, M., Ayupova, D.A., Lindhout, D.A., Higbee, J., Kutach, A., Joo, W., Gao, Z., Fu, D., et al. (2017). Non-homeostatic body weight regulation through a brainstemrestricted receptor for GDF15. Nature 550, 255–259. Luan, H., Wang, A., Hilliard, B., Carvalho, F., Rosen, C., Ahasic, A., Herzog, E., Kang, I., Pisani, M., Yu, S., et al. (2019). GDF15 is an inflammation-induced central mediator of tissue tolerance. Cell 178, 1231–1244. Medzhitov, R., Schneider, D.S., and Soares, M.P. (2012). Disease tolerance as a defense strategy. Science 335, 936–941. Nestel, P.J. (1964). Plasma triglyceride concentration and plasma free fatty acid changes in response to norepinephrine in man. J. Clin. Invest. 43, 77–82. Rao, S., Schieber, A.M.P., O’Connor, C.P., Leblanc, M., Michel, D., and Ayres, J.S. (2017). Pathogen-mediated inhibition of anorexia pro-
Sanchez, K.K., Chen, G.Y., Schieber, A.M.P., Redford, S.E., Shokhirev, M.N., Leblanc, M., Lee, Y.M., and Ayres, J.S. (2018). Cooperative metabolic adaptations in the host can favor asymptomatic infection and select for attenuated virulence in an enteric pathogen. Cell 175, 146– 158.e15. Schieber, A.M., Lee, Y.M., Chang, M.W., Leblanc, M., Collins, B., Downes, M., Evans, R.M., and Ayres, J.S. (2015). Disease tolerance mediated by microbiome E. coli involves inflammasome and IGF-1 signaling. Science 350, 558–563. Schneider, D.S., and Ayres, J.S. (2008). Two ways to survive infection: what resistance and tolerance can teach us about treating infectious diseases. Nat. Rev. Immunol. 8, 889–895. Wang, A., Huen, S.C., Luan, H.H., Yu, S., Zhang, C., Gallezot, J.D., Booth, C.J., and Medzhitov, R. (2016). Opposing effects of fasting metabolism on tissue tolerance in bacterial and viral inflammation. Cell 166, 1512–1525.e12.
Alternate-Day Fasting Gets a Safe Bill of Health Leonie K. Heilbronn1,2,* and Satchidananda Panda3,* 1Faculty
of Health Sciences, The University of Adelaide, Adelaide, SA, Australia Health Theme, South Australian Health and Medical Research Institute, Adelaide, SA, Australia 3The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA *Correspondence: [email protected] (L.K.H.), [email protected] (S.P.) https://doi.org/10.1016/j.cmet.2019.08.006 2Lifelong
Various forms of fasting improve health and longevity in preclinical models. However, safety, outcomes, and the molecular changes underpinning human fasting are unclear. Stekovic et al. (2019) report improved markers of health for up to 6 months and associated metabolic changes among healthy adults who followed alternate-day fasting. Reducing daily caloric intake by 20%– 40% (a.k.a. caloric restriction or CR) in animals increases total and healthy lifespan—a feat rarely achieved by any drug. However, counting calories for CR in humans can be challenging, and chronic CR in rodents can dampen immune function and bone metabolism. Intermittent fasting (IF) is an alternative strategy involving 2–4 days per week when food intake is severely reduced or completely withheld, interspersed with ad libitum eating the remainder of the week. Mice subjected to alternate-day fasting (ADF)—a form of IF—double their caloric
intake on feast days, maintaining body weight close to their ad lib cohorts. Yet ADF mice enjoy pleiotropic health benefits found among CR mice who weigh much less (Mattson et al., 2014). This observation raised the possibility that prolonged fasting elicits a pro-longevity metabolic state. Thus, testing the feasibility of IF in humans and its health impacts, and assessing accompanying molecular changes are crucial to devise optimum nutrition to support long healthspan. Few human trials on IF have been conducted: they are mostly in obese individuals and are modified to allow consump-
tion of 500 kcals during the fasting day (Harvie et al., 2011, 2013; Trepanowski et al., 2017). Whether ADF imparts any benefit to healthy people and unbiased omics-based molecular changes in ADF have not been addressed. As ADF can involve fasting for up to 36 h, there was also concern that it might trigger excessive muscle loss among healthy cohorts. In a study published in this issue of Cell Metabolism (Stekovic et al., 2019), healthy individuals who had been under a selfimposed complete ADF for >6 months (long-term ADF) were matched to a comparator group who were naive to IF. This comparator group was then
Cell Metabolism 30, September 3, 2019 ª 2019 Elsevier Inc. 411
Cell Metabolism
Previews
Figure 1. Alternate-Day Fasting Is Safe for Normal Weight Healthy Adults Ad libitum eating every other day leads to both a prolonged period of fasting and an overall reduction in caloric intake. This results in several metabolic changes that correlate with healthy lifespan. ADF does not change the immune cell composition or bone mineral density.
randomized to 4 weeks of ADF (shortterm ADF) or ad lib control. The ADF group was in marked CR (37% in short-term ADF and 28% in long-term ADF) versus control (8%); i.e., the ADF group did not double their caloric intake, but modestly increased calorie intake to >125% on feast days. This inability to consume enough calories to maintain weight is observed in other studies of ADF/IF (Hutchison et al., 2019). Significant weight loss was observed in ADF, which was due to reductions in both lean (2.7%) and fat mass (8.9%). The reduction in fat mass occurred from the trunk area and particularly the android area, which is considered lipotoxic (Figure 1). The expected fasting-induced surge in ketone bodies (specifically beta hydroxybutyrate or BHBA), which are products of fatty acid oxidation, was observed. Keeping in mind that the ADF group ate >125% of caloric intake, the increase in ketones following feast days was unexpected. There was also no reduction in resting metabolic rate, despite weight loss. These findings raise important questions about whether postprandial metabolic rate, relative fuel utilization, or substrate interconversion is altered by ADF in healthy adults. In mice that undergo 16 h of fast every day, post-prandial energy expenditure was increased without any change in energy expenditure during the rest or fast period (Hatori et al., 2012). Among hormonal changes, the chronic ADF group showed relatively less circulating fT3, although the TSH and free thyroxine did not change. This reflects 412 Cell Metabolism 30, September 3, 2019
normal thyroid function even after 6 months of ADF. However, there was a marked increase in parathyroid hormone, which may partly explain the preservation of bone mineral density (BMD) following ADF. This is an important finding as a slight reduction in BMD was reported in a relatively younger cohort following chronic CR, in which participants reduced calories by 12% for more than 12 months (Villareal et al., 2016). Did ADF improve metabolic health? Since the participants were healthy to begin with, the authors assessed metabolic changes that are predictive of cardiometabolic health. Long-term ADF induced multiple beneficial health effects including reduced cholesterol and inflammatory markers and reduced risk for cardiovascular disease versus the comparator group. How comparable are these metabolic benefits with those observed in other ADF/IF studies? While there is general agreement that ADF/IF imparts metabolic health benefits, protocol differences often hinder direct comparison. For example, many IF studies prescribe a reduced caloric intake on ‘‘fast’’ day, so participants don’t experience 24 h of true fast. Other groups prescribed 24 h fasts initiated immediately after breakfast (Hutchison et al., 2019) or dinner (Halberg et al., 2005), or advocate for ‘‘breaking’’ the fast in the middle of the day (Trepanowski et al., 2017). There is accumulating evidence that timing of nutrition impacts peripheral clocks, which modulate several aspects of metabolism and physiology (Skene et al., 2018).
Uniquely, the authors used omics approaches to assess molecular changes after 12 or 36 h fast among the ADF group. Approximately 20% of the plasma metabolome was transiently elevated after 36 h fast, with lipids contributing a major portion. Another 20% of the metabolites were lowered by the extended fast, and amino acids were the major contributor. These plasma metabolite changes may reflect changes in adipose tissue and liver metabolism. The liver is a key player in extended fasting, converting fatty acids released from adipose tissue to ketones, and amino acids as substrates for gluconeogenesis. ADF also reduced the pro-aging metabolite methionine, which may have prolongevity effects (Richie et al., 1994). The proteome, measured from peripheral blood mononuclear cells (PBMCs) in fasted state, showed inactivation of pathways involved in lipid metabolism, mitochondrial biogenesis, and stress response. Although there was no significant compositional change in blood cells, the proteomic changes suggest a distinct molecular state of PBMCs. So it remains to be tested whether people under ADF mount a different immune response upon challenge. The similarity of the metabolome changes with those seen in chronic CR (Mitchell et al., 2016) either implies a common mechanism or was due to unintended CR in the current study. Whether the reduced caloric intake accompanied disproportionate changes in the source of macro- and micro-nutrient (e.g., processed or raw) cannot be ruled out. Further, a disproportionate reduction in carbohydrate relative to fat intake might also explain the observed increase in BHBA after an overnight fast. Similarly, there was some indication of slight, albeit clinically insignificant, reduction in iron, RBC, and hematocrit. Altogether, this study gives a safe bill of health to relatively long-term ADF in healthy adults and highlights that a combination of fasting with optimum nutrition may ultimately increase healthy human lifespan. ACKNOWLEDGMENTS Research in the Panda Lab is supported by the NIH (DK115214 and EY 016807), the Department of Homeland Security (EMW-2016-FP-00788), and the Department of Defense (W81XWH1810645). Research in the Heilbronn Lab is supported by
Cell Metabolism
Previews the National Heath and Medical Research Council (APP1143092). DECLARATION OF INTERESTS S.P. authored The Circadian Code, for which he collects author royalty. L.K.H. declares no competing interest. REFERENCES Halberg, N., Henriksen, M., So¨derhamn, N., Stallknecht, B., Ploug, T., Schjerling, P., and Dela, F. (2005). Effect of intermittent fasting and refeeding on insulin action in healthy men. J. Appl. Physiol. 99, 2128–2136. Harvie, M.N., Pegington, M., Mattson, M.P., Frystyk, J., Dillon, B., Evans, G., Cuzick, J., Jebb, S.A., Martin, B., Cutler, R.G., et al. (2011). The effects of intermittent or continuous energy restriction on weight loss and metabolic disease risk markers: a randomized trial in young overweight women. Int. J. Obes. 35, 714–727. Harvie, M., Wright, C., Pegington, M., McMullan, D., Mitchell, E., Martin, B., Cutler, R.G., Evans, G., Whiteside, S., Maudsley, S., et al. (2013). The effect of intermittent energy and carbohydrate restriction v. daily energy restriction on weight loss and metabolic disease risk markers in overweight women. Br. J. Nutr. 110, 1534–1547.
Hatori, M., Vollmers, C., Zarrinpar, A., DiTacchio, L., Bushong, E.A., Gill, S., Leblanc, M., Chaix, A., Joens, M., Fitzpatrick, J.A., et al. (2012). Timerestricted feeding without reducing caloric intake prevents metabolic diseases in mice fed a highfat diet. Cell Metab. 15, 848–860. Hutchison, A.T., Liu, B., Wood, R.E., Vincent, A.D., Thompson, C.H., O’Callaghan, N.J., Wittert, G.A., and Heilbronn, L.K. (2019). Effects of intermittent versus continuous energy intakes on insulin sensitivity and metabolic risk in women with overweight. Obesity (Silver Spring) 27, 50–58. Mattson, M.P., Allison, D.B., Fontana, L., Harvie, M., Longo, V.D., Malaisse, W.J., Mosley, M., Notterpek, L., Ravussin, E., Scheer, F.A., et al. (2014). Meal frequency and timing in health and disease. Proc. Natl. Acad. Sci. USA 111, 16647–16653. Mitchell, S.J., Madrigal-Matute, J., ScheibyeKnudsen, M., Fang, E., Aon, M., Gonza´lez-Reyes, J.A., Cortassa, S., Kaushik, S., Gonzalez-Freire, M., Patel, B., et al. (2016). Effects of sex, strain, and energy intake on hallmarks of aging in mice. Cell Metab. 23, 1093–1112. Richie, J.P., Jr., Leutzinger, Y., Parthasarathy, S., Malloy, V., Orentreich, N., and Zimmerman, J.A. (1994). Methionine restriction increases blood glutathione and longevity in F344 rats. FASEB J. 8, 1302–1307.
Skene, D.J., Skornyakov, E., Chowdhury, N.R., Gajula, R.P., Middleton, B., Satterfield, B.C., Porter, K.I., Van Dongen, H.P.A., and Gaddameedhi, S. (2018). Separation of circadianand behavior-driven metabolite rhythms in humans provides a window on peripheral oscillators and metabolism. Proc. Natl. Acad. Sci. USA 115, 7825–7830. Stekovic, S., Hofer, S.J., Tripolt, N., Aon, M.A., Royer, P., Pein, L., Stadler, J.T., Pendl, T., Prietl, B., Url, J., et al. (2019). Alternate day fasting improves physiological and molecular markers of aging in healthy, non-obese humans. Cell Metab. 30, this issue, 462–476. Trepanowski, J.F., Kroeger, C.M., Barnosky, A., Klempel, M.C., Bhutani, S., Hoddy, K.K., Gabel, K., Freels, S., Rigdon, J., Rood, J., et al. (2017). Effect of alternate-day fasting on weight loss, weight maintenance, and cardioprotection among metabolically healthy obese adults: a randomized clinical trial. JAMA Intern. Med. 177, 930–938. Villareal, D.T., Fontana, L., Das, S.K., Redman, L., Smith, S.R., Saltzman, E., Bales, C., Rochon, J., Pieper, C., Huang, M., et al.; CALERIE Study Group (2016). Effect of twoyear caloric restriction on bone metabolism and bone mineral density in non-obese younger adults: a randomized clinical trial. J. Bone Miner. Res. 31, 40–51.
Cell Metabolism 30, September 3, 2019 413