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Biological Rhythm Research

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The role of time of food intake on upcoming liverdisease in male Wistar rat

Nazanin Asghari Hanjani, Negar Zamaninour, Narjes Najibi, Agha FatemehHosseini, Tayebeh Rastegar & Mohammad Reza Vafa

To cite this article: Nazanin Asghari Hanjani, Negar Zamaninour, Narjes Najibi, AghaFatemeh Hosseini, Tayebeh Rastegar & Mohammad Reza Vafa (2018): The role of time offood intake on upcoming liver disease in male Wistar rat, Biological Rhythm Research, DOI:10.1080/09291016.2018.1478635

To link to this article: https://doi.org/10.1080/09291016.2018.1478635

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Biological Rhythm ReseaRch, 2018https://doi.org/10.1080/09291016.2018.1478635

The role of time of food intake on upcoming liver disease in male Wistar rat

Nazanin Asghari Hanjania, Negar Zamaninourb, Narjes Najibia, Agha Fatemeh Hosseinic, Tayebeh Rastegard and Mohammad Reza Vafaa#

aResearch center of Prevention of cardiovascular Diseases, institute of endocrinology and metabolism, iran University of medical sciences, tehran, iran; bminimally invasive surgery Research center, iran University of medical sciences (iUms), tehran, iran; cDepartment of statistics and mathematics, school of health management and information science, iran University of medical sciences, tehran, iran; dFaculty of medicine, Department of anatomy sciences, tehran University of medical sciences, tehran, iran

ABSTRACTInterventions against obesity, are mainly around changing calorie intake and energy expenditure. Recently, some studies focused on the influence of circadian time of food intake on metabolic status. Here, we compare the role of calorie restriction and time restricted feeding followed by high-fat diet started post weaning, First, 52 male Wistarrats (3 weeks old) were divided into two groups: the high-fat diet (HFD, n = 42) and the control group (CON1, n = 11). After 17 weeks, five rats were randomly selected from each group for sample preparation. In the second phase, the animals in HFD group were assigned into four groups (n = 9): (1) 30% calorie restriction (CR), (2) day intermittent fasting (DIF), (3) night intermittent fasting (NIF), (4) adlibitum food intake (AL), (5) remained animal from the first phase control (CON2). Seventeen weeks of HFD started post-weaning did not cause fatty liver but it caused a significant difference in the body and the adipose tissue weight (P0.05). The results showed that longtime HFD did not lead to liver steatosis while the incorrect time of food intake predisposes the animal to the upcoming liver disease. This data indicate a significant role of timing of food intake rather than nutrition composition itself.

Abbreviations: HFD: high-fat diet; CON1: first-phase control group; CR: calorie restriction; NIF: night intermittent fasting; DIF: day intermittent fasting; CON2: second-phase control group; SCN: suprachiasmatic nucleus; ZT: zeitgeber; ANONA; ALT: alanine aminotransferase; AST: aspartate aminotransferase; ANOVA: one-way analysis of variance

Introduction

One of the most serious public health issues is childhood obesity, which is associated with a long-term health outcome. According to WHO recent estimation, more than 42 million

© 2018 informa UK limited, trading as taylor & Francis group

KEYWORDSNaFlD; calorie restriction; circadian food intake; time-restricted feeding; juvenile obesity; visceral fat

ARTICLE HISTORYReceived 5 april 2018 accepted 5 may 2018

CONTACT mohammad Reza Vafa vafa.m@iums.ac.ir#Present address: school of Public health, iran University of medical sciences, tehran, iran.

supplemental data for this article can be accessed https://doi.org/10.1080/09291016.2018.1478635.

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people under the age of 5 are overweight or obese (organization 2016; Gies et al. 2017). Owing to the rapid increase in obesity, there has been a concurrent increase in the spectrum of metabolic diseases including dyslipidemia, insulin resistance, NAFLD, and many other adverse effects (Rizza et al. 2014; Sweat et al. 2017). Among them, fatty acid accumulation in the liver which is called NAFLD is the most common chronic liver disease and is the con-sequence of excess high glycemic carbohydrate and saturated fatty acids intake (Bezerra Duarte et al. 2014; Rinella 2015).

Since 1949, high-fat diet is used to induce obesity, which was later called dietary obesity. The combination of fat and sugar (cafeteria diet) or adding a particular fat (30–78% of total energy) is usually used to induce obesity (Buettner et al. 2007). Since then different studies show that high-fat diet which provides about 60% of energy from fat can cause obesity, hepatic steatosis, inflammation, and liver damage, which are considered as an early stage of NAFLD (Dhibi et al. 2011). Beside, perturbation of circadian clock which is under the tight control of suprachiasmatic nucleus in the hypothalamus can cause energy imbalance and metabolic disorder later in life (Ingenwerth et al. 2016).

Attempts, which focused on finding therapeutic interventions against obesity and related metabolic disease, are mainly around changing calorie intake and energy expenditure. Recently, a number of researchers are dedicated to studying the influence of circadian time of food intake on a wide variety of metabolic, hormonal, and physiological function. For example, metabolic syndrome has been observed following mutation of Clock genes in mice (Arble et al. 2009). Conversely, disruption of circadian rhythm is commonly found in the animal model of obesity (Hatori et al. 2012). Though it is postulated that, interventions, which can disrupt or restore diurnal rhythms may have high impact in combating obesity and related metabolic disease like NAFLD.

According to the U.S. Practice Guideline for the Diagnosis and Management of NAFLD, interventions, which lead to weight loss like hypocaloric diet, can reduce hepatic steatosis as well as obesity. Hence, to test whether high-fat diet can induce obesity or NAFLD starting post weaning and subsequently investigating the effect of calorie restriction (CR) and circa-dian intake of food on juvenile obesity and liver status, we used a diet-induced obesity rodent model. The underlying question was identifying the effect of high-fat diet, time restricted feeding and CR on juvenile obesity and liver status.

Material and method

Animals and diet

The study conducted in two-phase includes obesity induction and calorie and time restric-tion period. Fifty-two male Wistar rats (three weeks old) were purchased from Pasteur Institute of Iran and individually housed in a cage at 22 ± 2 °C under 12 h light/12dark cycle and allowed free access to food and water on accommodation period. The study protocol was in accordance with National Institutes of Health guide for the care and use of Laboratory animals. At the first phase of the study and one-week accommodation, animals were ran-domly divided into two groups. High-fat diet (HFD n: 41) receiving semi-purified high-fat diet with 60% of energy from milk butter and control (CON n:11) which fed standard chow for 17 weeks (contains 4 kcal/g with 20.10% as protein 4.02% as fat, 62.33% as carbohydrate, 9.27% as sucrose, 4.27% as fiber and other necessary nutrients for the growth of rats). The

BIOLOGICAL RHYTHM RESEARCH 3

experimental diet was prepared freshly every three days and was kept at -20 °C to maintain nutrition composition. Rats had free access to tap water throughout the study. At the end of this phase, five animals per group randomly selected for sample preparation. The HFD randomly assigned to four groups (each group: 9) while the mean body weight was equiv-alent in all groups. This phase continued with the standard diet. Calorie restricted group (CR) received 70% calorie compared to adlibitum, day intermittent fasting (DIF) which had access to food between zeitgeber time (ZT) 13 and ZT1 and night intermittent fasting (NIF) which received food between ZT1 and ZT13 (figure1), adlibitum (AL) which had free access to food and six animals from control group (CON). These dietary interventions continued for ten weeks. In CR group, the food was provided at ZT13 in order to inhibit circadian disruption. The health status of all animals was monitored regularly by a veterinarian and body weight and food intake recorded on a specific day of the week.

Sample preparation

After an overnight food deprivation, animals were anesthetized by intraperitoneal injection of a mixture of ketamine (ketamine 10%, 100 mg ml−1, Alfasan, Holand) and xylazine (xylazine 2%, 20 mg ml−1, Alfasan, Holand) in a 1.37:1 ratio and blood sample were collected from the heart. Then samples centrifuged at 1500 × g for 20 min at 4 °C and stored at −80 °C for analyses.

Histological analyses

Sections measuring 0.5 × 0.5 cm were taken from fresh liver and fixed in formalin 10% for 24 h. They were dehydrated through different solutions of alcohol and embedded into par-affin wax. Subsequently, tissue samples were sectioned by rotary microtome at thicknesses of 5 μm and stained with hematoxylin and eosin. Visceral fat-pads (mesenteric, perirenal, epididymal and retroperitoneal fat) was also collected and weighed after being rinsed with normal saline.

Biochemical analyses

Serum concentration of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assessed by an automated biochemical analyzer and biochemical kits (Bionic, Dialab, Australia) (Biotecnica instrument BT1500, U.S.A.).

Statistical analyses

The normality was checked by Kolmogorov–Smirnov test. In the first phase, independent T-test or its non-parametric equivalent (Mann–Whitney) and in the second phase, one-way analysis of variance (ANOVA) and Tukey post hoc test or Kruskal–Wallis and Bonferroni were used for data analysis. Data presented as mean ± SD. SPSS software (Released 2013. IBM SPSS Statistics for Windows, Version 22.0. Armonk NY: IBM Corp) was used for data analysis. p < 0.05 considered significant.

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Figure 1. the study protocol in the second phase of the study. in cR group, food was provided at the beginning of the dark cycle, in order to minimize diurnal rhythm disruption.

Figure 2. Body weight changes in rats fed either standard chow or high-fat diet (60% of energy from fat). Data presented as mean ± sD. *p < 0.05 (from weeks 5–17). hFD, high-fat diet n:41; coN1, the first-phase control group, n:9.

Figure 3. mean body weights of rats during the second phase of the study and restrictions protocols for 10 weeks. Values are presented as mean ± sD. cR, 30% calorie restriction; al, n = 9 adlibitum food access; DiF n = 9, day intermittent fasting; NiF n = 9, night intermittent fasting; coN2 n = 6, second-phase control. *p:0.08, **p:0.046, ***p:0.01, ****p:0.008.

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Results

Prior to each phase, there was not a significant body weight difference between groups. Average body weight in 17 weeks of the first phase is summarized in Figure 2. At the end of the first phase, HFD weighed 12.5% more than the control while the epididymal tissue weighed twofold more than the control) CON 341.72 ± 36.95, HFD 382.90 ± 48.14, P:0.011 CON 5.27, HFD10.73, body weight and adipose tissue weight, respectively, see figure2 and 3, P: 0.009). In the second phase, we wanted to test whether reducing calorie intake (CR) is effective in weight control or time-restricted feeding. We compared CR with NIF, which is aligned with these nocturnal animals circadian rhythm. Dietary interventions in the second phase, indicated that CR is more effective intervention in weight control compared with NIF (Figure 3). Since CR weighed significantly, less body weight compared with AL from 9th week to 11th week. Meanwhile, DIF and NIF demonstrated similar weight gain, which was not significant throughout the second phase. Adipose tissue weight (Figure 4) significantly decreased in HFD derivatives at the end of the second phase of the study compared to the beginning of this phase except for AL group (p: 0.0001). While adipose tissue weight/ body weight ratio reduced only in CR compared to the HFD (p: 0.016).

Figure 4. mean adipose tissue and adipose tissue weight /body weight ratio changes in the first and second phase of the study. Values are presented as mean ± sD. cR, 30% calorie restriction; al, n = 9 adlibitum food access; DiF n = 9, day intermittent fasting; NiF n = 9, night intermittent fasting; coN2 n = 6, second phase control. similar letters indicate a significant difference. p < 0.05*.

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As it is shown in Figure 5, seventeen weeks of HFD did not lead to significant liver weight changes. In the same way, serum concentration of ALT and AST did not alter between HFD and CON1 (Table 1). Moreover, histological analyses did not show a lipid droplet in the liver, while hepatocytes arrangement around central vein was a bit disturbed (Figure 6). In the second phase, liver weight and liver to body weight ratio did not change significantly between groups however, liver to body weight ratio was higher in DIF (Figure 5). Surprisingly, food intake in the rest phase (DIF) leads to the significant higher liver to body weight ratio compared to HFD in the first phase (p: 0.036). On the other hand, NIF decreased the serum concentration of ALT (P:0.043) while DIF increases it (p:0.011) in comparison to HFD. Between groups analyses in the second phase, revealed that ALT concentration is higher in DIF com-pared to NIF and AL (Table 1). These data indicated a vital role of circadian food intake on NAFLD even in comparison with HFD and CR. Histological analyses revealed lipid droplet in hepatocytes in AL and DIF group however, it was more significant in DIF group as well as hepatocytes disarrangement and nucleus picnozation in DIF (Figure 6, the blue arrow).

Figure 5. mean liver and liver to body weight ratio in the first and second phase of the study. Values are presented as mean ± sD. cR, 30% calorie restriction; al, n = 9 adlibitum food access; DiF n = 9, day intermittent fasting; NiF n = 9, night intermittent fasting; coN2 n = 6, second phase control. *P:0.036.

BIOLOGICAL RHYTHM RESEARCH 7

Discussion

Despite all of the efforts that are dedicated to studying the underlying mechanism of obesity and the possible ways which may prevent it, it is still a major health problem. Epidemiological studies indicated that the prevalence of obesity in all ages increased between 1998 and 2014 (Dietz 2016). Almost all organs are influenced by obesity while some systems like endocrine and cardiovascular are affected to a greater degree, hence pediatric liver disease, as well as childhood obesity, is rising up (Mathur et al. 2007).

In most studies, modest obesity is considered as 10–20% higher weight gain while 40% greater weight gain is considered as severe obesity. In the present study 17 weeks, fatty diet leads to 12.5% higher weight in HFD compared to their age-matched control group while the difference in epididymal tissue weight was about 100% which indicates the importance of adipose tissue measurement during obesity induction in animal models. Previously Woods

Figure 6. histological analysis of liver section in the first and second phase of the study (h&e × 400) which was performed by a pathologist who was blind to the experimental diets. A1,hFD; B1,coN1, A, coN2; B, cR; C, al; D, DiF; E, NiF.

Table 1. serum concentrations of alt and ast.

Notes: the data value is presented as mean ± sD. p-value in the first phase was determined by independent sample t-test and in the second phase by one-way aNoVa followed by tukey post hoc test. similar symbols are used to show a signifi-cant difference. p-value < 0.05 considered as significant.

First phase Second phase

AST (U/l) p-value ALT(U/L) p-value ALT(U/l)The

p-value AST(U/l) p-valuecoN1 49.40 ± 14.53 0.57 89.80 ± 32.75 0.48 coN2 86.33 ± 21.27 0.002 53 ± 9.83 0.58hFD 66.60 ± 24.65 108.2 ± 25.15 al 81.88 ± 17.25† 59.22 ± 18.99

DiF 107.66 ± 18.49*† 57.88 ± 10.44NiF 72.44 ± 10.86* 51.77 ± 12.79cR 85.11 ± 20.91 60.11 ± 8.49

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et al. reported that, the 10 weeks high-fat diet (40% of energy) caused 10% higher body weight while 35–40% increase in total body fat which showed body fat as a sensitive criterion for obesity. Here prolonged time and higher fat percentage still don’t lead to a greater dif-ference in body weight which points out an importance of body fat measurement (Levin and Dunn-Meynell 2002; Woods et al. 2003).

Despite changes in the body weight and fat depot, we did not observe any sign of typical hepatic lesion of steatosis or NAFLD which may be due to the ability of younger animal in the dissipation of energy as heat. Analyzing the hepatocytes of young rats who fed cafeteria diet showed increased gluconeogenesis and fatty acid oxidation which was not correlated with hepatic demand for adenosine triphosphate (ATP) (Berry et al. 1985). Romestaing et al. also didn’t indicate any sign of liver steatosis in response to long-term saturated fatty acid intake. It is hypothesized that the liver ability to export fat in triglyceride form is the main reason for these observations. Moreover, long chain fatty acids which are found in milk butter have a higher tendency for peripheral accumulation (Romestaing et al. 2007).

In the second phase of the study food intake in the rest, phase leads to an elevated level of ALT, liver weight and fat deposition in the liver, which indicates an important role of meal-time as well as nutritional quality. Recently, emerging data suggest a principal role of circadian time of food intake and diurnal rhythm in predisposing the organism to metabolic disease. Rodents are nocturnal animals and are more active during the night, hence diurnal food intake can disrupt their circadian rhythm which is regulated by suprachiasmatic nucleus (SCN) and circadian oscillation in peripheral tissues like liver. In more details, inappropriate food intake leads to desynchronization of SCN which is stimulated by light and peripheral clocks that is mainly regulated by food intake (Challet 2010). We confirm here that the time of food intake and circadian disruption is more important in fat accumulation in the liver than receiv-ing 60% of calorie from fat. While high-fat diet itself can reduce the amplitude of diurnal rhythm and alter liver molecule clock that is followed by weight gain and metabolic changes (Branecky et al. 2015), It didn’t lead to steatosis or fat accumulation in the liver. It is also reported that daytime feeding is responsible for decreased body temperature in the night that is followed by higher weight gain. Genetic studies have been reported that HFD change the circadian clock gene expression and dysregulate metabolic genes in peripheral and central tissue that predispose an organism to obesity and metabolic syndrome. Nevertheless, 17 weeks of HFD itself did not lead to significant fat accumulation in the liver while when it was followed by DIF (standard diet) changed cell arrangement and increased ALT. In other words, normal body metabolic adaption which is occurred in response to HFD, may be dis-rupted by DIF to a greater degree even with standard diet [20]. Although we couldn’t analyze liver triglyceride content and it is suggested to be evaluated in the future studies.

Among different dietary interventions in the second phase of the study, CR significantly reduced both body weight and tissue/ body weight ratio compared to AL group. Picard et al., previously reported that during CR, SIRTUIN1 content increase in many tissues which repress genes controlled by peroxisome proliferator-activated receptorγ and enhance fat mobilization from adipose tissue (Picard et al. 2004). In this work, a significant reduction of adipose tissue weight in parallel with body weight was seen in CR. A mild increment of AST and ALT in CR group in comparison to AL and NIF may be due to the mentioned fat mobili-zation and oxidation. Despite all groups, average body weight in NIF and DIF had a constant trend. Sutton et al. indicated that mice with 4 h food access for 12 consecutive days lose 10% of initial body weight (Sutton et al. 2008), while in this study, access food period was 12 h,

BIOLOGICAL RHYTHM RESEARCH 9

to make sure they have sufficient time for food access without restricting the whole calorie. So we didn’t expect significant body weight changes. Meanwhile, it is suggested to investi-gate the effects of more restricted feeding period and compare that with the effect of CR and NIF. Analyzing the liver’s circadian molecule (Damiola et al. 2000; Acharyya et al. 2004) in these different dietary interventions is helpful to find the best dietary pattern.

Providing hypocaloric food in light cycle makes animal partially diurnal because it has been shown that they eat all of the food in first three hours of food access (Mendoza et al. 2005). Hence in this study, we provided the food at ZT1, at the beginning of the dark cycle.

Conclusion

Here we demonstrated that long-term consumption of high-fat diet resulted in mild weight gain while the adipose tissue changes were about twofold in response to 60% of calorie from fat. Subsequently, between the diverse dietary interventions, including restrictions (time or calorie) and AL food access with normal fat, food intake during the rest time, resulted in elevated liver enzyme and weight gain, the effect which was not observed by 17 weeks of high-fat diet. Additionally, CR resulted in significant weight and adipose tissue loss which was not observed in other groups, specifically time-restricted feeding(night). Together, this data indicated the vital role of time of food intake beside than nutritional quality. Moreover, calorie restriction is more efficient in weight loss than time-restricted feeding which is high-lighted recently.

Authors contributions

VR and AHN made a substantial contribution including; planning the study, conception, and writing manuscript, HAF analyzed and interpret the data, ZN and NN made a contribution on histological examination and changing food access in light/dark cycle during the inter-vention. RT, as an experienced histopathologist read the samples, while was blinded to the group’s assignments and helped in tissue preparation. All of the authors have been read and approved the final proof.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

The study was conducted under the grant of Research Center of Prevention of Cardiovascular Diseases, Institute of Endocrinology and Metabolism,Iran University of Medical Sciences [grant num-ber 96-03-200-32101].

References

Acharyya S, Ladner KJ, Nelsen LL, Damrauer J, Reiser PJ, Swoap S, Guttridge DC. 2004. Cancer cachexia is regulated by selective targeting of skeletal muscle gene products. J Clin Investig. 114(3):370.

Arble DM, Bass J, Laposky AD, Vitaterna MH, Turek FW. 2009. Circadian timing of food intake contributes to weight gain. Obesity. 17(11):2100–2102.

10 N. ASGHARI HANJANI ET AL.

Berry M, Clark D, Grivell A, Wallace P. 1985. The contribution of hepatic metabolism to diet-induced thermogenesis. Metabolism. 34(2):141–147.

Bezerra Duarte SM, Faintuch J, Stefano JT, Sobral de Oliveira MB, de Campos Ferraz, Mazo D, Rabelo F, Vanni D, Aydar Nogueira M, Carrilho FJ, et al. 2014. Hypocaloric high-protein diet improves clinical and biochemical markers in patients with nonalcoholic fatty liver disease (NAFLD). Nutr Hosp. 29(1).

Branecky KL, Niswender KD, Pendergast JS. 2015. Disruption of daily rhythms by high-fat diet is reversible. PLoS One. 10(9):e0137970.

Buettner R, Schölmerich J, Bollheimer LC. 2007. High-fat diets: modeling the metabolic disorders of human obesity in rodents. Obesity. 15(4):798–808.

Challet E. 2010. Interactions between light, mealtime and calorie restriction to control daily timing in mammals. J Comp Physiol B. 180(5):631–644.

Damiola F, Le Minh N, Preitner N, Kornmann B, Fleury-Olela F, Schibler U. 2000. Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus. Genes Dev. 14(23):2950–2961.

Dhibi M, Brahmi F, Mnari A, Houas Z, Chargui I, Bchir L, Gazzah N, Alsaif MA, Hammami M. 2011. The intake of high fat diet with different trans fatty acid levels differentially induces oxidative stress and non alcoholic fatty liver disease (NAFLD) in rats. Nutr Metab. 8(1):65.

Dietz WH. 2016. Are we making progress in the prevention and control of childhood obesity? It all depends on how you look at it. Obesity. 24(5):991–992.

Gies I, AlSaleem B, Olang B, Karima B, Samy G, Husain K, Elhalik M, Miqdady M, Rawashdeh M, Salah M. 2017. Early childhood obesity: a survey of knowledge and practices of physicians from the Middle East and North Africa. BMC Pediatrics. 17(1):115.

Hatori M, Vollmers C, Zarrinpar A, DiTacchio L, Bushong EA, Gill S, Leblanc M, Chaix A, Joens M, Fitzpatrick JA. 2012. Time-restricted feeding without reducing caloric intake prevents metabolic diseases in mice fed a high-fat diet. Cell Metab. 15(6):848–860.

Ingenwerth M, Reinbeck AL, Stahr A, Partke H-J, Roden M, Burkart V, von Gall C. 2016. Perturbation of the molecular clockwork in the SCN of non-obese diabetic mice prior to diabetes onset. Chronobiol Int. 33(10):1369–1375.

Levin BE, Dunn-Meynell AA. 2002. Defense of body weight depends on dietary composition and palatability in rats with diet-induced obesity. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 282(1):R46–R54.

Mathur P, Das MK, Arora NK. 2007. Non-alcoholic fatty liver disease and childhood obesity. Indian J Pediatrics. 74(4):401–408.

Mendoza J, Angeles-Castellanos M, Escobar C. 2005. A daily palatable meal without food deprivation entrains the suprachiasmatic nucleus of rats. Eur J Neurosci. 22(11):2855–2862.

organization wh. 2016. Childhood overweight and obesity. [accessed]. http://www.who.int/dietphysicalactivity/childhood/en/.

Picard F, Kurtev M, Chung N, Topark-Ngarm A, Senawong T, Machado de Oliveira R, Leid M, McBurney M, Guarente L. 2004. Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-gamma. Nature. 429(6993):771–776.

Rinella ME. 2015. Nonalcoholic fatty liver disease: a systematic review. JAMA. 313(22):2263–2273.Rizza W, Veronese N, Fontana L. 2014. What are the roles of calorie restriction and diet quality in

promoting healthy longevity? Ageing Res Rev. 13:38–45.Romestaing C, Piquet M-A, Bedu E, Rouleau V, Dautresme M, Hourmand-Ollivier I, Filippi C, Duchamp

C, Sibille B. 2007. Long term highly saturated fat diet does not induce NASH in Wistar rats. Nutr Metab. 4(1):4.

Sutton GM, Perez-Tilve D, Nogueiras R, Fang J, Kim JK, Cone RD, Gimble JM, Tschöp MH, Butler AA. 2008. The melanocortin-3 receptor is required for entrainment to meal intake. J Neurosci. 28(48):12946–12955.

Sweat V, Yates KF, Migliaccio R, Convit A. 2017. Obese adolescents show reduced cognitive processing speed compared with healthy weight peers. Childhood Obesity. 13(3):190–196.

Woods SC, Seeley RJ, Rushing PA, D’Alessio D, Tso P. 2003. A controlled high-fat diet induces an obese syndrome in rats. J Nutr. 133(4):1081–1087.