Time-Restricted Feeding Improves Body Weight Gain, Lipid Profiles, and Atherogenic Indices in Cafeteria-Diet-Fed Rats: Role of Browning of Inguinal White Adipose Tissue

Obesity is defined as abnormal or excessive fat accumulation that may impair health [1]. The etiology of obesity is quite complicated owing to the involvement of both genetic and environmental factors. Among the environmental factors, diet plays a significant role in the development of obesity [2]. Modern nutritional patterns, termed as “Western diets”, including high-fat and -cholesterol, high-protein, high-sugar, and excess salt intake, as well as frequent consumption of processed and fast foods, cause a high prevalence of obesity in Western societies [3,4]. According to the data published by the World Health Organization, the worldwide prevalence of obesity increased threefold over the last 40 year sand is expected to reach ~30.3 billion by 2030 [1]. Obesity is often associated with a condition known as metabolic syndrome and characterized by insulin resistance, dyslipidemia, hypertension, and systemic inflammation, as well as cardiovascular diseases [5]. Classical attempts to develop therapeutic strategies have mostly focused on limiting caloric intake by diet and increasing energy expenditure by physical exercise; however, these strategies are often not effective, withunsatisfactory results and success limited to a small percentage of individuals [6]. Today, TRF, an eating pattern that limits food intake to a specific number of hours (≤12 h) without altering nutrient quality or quantity, is emerging as a promising therapeutic strategy against obesity and associated metabolic disorders [7]. One outstanding study pioneered by Panda’s groupdemonstrated that mice fed a high-fat diet (HFD) under TRF paradigm of 8 h per day during the active phase were protected against obesity, hyperinsulinemia, hepatic steatosis, and inflammation, even though they consume equivalent calories to those with ad libaccess to the HFD [8]. Interestingly, another study by the same group showed thata TRF of 8–12 h without reducing caloric intake in mice prevented and even reversed obesity and related metabolic disorders arising from a variety of obesogenic challenges, including high-fructose and high-fat, high-sucrose diets [9]. To date, there have been extensive studies in both rodents and humans reporting that TRF reduces body weight, improves glycemic control and insulin resistance, prevents atherogenic dyslipidemia, reduces blood pressure, prevents hepatic steatosis, and improves inflammatory markers in diet-inducedobesity models (DIO) [10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25]. It should be noted that most rodent studies have attempted to mimic the unhealthy human Western diet through the use of numerous obesogenic diets, which generally focused on a single or a combined macronutrient (s), in particular, fat and/or sugar. However, these commercial high-fat, high-sugar or high-fat, high sugar rodent diets are not analogous to the highly processed food common in Western societies and associated with increased global obesity rates. The closest comparable to human Western foods is the cafeteria diet (CAF), as it provides animals with nutritionally varied diet of high-energy, palatable human foods that are low in fiber and contain substantial amounts of fat, sugar, and salt (for example, cheese, chocolate, processed meat, chips, and cookies), thereby recapitulating the key obesogenic features of the unhealthy human diet [26,27]. Furthermore, an extended CAFdiet in rodents produced an exaggerated phenotype of obesity with excessive body weight gain, pronounced adiposity, dyslipidemia, and liver inflammation [28,29,30] and has been argued to model modern human obesity and related metabolic disorders more severely than the classical HFD mode [28]. In addition, a CAFdiet exerted a robust impact upon appetite and energy intake due to its high amount of sugar, salt, and additives, such as appetizers and flavor enhancers, compared to the traditional HFD [31]. To the best of our knowledge, to date no study has reported on whether a TRF has the potential to prevent obesity and related metabolic disarrangements in a CAF-diet-induced obesity model. Furthermore, the mechanism that underlies the beneficial effect of the TRF paradigm on obesity is still not welldefined.Various mechanisms have been proposed to explain the anti-obesity effect of a TRF regimen, includingentraining the circadian clock to a fixed feeding time [32], altering gut microbiome [33], and increasing free fatty acid mobilization and fat oxidation [8]. However, it remains to be determined whether TRF can trigger white adipose tissue (WAT) browning. WAT browning, a process of formation of brown-like adipocytes within WAT, is regarded as a potential therapeutic target for obesity due to its unique capacity to up-regulate thermogenesis and thusincrease energy expenditure through glucose and fatty acidoxidation.Brown-like adipocytes, termed “beige cells”, are characterized by multilocular lipid droplets and high amounts of UCP1-positive mitochondria, and they switch from an energy storage to an energy expenditure state by expressing thermogenic markers, including UCP1 and PGC1α, which constitute the molecular signature of brown and beige adipocytes and play an important role in metabolic thermogenesis [34]. Classically, browning of WAT can be induced in animals and humans by various physiological cues, such as exercise and cold, and pharmacological agents, suchasan active ingredient like melatonin, which has previously been demonstrated by our group to have an iWAT browning potential in obese diabetic animals [35]. Based on previous findings showing that TRF regimen reduced body weight gain in DOI without altering caloric intake or activity level [8,12,19], we hypothesized that a TRF regimen would prevent obesity via promoting WAT browning. Hence, the present study was aimed to address whethera TRF schedule of 8 h per day during the active phase can attenuate excessive body weight gain inCAF-diet-induced obeserats. There arescarce data available in the literature regarding the effects of TRF on lipid profile and atherogenicity, in animal models of obesity, and therefore, we determined whether TRF can prevent the dyslipidemia produced in response to a CAFdiet. Additionally, the potential role of TRF paradigm as a white fat browning inducer was also explored.

Herein, we report, for the first time, that a TRF regimen prevents obesity and associated dyslipidemia in CAF-diet-induced obese rat model. This effect occurred probably via browning of the iWATwithout changing the calorie intake or locomotor activity.

Our results showed that consumption of calorically dense, palatable human foods induced an excessive caloric intake, resultingin rapid and drastic weight gain, abdominal adiposity, and dyslipidemic state in thead lib-fed group, suggesting that the CAF-diet used in the present study successfully induced an animal model of obesity. As shown in Table S1, the CAFdiet contained foods that are high in fat and saturated fatty acids; hence, the weight gain in the CAF-ad lib group could be due to the high rate of acylation of saturated fatty acids into triglycerides that are subsequently stored in the adipose tissue [44]. Besides, post-ingestive effects of high-fat food contribute to weight gain through reduction of satiety signals [45] and attenuation of fatty acid oxidation [46]. Interestingly, TRF significantly reduced the body weight gain in our rats fed on a CAF-diet, suggesting thatTRF is an effective strategy for weight reduction with aCAF-style diet that models the Western pattern diet. Similar findings have also been previously reported in different animal strains after exposure to TRF of other obesogenic diets [8,9,12,19,20,21,23]. This has also been observed among some overweight or obese humans undergoing a TRF eating pattern [14,15]. It should be noticed that the body weight gain-lowering effect of TRF in the current study took place in the absence of changes in caloric intake and locomotor activity, which is consistent with previous studies [8,9,12,19]. Therefore, the mechanism whereby TRF reduced body weight cannot simply be explained by changes in quantity of diet or physical activity, which represent the locomotor activity of 5 min duration of the dark phase (phase of activity) of the 15th week of the experiment, but rather by other mechanisms, as discussed below. Our present data of calorie intake are not consistent with another previous report in which mice fed an HFD under TRF of 12 h during the dark or light phase were unable to fully compensate for the limited availability of food, and calorie intake was significantly reduced compared with their ad lib counterparts [47]. The reason for this discrepancy is uncertain and requires further investigation.

One potential explanation to account for the attenuated body weight gain without any overall change in calorie intake or physical activity upon TRF is through switching lipid metabolism from storage to oxidative as a strategy to enhance energy expenditure. Although energy metabolism was not measured in the current study, the study by Panda’s group showed that 8hTRF during the active phase attenuated the HFD-induced dampening of the daily rhythm of the respiratory exchange ratio and led to an overall increase in oxygen consumption and energy expenditure in mice, indicating lipid utilization for energy metabolism [8]. In another study by the same group, TRF of HFD or normocaloric diet increased PGC1α gene expression in WAT of mice, leading to increased β-oxidation [9], which corroborates our data of PGC1α.

Ourfindingsconcerning body weight gain paralleled those of fat accumulation measurements. Notably, rats fed a CAFdietad lib exhibited drastic increases in fat pad weights (inguinal, mesenteric, epididymal, retroperitoneal) compared to rats fed an NCdietad lib. These observations suggest that the exposure of rats to calorically dense diets facilitated fat accumulation in the abdominal regions due to the high effective energy content of the high-fat foods [48]. Previous studies have observed a marked reduction in adipose tissue mass in rodents under a TRF regimen [8,9,23], which is in agreement with the present finding showing that TRF of NC- or CAF-diet reduced fat accumulation in subcutaneous and visceral fat, resulting in a significant decrease in total adiposity. The reduction of fat accumulation in WAT under TRF may be due to an overall increase in fatty acid oxidation and a decrease in free fatty acid synthesis [9], which paralleledthe current data showing that adipocytes size of iWATinTRF rats were smaller than thatin diet-matched ad lib rats.

The excessive weight gain in the CAF–Agroupwas associated with a dyslipidemic condition, as evidenced by abnormally elevated serum levels of TG, TC, and LDL-c and reduced serum level of HDL-c, which is consistent with the findings of a previous study using a CAFdiet [49]. The dyslipidemia was probably evident due to the overall higher consumption of the CAFdiet, which, while being high in fat, is also high in saturated fatty acids, known to increase the production of TG and LDL-c by the liver [50], and in cholesterol, which can result in a hypercholesterolemia [51] and reduced HDL production or HDL clearance from plasma [52]. Dyslipidemia may also beexplained bya hepatic lipid accumulation, which was associated with disrupted circadian rhythms and food intake patterns in response to high caloric diet [53]. Interestingly, the TRF regimen improved the negative lipid profile in CAF-fed rats by decreasing serum levels of TC, TG, and LDL-c and increasing serum level of HDL-c, suggesting its anti-hyperlipidemic effect.However, TRFunexpectedly had no effect on dyslipidemic parameters when rats were fed with anNCdiet, in spite of a marked effect on body weight. The lack of the TRF effect on dyslipidemic parameters in normocaloric-fed rats may indicate that the influence of TRF may be negligible when the overall content of diet is less caloric, which would require future investigation.To our knowledge, only three studies have been reported so far on the anti-hyperlipidemic potential of TRF in DIO models(Chaix et al. [9], Sherman et al. [23], and Sun et al. [12]). It is important to note that there were considerable variations in experimental design, such as obesogenic diets, animal strain, feeding window, and experimental durations between the present study and the aforementioned studies, which make direct comparison difficult.The mechanism wherebyTRF underlies lipid parameters is unclear. Typically, weight loss is expected to promote lipidimprovements [54]; however, outcomes from thecurrent studydo not consistently support improvements in body weight as, despite reduced body weight gain, no changes in lipid panel were recorded in NC-fed rats. Since the liver is a central organ in lipid metabolism, we suggest that the beneficial effect of a TRF regimen on lipid profiles in CAF-fed ratsmay be due to the enhancement of hepatic lipid metabolism. This is supported by several studies in which TRF has been found to enhance lipid oxidation over lipogenesis [8,12,21,55]. Indeed, TRF has been shown to reduce hepatic expression of PPARγ and SREBF1, the key lipogenic genes, in HFD-fed rats [55]. In addition, TRF regimen reduced lipid droplet associated and lipolysis inhibitor gene Cidec expressions, TG storage-associated protein CD36, and plasma TG marker ApoA 4 in HFD-fed mice [8].Decreased TG in the liver results in reduced serum levels of LDL and very-low-density lipoprotein (VLDL), leading to loss of transported cholesterol and TG within them [54]. The TC-lowering effect of TRF may also be attributed to enhanced cholesterol catabolism to bile acids biosynthesis in theliver, as indicated by increased expression of two rate-limiting enzymes of bile acids biosynthesis, namelyHmgcs 2 and Cyp7α1, in the liver of time-restricted mice [9].

It is commonly thought that unfavorable lipid profiles, synonymous with intake of dietary saturated fat, are the hallmark for the progression of cardiovascular diseases (CVD) and coronary heart diseases [56].Observations from epidemiological studies have confirmed that high TC, LDL-c, and TG and low HDL-c levels are associated with increased risk of CVD [56,57,58,59]. Together, thesedatacorroborate the present findings showing marked increases in AIP, AC, and CRI in CAF–A rats compared to NC–A rats. These indices are strong indicators of the CVD risk in clinical practices by their expression of imbalance between atherogenic and anti-atherogenic lipoproteins [60]. A recent human pilot study reported the effectiveness of 10 h time-restricted eating intervention for 12 wks to lower atherogenic lipids in obese subjects with metabolic syndrome, leading to improved cardiometabolic health [61]. Herein, TRF reduced the calculated values of AIP, AC, and CRIin CAF-fed rats, suggesting its very interesting cardioprotective potential. Since this is the first study to examine the effect of TRF onatherogenic indices in an animal model of obesity, these results should be considered preliminary; therefore, further in-depth investigationsare warranted to confirm the cardioprotective properties of TRF intervention in DIO models.

Since white fat browning is associated with body weight loss and metabolic improvements, we aimed to characterize the morphological and molecular features of iWAT. As hypothesized, we found that, in TRF rats, there was an obvious appearance of multilocular brown adipocytes and increased UCP1 and PGC1α protein expressions in iWAT of NC- and CAF-fed rats, indicating its potential to promote WAT browning.In one interesting study, intermittent fasting, an eating pattern that involves a 24 h fasting period on 2–3 days per week, or on alternate days, was reported to promote inguinal and gonadal fat browning in lean and obese mice [62]. In support of these findings, results from the current study showed that 8hTRF induced iWAT browning in normocaloric and CAF-fed rats. To the best of our knowledge, this report is the first to show the potential of TRF to promote WAT browning.Direct experimental evidence for the role of UCP1 in counteracting obesity has been reported previously in many studies. Of note, an animal model of genetic obesity using adipose tissue-targeted overexpression of UCP1 resulted in a reduced level of obesity [63]. In addition, UCP1 ablation resulted in increased obesity and metabolic deficiency in obesity-resistance mouse strain [64]. In humans, UCP1 gene polymorphism was significantly associated with increased body mass index in obese subjects [65]. Therefore, the present data concerning the iWAT browning and UCP1 expressionmay decipher the possible mechanism behind the body weight gain-lowering effect of a TRF regimen. Although the current data support the possibility that a TRF regimen could act as a WAT browning inducer, further in-depth studies are warranted to understand whether a TRF regimen could promote browningin other WAT depots and to investigate the expression of browning-related genes. Furthermore, the present study is limited by the fact that we did not measure whole-body energy expenditure due to practical reasons; hence, to what extent the effects of a TRF regimen on WAT browning andUCP1 content could be translated into increased whole-body energy expenditure is unclear, and future studies will address this shortcoming.A further limitation is that we did not measure a 24-h profile of locomotor activity. Therefore, the 5-min locomotor activity, which was measured at different times between groups, could induce time effects rather than TRF effects. However, as the locomotor activity for all groups was assessed within a short period of 2 h during the dark phase, it suggests that the locomotor activity was not affected by different times between groups but rather by TRF, although this hypothesis needs to be confirmed.

This present study showed that TRF regimen improved body weight gain and dyslipidemia in CAF-diet model and acted as white fat browning inducer with thermogenic properties, which may explain the anti-obesity effect of TRF.

We would like to express our appreciation to all staff from Diabesity-UGR group (AnaTorrices, DiegoSalagre, and Juan Miguel Leiva) for their technical support and for being attentive to answer our research questions and to AsmaAmalou from Physiopathology-USTHB group for the animal sacrifice assistance. We would also like to express our warmest thanks to NemchaLebaili and her staff from ÉcoleNormaleSupérieure (Kouba, Algiers) for the use of their animal facilities.

The following are available online at https://www.mdpi.com/2072-6643/12/8/2185/s1↗, Table S1: normal chow and cafeteria food items compositions as given by the product manufacturers.

The authors’ responsibilities are as follow: Research design conceptualization, A.A. and S.A.; animal experiment, S.A.; data analysis and interpretation, S.A.; histological techniques, M.C.; writing—original draft preparation, S.A.; writing—review and editing, A.A. and S.B.-A.; supervision, A.A. and S.B.-A. All authors have read and agreed to the published version of the manuscript.

This research was funded by SAF2016-79794-R grant from the “Ministerio de Economía y Competitividad (Spain)” and by Erasmus + Mobility Program DimensiónInternacional (grant number KA 107) from “European Commission”.

The authors declare no conflict of interest.