**5. Effects of Diet on Metabolic Syndrome**

The Western diet, characterized by a high intake of red and processed meat, refined grains, sweets, and sugary beverages, is associated with an increased risk of developing MetS [60,61]. This diet is calorie-dense, rich in small-chain fatty acids (SFA), simple carbohydrates, and other nutrients that feature pro-inflammatory properties, disrupt the gut microbiota, and dampen insulin sensitivity [61–63]. A meta-analysis by Fabiani et al. has shown that the "Meat/Western" pattern leads to a 19% increase in MetS risk, while a "Healthy" dietary pattern (fruit, vegetables, whole grains, fish, no processed food/high content of vitamins, minerals, antioxidants, fiber, MUFA, and n-3 fatty acids) is associated with a 15% decrease in MetS risk [60]. Similar results were obtained in another meta-analysis that also studied the relationship between a posteriori dietary patterns and MetS: a healthy/prudent diet was associated with a lower prevalence of MetS, while an unhealthy/Western pattern was associated with an increased risk of developing MetS [64]. Another popular dietary pattern, the Mediterranean diet, has also shown benefits regarding MetS [65,66]. In a meta-analysis by Kastorini et al., the combined effect of prospective studies and clinical trials showed that the Mediterranean diet is associated with a reduced risk of MetS (log hazard ratio: −0.69, 95% confidence interval (CI): −1.24 to −1.16) [67]. Comparable results were achieved with the dietary approaches to stop hypertension (DASH), where multiple studies showed how the DASH diet led to a reduction in systolic and diastolic blood pressure, a reduction in BMI and waist circumference, an improvement in cardiometabolic profile, and a reduction in T2DM incidence [68–72].

Therefore, there is substantial evidence that diets such as the Mediterranean diet and DASH exert a beneficial effect on cardiometabolic risk factors, with a common theme in which foods such as vegetables, fruit, whole grains, and fish are associated with these benefits. Nevertheless, in addition to the diet itself, dietary regime adjustments may provide metabolic benefits regardless of the amount and type of food ingested.

### **6. Metabolic Syndrome and Fasting**

#### *6.1. Evidence on Animal Models*

A number of studies have exhibited the beneficial effects of fasting/time-restricted feeding on animal models. A number of studies have exhibited the beneficial effects of IF/TRF on animal models. In a study by Hatori et al., the authors demonstrated the favorable effects of TRF in mice fed with a high-fat diet (HFD, 61% energy from fat). The group fed during an 8 h period lost weight and exhibited better insulin sensitivity, less hepatosteatosis, less inflammation, and improved motor coordination while consuming the same number of calories as the group fed ad libitum (AL) [73]. The beneficial effect of TRF was substantiated from a biochemical standpoint as well, since the TRF regimen improved CREB, mTOR, and AMPK (metabolic master regulator) pathway function [73]. Similar results were achieved in another study where, in comparison to mice on high-fat (HF) AL diets, mice on a time-restricted HF diet experienced an 18% reduction in body weight, a 30% reduction in cholesterol levels, 10% lower TNF-α levels, and a 3.7 fold increase in insulin sensitivity [74]. Moreover, in the same study, the authors compared mice on a time-restricted HF diet with mice on an AL low-fat diet consuming the same number of calories. In this comparison, the TRF mice showed a 2% bodyweight reduction, a 21% reduction in cholesterol levels, and a 1.4 fold increase in insulin sensitivity [74].

Furthermore, in a recent study, Pak et al. used a series of feeding regimens to dissect the effects of caloric restriction (CR) and fasting [75]. Four groups of mice were examined: (1) mice given AL access to a regular rodent diet; (2) mice provided with AL access to chow that was 50% diluted with indigestible cellulose (corresponding to a 30% CR); (3) mice fed 30% less food than AL-fed mice using an automatic feeder to release food in three equal portions during the 12 h dark period; (4) mice fed once per day in the morning, with 30% restriction compared to the AL group; (5) mice that were trained to consume approximately the same quantity of the food as AL-fed mice, but within three hours. The crucial comparison was between mice on a diet according to which they consumed fewer calories without imposed fasting (AL chow diluted with 50% indigestible cellulose) and mice that were fed only once per day in the morning (without CR). The study showed that fasting is needed for CR-induced improvements in glucose metabolism, frailty, and lifespan in C57BL/6J male mice. In addition, the results elucidate how fasting alone, without the reduced intake of calories, is sufficient to realize the metabolic phenotypes and transcriptional signature of a CR diet [75]. The importance of these studies that they show the benefits of TRF whilst controlling for caloric intake, thus decoupling the effects from caloric restriction.

Other studies have proven the benefits of fasting in alleviating circadian clock disruptions and improving metabolic homeostasis [29,76]. As reviewed in a paper by Swi ˛ ´ atkiewicz et al., a proper TRF cycle sustains circadian rhythms and restores normal daily rhythms in many mRNAs, proteins, and metabolites that are involved in the homeostasis of carbohydrates and lipids [29]. TRF also regulates circulating leptin and adiponectin levels, and exerts multiple valuable effects on the main organs involved in metabolic homeostasis (liver, muscle, white and brown adipose tissue, gut, and brain) [29,76–78]. Interestingly, a study on mice has shown the effects of IF (every-other-day fasting – EOFD) on white adipose tissue browning [79]. The EOFD mice exhibit significantly more beige fat development within white adipose tissue, were less obese, better insulin sensitivity, and less hepatic steatosis. The researchers also pointed out how these beneficial effects are most probably exerted through the shaping of gut microbiota because the microbiota-depleted mice were resistant to EODF-induced beiging, while microbiota transplantation from EODF-treated mice to microbiota-depleted mice activated beiging and improved metabolic homeostasis [79]. Moreover, a study on a drosophila melanogaster (fruit fly) model of obesity, in which flies were subjected to a 12 h TRF, exhibited improved muscular function (fewer intramuscular fat deposits), improved phosphorylated AKT levels, fewer mitochondrial aberrations, and better insulin sensitivity [80].

Importantly, not all fasting protocols exhibit beneficial effects, as was shown in a study investigating alternate-day fasting in young female Wistar rats (24 h fasts intercalated with 24 h of free access to the same chow) [81]. Alternate-day IF decreased weight gain and food intake, but it led to increased fat reserves, elevated plasma insulin concentrations, and reduced muscle mass. Although the study was conducted on young and healthy animals with no MetS, these findings suggest the potential adverse effects of alternate-day fasting and promote caution in thinking that all fasting methods are similar and beneficial.

Overall, the above-noted animal studies have set the biochemical and pathophysiological evidence and groundwork (positive effects on weight, obesity, gut microbiota, energy metabolism, and circadian rhythms) for further human studies on patients with obesity and MetS (Figure 1, Table 1).


**Table 1.** Studies examining fasting on animal models of MetS and obesity.

Abbreviations: HFD = high-fat diet, CR = caloric restriction, CREB = cAMP response element-binding protein, mTOR = mammalian target of rapamycine, AMPK = 5' AMP-activated protein kinase, TNFα = tumor necrosis factor alpha, TRF = time-restricted feeding, EODF = every-other-day fasting.
