Time restricted feeding attenuates metabolic dysregulation and demonstrates legacy effect in high fat diet rat model

Obesity is a major contributing factor in the development of many chronic conditions such as type 2 diabetes and hypertension. Intermittent fasting (IF) isa pattern of feeding that cycles between periods of fasting and eating. It allows eating within a specific time period, and fast during the rest of the day. Intermittent fasting appears to be a promising approach to prevent or reverse obesity. Our project involved the induction of obesity using a high fat diet (HFD; D12492‐60% kcal diet) in male Sprague Dawley rats. Blood glucose and ketone levels were measured twice a week and daily body weights were monitored. The rats being fed on the HFD gained 12% more weight than those on a standard diet (SD) and 14% weight gain compared to the growth chart of the animal supplier within seven weeks of feeding this special diet. Blood ketone and glucose levels remained unchanged between these groups. The animals with HFD, now referred to as the obese (OB) group and those with SD referred to as non‐obese (non‐OB) rats were placed on IF next. Intermittent fastingin this experiment was designed to allow animals to eat for 6 hours during their nocturnal window of time and fast during the remaining 18 hours. The OB and non‐OB groups were subdivided into five groups during the IF study: (1) OB rats on IF who were given HFD (OB‐IF‐HFD; N=3), (2) OB rats on IF given SD (OB‐IF‐SD; N=3), (3) OB rats maintained on HFD ad libitum (OB‐non‐IF; N=4), (4) non‐OB rats on IF with SD (non‐OB‐IF; N=3), and (5) non‐OB rats maintained on SD ad libitum (non‐OB‐non‐IF; N=3). Within three weeks of exposure to IF, there was a significant (p<0.05) decrease in body weight in the IF groups compared to the non‐IF groups receiving food ad libitum. However, blood ketone levels were significantly increased in the non‐OB‐IF rats compared to non‐OB‐non‐IF group, blood glucose levels were significantly decreased. A significant decrease in blood glucose levels was also noted in the OB‐IF‐SD group compared to OB‐non‐IF animals. This study suggests that HFD induces obesity as measured by body weight in approximately seven weeks, which can be reversed by subsequent practice of IF for three weeks but depends on the nature of the diet being used during IF i.e., IF‐induced weight loss with HFD is significantly less than with SD.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

We sincerely appreciate the opportunity to respond to the editorial by Mohr and Stine discussing our study on the effects of a 12-week Mediterranean-type time-restricted feeding (TRF) protocol in patients with metabolic dysfunction-associated steatotic liver disease (MASLD) [1, 2]. We are grateful for their insightful commentary and for highlighting the strengths of our randomised controlled trial (RCT). Their analysis underscores the emerging role of chrononutrition in managing MASLD while also highlighting key questions regarding the independent contribution of TRF and caloric restriction to metabolic improvements. There is indeed a great need for studies that directly compare ad libitum TRF protocols with caloric restriction to evaluate their differentiative impact on several metabolic parameters. Most of the studies in MASLD have compared either ad libitum TRF protocols with the usual dietary habits of the participants or hypocaloric diets in both TRF and control groups, as in our study. Our study was the first RCT that used the Mediterranean Diet (MD) as a control group, the gold standard for patients with MASLD [3]. The MD has been extensively documented as an effective intervention for MASLD [4]. Our study adds to this body of evidence by demonstrating that a hypocaloric MD, even over a short-term period, yields significant improvements in body weight, body fat, blood pressure and liver fat content [1]. Regarding the comment on the generalizability of the results [2], it is true that Greece is a Mediterranean country, and as described in previous studies, Greeks' adherence to the MD is moderate [5]. Our results [1] agree with these studies [5] and this may have enhanced our participants' adherence. The TRF interventions (early or late) did not seem to improve the metabolic parameters mentioned above further in this population [1]. However, insulin resistance and haemoglobin A1c (HbA1c) were only improved in the early but not in the late TRF group. The reduction in HbA1c in the early TRF group (0.3% in total, 0.37% in those with T2DM under early TRF) in our study [1] was greater than in other similar studies, for example, 0.2% in the study by Wei et al. (early TRF + caloric restriction group) [6], whilst this grade of improvement has been associated with lower mortality in individuals with T2DM [7] and reduction in diabetic complications [8]. Prior studies suggest that aligning food intake with circadian rhythms and the light/dark cycle via TRF may enhance glucose metabolism independent of caloric restriction as humans are diurnal [9]. This is particularly relevant for MASLD patients, where insulin resistance is a pivotal driver of disease progression [10]. That means that the differences in glucose metabolism observed in our study were probably due to the early TRF intervention, as all groups had the same caloric restriction. We would like to thank the authors for their thoughtful comments, which have allowed us to refine our interpretation and highlight the robustness of our findings. Future research will provide answers to all the raised concerns. Sofia Tsitsou: writing – original draft, investigation, methodology, data curation. Magdalini Adamantou: writing – original draft, data curation, investigation. Triada Bali: investigation, data curation. Aristi Saridaki: data curation, investigation. Kalliopi-Anna Poulia: methodology. Dimitrios S. Karagiannakis: methodology. Emilia Papakonstantinou: methodology. Evangelos Cholongitas: conceptualization, investigation, methodology, writing – review and editing, project administration, supervision, visualization, writing – original draft. The authors declare no conflicts of interest. This article is linked to Tsitsou et al papers. To view these articles, visit https://doi.org/10.1111/apt.70044 and https://doi.org/10.1111/apt.70078. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

Irregular timing of food intake increases hypertension and cardiometabolic disease risk. A chronic high fat diet (HFD) also disrupts circadian rhythms. We hypothesized that active period time restricted feeding (TRF) during the last 2 weeks in mice on a chronic HFD will improve blood pressure rhythm, diurnal variation of circulating plasma factors, and vascular metabolism. Mice (male 8-week old, C57BL/6J) were fed a normal diet (ND; 10% fat) or HFD (45% fat) for 20 weeks ad libitum. For the final 2 weeks, half of the HFD mice were subjected to TRF. Mean arterial pressure (MAP), heart rate (HR), and locomotor activity were assessed by telemetry. TRF significantly increased the active-inactive period difference in MAP and HR in in mice fed a HFD (ΔMAP: ND: 16±0.7 mmHg, HFD: 15±0.8 mmHg, HFD+TRF: 18±0.9 mmHg, n=6-8, p=0.01; ΔHR: ND: 68±5.1 bpm, HFD: 69±6.5 bpm, HFD+TRF: 113±7.9 bpm, n=6-8, p<0.01). Diurnal changes in locomotor activity are not different between groups. At the end of the study, plasma was collected at 4 hour intervals over a 24 hour period (ZT0 at 7AM; ZT12 at 7PM). Circulating levels of liver-derived mediators β-hydroxybutyrate (βHB) and insulin-like growth factor-1 (IGF-1) showed significant differences due to diet but not TRF (βHB, ZT21: ND: 0.16±0.01 mM, HFD: 0.20±0.02 mM, HFD+TRF: 0.19±0.01 mM, n=5-6, p=0.02; IGF-1, ZT5: ND: 232±18 ng/mL, HFD: 292±34 ng/mL , HFD+TRF: 371±14 ng/mL, n=5-6, p<0.01). Plasma leptin was significantly higher in mice on HFD and reduced by TRF at ZT12 (ND: 5.3±1.3 ng/mL, HFD: 22.5±2.9 ng/mL, HFD+TRF: 10.3±3.5ng/mL, n=5-6, p<0.01) and ZT17 (ND: 6.7±1.1 ng/mL, HFD: 32.5±3.0 ng/mL, HFD+TRF: 25.0±1.3 ng/mL, n=5-6, p<0.01). Plasma adiponectin was unchanged between all groups. TRF in HFD mice increased NAD + , important for metabolism, in renal vessels at ZT17 (HFD: 0.10±0.02 pmol/μg; HFD+TRF: 0.19±0.03 pmol/μg; n=5, p=0.03). Aortic NAD + at ZT1 was not affected by TRF in HFD mice (HFD: 1.83±0.35 pmol/μg, HFD+TRF: 1.35±0.35 pmol/μg, n=4, p=0.37). Our results indicate that TRF in mice on HFD increases the active-inactive period difference in MAP and HR and alters plasma metabolites, suggesting the timing of food intake on a chronic HFD improves cardiovascular rhythms with increased renal vascular metabolism and reduced leptin levels.

Obesity, where there is enhancement of stored body fat in adipose tissues, is associated with cardiovascular complications that are mainly related to atherosclerosis. Time-restricted feeding (TRF) is a form of restricted eating aimed at reducing weight in obese subjects. The present study aims to investigate changes in vascular endothelial function, endothelial nitric oxide synthase (eNOS), and protein kinase B (Akt) protein expressions with TRF in obese and normal rats. Male Sprague Dawley rats were divided into two normal and three obese groups; obesity was induced in the obese groups by feeding with a high-fat diet (HFD) for six weeks. After six weeks, rats were equally divided into five groups (n = 7 per group): Normal group (NR) which continued on a standard diet for six more weeks, normal group switched to TRF with a standard diet for six weeks (NR + TRFSD), obese group (OR) which continued on HFD for six more weeks, obese group switched to TRF of HFD (OR + TRFHFD), and obese group switched to TRF of a standard diet (OR + TRFSD). TRF was practiced for six weeks, after which the rats were sacrificed. Aortic endothelium-dependent and endothelium-independent relaxations and contractions were assessed using the organ bath. Aortic eNOS and Akt protein expressions were determined using immunoblotting. Fasting blood glucose, body weight, body mass index (BMI), serum lipid profile, Lee’s index, serum insulin levels, and sensitivity (HOMA-IR) were also measured. Endothelium-dependent relaxation was significantly impaired, while endothelium-dependent contraction increased in obese rats compared to that in normal rats. Both obese groups which underwent TRF with a HFD and standard diet improved their impairments in endothelium-dependent relaxation and reduced endothelium-dependent contraction; these were associated with increased expressions of aortic eNOS and Akt protein. Both obese groups with TRF reduced body weight, BMI, Lee’s index, total cholesterol, triglycerides, low-density lipoprotein cholesterol, and improved insulin sensitivity. TRF improved endothelium-dependent relaxation and reduced endothelium-dependent contraction, thus attenuating endothelial dysfunction in obese rats. These were associated with increased aortic eNOS and Akt protein expressions.

Cardiovascular disease remains a leading global cause of death, highlighting the need for new strategies to improve cardiovascular health. Time-restricted feeding (TRF), which limits daily food intake to a specific window, has shown promise in improving metabolic health and supporting weight control. This study investigated the effects of TRF in an obese rat model induced by a high-fat diet (HFD), focusing on early vascular, liver, and kidney structural changes, as well as oxidative stress and inflammation. Thirty male Sprague Dawley rats were assigned to five groups: a normal diet group (NOR), a normal chow with TRF (NOR + TRFNC), a continued HFD group (OB), an HFD with TRF group (OB + TRFHFD), and a group switched to TRF with normal chow (OB + TRFNC). Obesity was induced in three groups over six weeks, followed by a six-week intervention phase. TRF involved fasting for 16 h daily (5:00 p.m. to 9:00 a.m.). TRF led to improved lipid profiles and atherogenic indices in obese rats, regardless of diet. Elevated liver enzymes, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in obese rats were normalized by TRF. Additionally, TRF increased vascular superoxide dismutase (SOD) and decreased malondialdehyde (MDA), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α). Histological analysis showed that fat infiltration and steatosis in the liver were reduced by TRF. Renal and vascular structures also showed improvement. In conclusion, TRF exhibits anti-atherosclerotic effects, likely due to reduced vascular oxidative stress, inflammation, improved liver and kidney function, and better atherogenic profiles. These benefits were supported by histopathological findings in hepatic and renal tissues.

The effect of lifestyle modifications in the form of exercise training and fasting intervention on glucose metabolism in subjects on a high-fat diet is not completely understood. The present study aimed to examine the effects of alternate-day fasting (ADF) and high-intensity interval training (HIIT) on serum levels of C-peptide, fructosamine, and glucose in rats under a high-fat diet. Twenty-eight male Wistar rats were initially fed a high-fat diet for 12 weeks, then randomised into the following four groups: HIIT, ADF, HIIT + ADF, and control (CON). The HIIT and ADF interventions were conducted 3 days per week for 6 weeks. The HIIT induced a significant reduction in serum fructosamine levels compared to other groups (), as well as there was a significant reduction in serum glucose levels compared to the ADF and HIIT + ADF groups (). ADF and HIIT + ADF did not cause any significant changes in fructosamine, glucose, and C-peptide serum levels compared to the CON group (). In subjects under a high-fat diet, HIIT but not ADF or HIIT + ADF may be associated with favourable improvements in glucose metabolism markers.

Obesity has become a prevalent global health issue, and recently it has been reported to be intimately associated with neuronal health. Obesity triggers peripheral inflammatory responses concomitant with neuroinflammation, elevated oxidative stress, and compromised autophagy. Intermittent fasting (IF) positively influences lowering body weight and improving the metabolic changes accompanying obesity. IF also has a beneficial impact on neuronal function; however, no studies have discussed this effect on high-fat diet (HFD)-induced cerebellar damage. This study examines the effect of IF on the cerebellum of HFD-fed rats. Male Wister Albino rats (n = 16) were fed HFD for 16 weeks (HFD group); half of them were subjected to IF alternating with HFD for 6 weeks starting at the 11th week till the end of the experiment (fasting + HFD group). The control group of rats (n = 8) was kept on a basal diet. The animals were euthanized after 16 weeks. Their tissue was harvested and processed for morphology using H&E, cresyl violet, and luxol fast stains, and immunohistochemical staining was carried out for inflammatory marker (TNF-α), gliosis marker (GFAP), and autophagy markers (LC3 II and P62). Oxidative stress markers (SOD, MDA) were measured, and protein expression of phosphorylated-AMP-activated protein kinase (p-AMPK) and phosphorylated-rapamycin complex (p-mTOR) in cerebellar tissue was detected via western blotting. IF mitigated HFD-induced cerebellar morphological changes, reduced cerebellar TNF-α expression, decreased oxidative stress markers, and balanced p-AMPK and p-mTOR with autophagy improvement. Moreover, a decrease in body weight and ameliorated obesity-induced metabolic changes in the serum levels of glucose, insulin, cholesterol, and triglyceride were seen. These observations suggest that IF can improve both peripheral and central changes prompted by HFD through attenuating inflammation, oxidative stress, and reestablishing the autophagy balance.

Intermittent fasting reduces body fat but exacerbates hepatic insulin resistance in young rats regardless of high protein and fat diets

Fatty liver disease has become increasingly prevalent. While intermittent fasting (IF) is gaining popularity for its potential metabolic benefits, its specific effects on liver function and related molecular pathways remain unclear. This study aimed to examine the impact of IF on liver function, histological changes, and transforming growth factor-B1 (TGF-B1) and matrix metalloproteins 9 (MMP 9) hepatic expressions. A total of 24 male Sprague Dawley rats were divided into four groups: Control group, Lean fasting group fed standard chow combined with IF, Fatty liver group fed high-fat diet (HFD), Fasting fatty liver group fed HFD combined with IF as 24-h alternate-day fasting for eight weeks. Finally, oxidative stress, hepatic histology, immunohistochemistry expression of TGF-β and MMP 9, liver function tests, and m RNA expression of inflammatory markers were evaluated. The results showed that IF combination with HFD has decreased the degree of degenerative alterations and fibrosis at the portal area (p < 0.001), along with a considerable reduction in liver enzymes (p < 0.001), glucose and cholesterol (p < 0.001), oxidative stress, and inflammation caused by IF. Furthermore, compared to the fatty liver group, there was a significant decrease in hepatic expression of MMP 9 (p < 0.001), and TGF-β1 (p < 0.001). In our conclusion, by regulating TGF-β1 and MMP 9 expression in HFD-fed rats and having anti-inflammatory and antioxidative stress capabilities, IF may lessen hepatic fibrotic alterations.

High-fat diet is considered as the main risk factor for the development of dyslipidemia and lipid peroxidation, which in turn results in serious health problems such as cardiovascular disease, type 2 diabetes, and some forms of cancers. Therefore, the purpose of this study was to investigate the combined effect of high-intensity interval training (HIIT) and intermittent fasting (IF) on lipid profile and peroxidation in Wistar rats under a high-fat diet. A total of 28 male Wistar rats went through a high-fat diet for 12 weeks. Then, they were randomly divided into four groups: (1) HIIT (3 days week−1 for 6 weeks), (2) IF (3 days week−1 for 6 weeks), (3) combined HIIT and IF (received both treatment for 6 weeks), and (4) control (CON). All groups were under a high-fat diet until the end of the study. According to the results, LDL levels significantly decreased in the HIIT group compared to the CON group, and in the IF and HIIT + IF groups, a non-significant increase in HDL was observed compared to the CON group. In HIIT + IF and HIIT groups, LDL/HDL ratio decreased significantly compared to the CON group (P < 0.05). Moreover, in IF group, a significant decrease in TG occurred in comparison with HIIT and HIIT + IF groups. A significant increase in serum MDA levels was observed in IF and HIIT + IF groups but not in the HIIT group in comparison with the CON group. Our results suggest that in subjects under a high-fat diet, both HIIT and IF may help to improve lipid profile, but their combination may not have any synergistic effect. Also, IF and HIIT + IF may increase lipid peroxidation in subjects under a high-fat diet.

Time-Restricted Feeding a High-fat Diet in Mice Elevates Hepatic Long-Chain Polyunsaturated Fatty Acid Content and Modifies the Triacylglyceryl Lipidome (P21-013-19)

Intermittent fasting (IF) is an alternating pattern of restricting eating. This study evaluated mental and physical fatigue secondary to IF (daily 18-hour fast, 7-days-a-week) in the high-fat diet (HFD)-induced male obese Sprague Dawley rats. Fifty-four rats were randomly assigned to a HFD (n = 28) or a standard diet (SD; n = 26). After six weeks, the HFD rats were divided into one of four groups: obese HFD ad libitum (OB-HFD-AL), obese HFD-IF (OB-HFD-IF), obese SD-AL (OB-SD-AL), and obese SD-IF (OB-SD-IF). Similarly, non-obese controls were grouped into HFD-AL (C-HFD-AL), non-obese HFD-IF (C-HFD-IF), non-obese SD-AL (C-SD-AL), and non-obese SD-IF (C-SD-IF). After 2 weeks of IF, mental and physical fatigue were measured using open field (OF) and novel object recognition (NOR) tests. Rats on IF gained weight at a slower pace (p<0.05) and had lower glucose levels (p<0.01) compared to the AL group. In non-obese rats, ketone levels were higher in the IF-HFD group than IF-SD (p<0.05) and AL-SD (p<0.01) animals. Obese rats exhibited elevated blood ketone levels in IF-SD conditions versus AL-SD rats (p<0.01). AL-HFD rats had higher ketone levels than AL-SD animals in both obese and non-obese groups (p<0.05). In conclusion, rats with higher blood ketone levels, whether they were on IF or AL, traveled a greater distance during OF suggesting a lack of physical fatigue. There was no significant difference between IF and AL during NOR indicating a lack of mental fatigue. Thus, IF results in reduced body weight and blood glucose levels but does not induce physical or mental fatigue.

Time-restricted feeding (TRF), a chrono-nutrition-based intervention, has shown promise in mitigating the adverse effects of high-fat diets (HFDs) on metabolic and hepatic health. This study evaluates the protective effects of TRF on hepatic gene expression, inflammatory markers, and serum adipocytokine levels in a rat model of HFD-induced hepatic steatosis. Forty male Wistar rats were divided into four groups: control, control + TRF, HFD, and HFD+TRF. The TRF was implemented as an 18-hour fasting period followed by a 6-hour feeding window for 12 weeks. Body composition, glucose homeostasis, lipid profiles, serum inflammatory markers, and adipocytokines (adiponectin, leptin, resistin) were measured. Hepatic oxidative stress markers (malodialdehyde, total antioxidant capacity) and gene expression of inflammatory mediators (nuclear factor-κB, tumor necrosis factor-α, interleukin-1β, interleukin-6, high-sensivity-C-reactive protein) were assessed. Histopathological analysis of liver tissue evaluated steatosis and p53 expression. TRF significantly reduced body weight in HFD-fed rats from 396.5±6.9 g to 350±6.4 g (p<0.0001). Homeostasis model assessment of insulin resistance (HOMA-IR) decreased from 2.03±0.06 to 0.72±0.05 (p<0.05), and hepatic malodialdehyde levels were reduced from 25 to 12.42 nmol/g (p<0.0001). TRF also downregulated inflammatory cytokines, including tumor necrosis factor-a and nuclear factor-κB, while increasing adiponectin levels and improving adiponectin/leptin and adiponectin/resistin ratios. Histological examination showed steatosis improved from grade 3 to grade 1 or 0. We found that RF effectively mitigates HFD-induced hepatic steatosis and metabolic dysfunction by reducing oxidative stress and inflammation and modulating adipocytokine balance. These findings highlight TRF's potential as a therapeutic strategy for managing hepatic and metabolic disorders.

Kidney function follows a strong circadian rhythm that is tightly regulated by clock genes. We previously reported that high salt diet induced dyssynchrony in renal clock gene expression in the cortex and medulla. However, whether changes in other dietary factors pose threats to renal circadian rhythms remain largely unknown. The current study was designed to test the hypothesis that high fat diet disrupts renal clock gene expression with 2 mouse models. First, C57Bl/6J male mice (8 weeks old, n=3) were fed normal fat (NF) or high fat (HF) diet for 20 weeks. During the last 2 weeks of the protocol, mice underwent either time restricted feeding, where food access was allowed only during lights‐off, active period (7 pm–7 am), or sham feeding procedure. Restricted feeding did not cause significant changes in body weight or food consumption. Renal cortex was collected in 4‐hour increments throughout a 24‐hour period and clock gene expression measured by qPCR. We found that Per2 expression has a robust circadian rhythm with a peak expression at zeitgeber time (ZT) 13. We did not observe any significant differences among groups. Second, Period2Luciferase (Per2Luc) mouse model was utilized to monitor real‐time molecular clock rhythm. Male and female Per2Luc mice (n=4) were fed NF or HF diet with sham or time restricted feeding. Kidneys were dissected into three parts (cortex, outer and inner medulla) at the end of study and cultured for 3 days to measure bioluminescent rhythms. We found that HF diet lengthened the period of luciferase rhythm by 2 hours (NF: 23.98 hours vs. HF: 26.17 hours, p=0.02) in the cortex. No significant differences were observed in the groups in renal outer medulla or inner medulla. Restricted feeding did not cause any significant changes in any groups. These data suggest that clock gene expression in the kidney follows a circadian rhythm that can be disrupted by chronic high fat diet.Support or Funding InformationP01 HL136267 Integrating novel mechanisms controlling sodium excretion and blood pressure UAB School of Medicine AMC21 Circadian Disruption and Susceptibility to Target Organ DamageThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Beneficial effects of intermittent fasting on steatosis and inflammation of the liver in mice fed a high-fat or a high-fructose diet.