1. Introduction
The Dietary interventions are broadly considered to be powerful non-pharmacological approaches not only for losing weight and adiposity but also for the prevention of metabolic and cardiovascular diseases in diverse model organisms [
1,
2,
3,
4].
Intermittent fasting (IF) has been promoted as an alternative dietary weight loss strategy to caloric restriction diets [
5]. The IF regimen encompasses several patterns, including every-other-day fasting (feeding no calories on fasting days), every-other-day modified fasting (feeding less than 25% of caloric demand on fasting days), cyclic fasting (fasting on two to three days per week), and time-restricted fasting (limiting the interval of daily food intake to specific daytimes).
Accumulating evidence from both human and animal studies suggests that IF, in particular every-other-day fasting, has ample potential to improve cardiometabolic health [
6,
7,
8,
9,
10].
Human studies have reported that IF interventions favour body weight loss. However, these studies have been primarily conducted in small populations including non-obese healthy individuals or metabolically healthy overweight or obese patients [
11,
12,
13,
14]. Moreover, Wilkinson
et al. recently reported that IF interventions in humans efficiently promote body weight loss but only slightly improve glucose metabolism [
15]. Yet IF significantly reduced several cardiovascular risk factors, such as blood pressure and plasma atherogenic lipids levels, including triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) in patients with metabolic syndrome [
15,
16]. However, the dietary regimen compositions were poorly documented in these studies, and no study to date has assessed the effect of IF on patients with mild or advanced atherosclerosis.
In mice, IF intervention extends lifespan independently of sex and calorie intake [
17]. In both sexes, IF protects mice from diet-induced obesity, glucose intolerance, insulin resistance, liver steatosis, systemic inflammation, and dyslipidemia [
18,
19]. These beneficial effects of IF on metabolic syndrome are mediated by diverse mechanisms including modulation of adipokine production [
20], improvement in insulin sensitivity, reduction in oxidative stress and inflammation, and enhancement of adaptive stress responses such as autophagy, proteostasis, and endogenous antioxidant systems [
17,
21].
Three studies have analysed the impact of IF in atherogenesis in atherosclerosis mouse models, apolipoprotein E- and low-density lipoprotein receptor-deficient mice (
Apoe-/- and
LDLr-/-, respectively) [
22,
23,
24], but they included only males and the differences in the diet compositions and IF regimen patterns used in these studies have yielded contradictory results regarding glucose homeostasis and atherogenesis.
In this context, the present study aimed to examine whether an IF intervention, in the form of an alternate-day fasting regimen, improves metabolic health and prevents atherogenesis in male and female
Apoe-/- mice fed chow or atherogenic diet by analysing lesions at early and advanced stages of atherosclerosis, respectively [
25].
4. Discussion
Clinical parameters of cardiovascular diseases such as levels of circulating cholesterol and triglycerides have been reported to be reduced in animals and humans on every-other-day fasting, called intermittent fasting (IF). However, the majority of the results from these studies were conducted in healthy and overweight humans or in healthy or obese and diabetic mice that do not present cardiovascular abnormalities [
39,
40,
41].
Only three studies to date have assessed the effect of IF in atherosclerosis-prone genetic mouse models [
22,
23,
24]. However, discrepancies in the results regarding the weight gain reduction, the improvement of glucose metabolism, and the development of atherosclerosis, were observed between these studies. Furthermore, only male mice were subjected to these interventions, and each study assessed IF in mice subjected to a single diet (chow diet, high-fat diet, or Western diet).
Our study evaluated the relevance of IF in the improvement of glucolipid metabolism and atherosclerosis development in male and female Apoe-/- mice fed either a chow diet (CD) or a high-fat and high-cholesterol atherogenic diet (HFCD). Our main findings are that the IF regimen prevented weight gain only in male Apoe-/- mice fed a chow diet, ameliorated glucose tolerance in a sex- and a diet-independent manner, and reduced hypertriglyceridemia and atherogenesis only when Apoe-/- mice were fed a chow diet.
Despite the overall reduction in cumulative calorie intake in all groups submitted to IF compared to
ad libitum ones, IF significantly reduced body weight gain and metabolic efficiency only in males fed CD, and it failed to reproduce the same effect in females fed CD or in mice fed HFCD. These findings suggest that the decrease in calorie intake alone cannot explain the observed decrease in body weight gain in males under CD. The sexual dimorphism regarding body weight benefits of IF in CD-fed
Apoe-/- mice was also described for wild-type C57BL/6J mice submitted to 10 h time-restricted feeding [
42]. Moreover, IF by every-other-day fasting, the regimen we used in our study, has been reported to affect the level of sex hormones and gonadal function in rats, with increasing testosterone levels in males but not in females, and a significant change in gene expression in the gonads of males compared to females [
43]. This could explain the beneficial effect of IF only in male mice fed CD. On the other hand, studies have shown that long-term feeding of a high-fat diet leads to similar cardiometabolic dysfunction in both male and female C57BL/6J mice [
44], which is consistent with our results that HFCD outweighs the beneficial IF effect as both male and female
Apoe-/- mice fed HFCD were resistant to a reduction in body weight gain by IF.
Metabolic efficiency is a hallmark of metabolic control of weight gain. While the bulk of energy intake is converted to heat by brown adipose tissue (BAT), metabolic efficiency constitutes a small part of the ingested energy that is stored as extra body energy deposits in white adipose tissue [
33]. Interestingly, the effect of IF on weight loss was reported to be consequent to its effect on WAT browning and BAT activation [
34].
We found that IF did not affect subcutaneous white adipose tissue (SC-WAT) weight irrespective of sex or diet. However, IF resulted in browning of WAT in mice fed chow diet independently of sex. This was accompanied by an increase in gene expression of browning markers and lipid metabolism enzymes. Deficiency in
Apoe results in a smaller adipocyte size due to impairment of lipogenesis and a defect in lipid acquisition from the circulation. This renders
Apoe-/- mice resistant to adiposity when fed an obesogenic diet [
35,
45]. Accordingly, we found that HFCD feeding did not have a significant effect on body weight gain compared to CD feeding. Furthermore, HFCD by itself induced transformation of WAT towards a BAT-like phenotype, as previously reported [
35,
36]. This masked the effect of IF on WAT morphology when mice were fed HFCD. Interestingly, among the browning markers and lipid metabolism genes explored, only
Ucp1 gene expression was dramatically upregulated by IF in WAT when mice were fed HFCD. Intriguingly, brown adipocytes from mice fed CD acquired a white-like unilocular adipocyte phenotype, when submitted to IF in a sex-independent manner, accompanied by a significant decrease in
Ucp1 gene expression. However, BAT from mice fed HFCD presented an increase in genes induced by BAT activation. These results suggested that when mice were fed CD, IF induced a reduction in BAT activity that was offset by an increase in WAT browning. This is in line with the previously reported fasting suppression of
Ucp1 expression-induced thermogenesis in BAT [
46,
47,
48], and the compensatory effect of white fat browning for defective BAT activity to maintain energy homeostasis metabolism [
49]. However, when mice were fed HFCD, IF resulted in browning of WAT and activation of BAT. Interestingly, activated BAT and increased browning of WAT in
Apoe-/- mice exacerbate atherosclerosis [
50,
51], and genetic deletion of
Ucp1 in
Apoe-/- mice has been reported to prevent atherosclerotic plaque growth [
50,
51]. Furthermore, in humans, improvements in coronary heart disease risk factors by every-other-day fasting involved modulation of adipose tissue parameters [
20].
IF effects on body fat remodelling were associated with an increase in glucose tolerance and improvement of insulin sensitivity [
52,
53,
54]. We found an increase in glucose tolerance by IF in
Apoe-/- mice irrespective of sex and diet. Unexpectedly, the improvement in glucose clearance was not a consequence of an enhancement in insulin sensitivity. Recent studies have demonstrated that glucose homeostasis could be maintained by non-insulin determinant pathways through a rearrangement of the gastrointestinal tract, glucose utilization in insulin-independent tissues/organs such as the brain and kidneys, or renal excretion of glucose, which is an insulin-independent process [
55,
56]. On the other hand, while glucose transporter GLUT4 is insulin-dependent and is responsible for the majority of glucose transport into muscle and adipose cells under anabolic conditions, GLUT1 is insulin-independent and is widely distributed in different tissues [
57]. Although we did not explore these mechanisms, they could explain the IF-improved glucose tolerance in our model.
We also found that IF decreased liver TG accumulation only in males fed chow diet. This was consistent with an increase in the expression of lipid catabolism genes in the liver. This effect was not observed in females fed CD. As the decrease in VLDL secretion was observed in both sexes, it could not explain the sexual dimorphism regarding liver TG content. This suggests that the decrease in TG content and VLDL secretion in males could be a result of increased liver lipid catabolism, while the absence of an IF effect in females could be a result of an equilibrium between lipid oxidation, synthesis, and secretion.
There is a degree of discrepancy regarding the role of Apoe in hepatic lipid accumulation upon high-fat feeding. Karvia et al. showed that Apoe deficiency has a protective effect on diet-induced nonalcoholic fatty liver disease in mice
through a mechanism involving a delay in post-prandial triglyceride (TG) clearance from their plasma [
58]. However, Lu
et al. demonstrated that
Apoe deficiency promotes nonalcoholic fatty liver disease in mice by a mechanism involving a decrease in autophagy [
59]. We found that IF exacerbated the mild HFCD-induced hepatic steatosis in a sex-independent manner in
Apoe-/- mice. We found that this increase in liver TG content by IF was not associated with the regulation of lipolytic genes. However, the increased expression of
Srebp-1c, which codes for a key
transcription factor that regulates lipogenesis, could partially explain the exacerbation of hepatic steatosis, although there was no modification of the expression level of SREBP-1c target genes by IF. Interestingly, VLDL secretion is also inhibited by IF in
Apoe-/- mice fed HFCD. These findings suggest that the high-fat diet
outweighs the benefits of IF and worsens liver steatosis by disrupting the equilibrium between degradation, synthesis, and secretion of TG.
Although the levels of cholesterol content in the liver in all IF groups and the AL groups were comparable, we found that IF resulted in a decrease in gene expression of HMG-CoA synthase (Hmgs), which is an enzyme involved in cholesterol synthesis in males fed CD. Furthermore, in the same mice, IF significantly increased the expression of genes involved in cholesterol uptake by the liver, cholesterol efflux, and cholesterol bile secretion. These changes were not observed when mice were fed HFCD. These data suggest that when mice were fed CD, IF promoted an increase in cholesterol transport and excretion, while it induced a decrease in cholesterol synthesis.
Interestingly, while we did not observe any effect of IF on plasma cholesterol levels (total, HDL, and non-HDL cholesterol) in CD-fed mice, we found that cholesterol excretion into the bile, either as unesterified (free) cholesterol or after conversion to bile acids, was increased in these mice compared to their AL control. This is in line with the increase in the expression of genes involved in the biliary excretion of cholesterol, including
Abcg5/Abcg8 responsible for reducing circulating cholesterol. These data suggest that IF increases the clearance of cholesterol by reverse cholesterol transport (RCT), which is considered to be a main way for the body to excrete cholesterol [
60]. We also found that the level of plasma TG was decreased by IF in CD-fed mice in a sex-independent manner. This could be explained by the observed decrease in hepatic VLDL-TG secretion. Although we did not measure lipoprotein lipase activity (LPL), the decrease in plasma TG concentration could also be attributed to an increase in LPL activity. Indeed, LPL, which is a key enzyme in lipid metabolism, hydrolyses triglycerides in circulating TG-rich lipoproteins and promotes the cellular uptake of chylomicron remnants, cholesterol-rich lipoproteins, and free fatty acids by adipose tissue and skeletal muscle [
61]. Contrary to the beneficial effect of IF in mice fed CD, IF exacerbated both the plasma TG and cholesterol levels, which were increased by HFCD, and had no effect on biliary lipid secretion. We did not explore the molecular mechanisms of this effect but we can hypothesize that IF in HFCD mice induced
a decrease in LPL activity, a delay in post-prandial TG-rich lipoprotein clearance from the plasma, and/or an increase in intestinal lipid absorption.
Finally, and in keeping with the beneficial effects of IF on lipid metabolism in Apoe-/- mice when fed CD, IF also reduced the development of aortic root sinus lesions in these mice in a sex-independent manner, but it failed to counteract atherosclerosis progression induced by HFCD.
The effect of IF on atherosclerosis development has also been explored in the other frequently used model of mouse atherosclerosis, the low-density lipoprotein receptor-deficient (
Ldlr-/-) model. The first study found that every-other-day fasting induced obesity and diabetes and exacerbated the development of spontaneous atherosclerosis in
Ldlr-/- male mice fed a chow diet [
24]. The difference with our results regarding glucose metabolism could be explained by the difference in genotypes, as LDL receptor but not apolipoprotein E deficiency increases diet-induced obesity and diabetes in mice [
62]. Furthermore, aside from the fact that they only studied the effect of IF in
Ldlr-/- male mice, the results were obtained by IF of chow diet feeding. Yet, on a chow diet,
Ldlr-/- mice do not readily develop atherosclerosis, and thus a high cholesterol diet with or without a high fat is needed to provide the hyperlipidemic drive for atherogenesis in these mice [
63]. Indeed, atherosclerosis development in
Ldlr-/- mice was very slow when they were fed a chow diet for 3 months [
64]. Therefore, under these conditions,
Ldlr-/- mice could display significant individual differences in lesion development, which could bias the results of the IF regimen.
A more recent study showed that intermittent fasting cycles of 3 days of
ad libitum feeding and 1 day of fasting ameliorated hypercholesterolemia, reduced atherosclerosis lesions, and increased plaque stability in
Ldlr-/- male mice fed a high-fat diet [
22]. Although we used a different mouse model and fasting cycles for IF intervention, our results with
Apoe-/- mice fed CD are comparable to the beneficial effect of IF on atherosclerosis development in
Ldlr-/- male mice fed a high-fat diet. Taking into account that
Apoe-/- mice exhibited higher plasma cholesterol and larger aortic root lesions with larger necrotic cores, and more smooth muscle cells and matrix at 3 months of atherogenic diet than did the
Ldlr-/- mice [
63], one can suggest that the beneficial effect of IF depends on the stage of atherosclerosis development and the lesion area. Future studies will be needed to determine the plasma and aortic inflammatory profile as well as the plaque stability in our model.
It was recently reported that time-restricted feeding of a Western diet in
Apoe-/- males mice limits adiposity but fails to inhibit atherosclerosis progression and glucose intolerance [
23]. Despite obtaining the same results regarding atherosclerosis development under HFCD, the effect of IF on glucose homeostasis and body weight gain is different. This discrepancy could be explained first by the difference in the fasting regimen treatment, as we used a more stringent intermittent fasting (every other day) compared to the 9 hours per day used in the above study. The origin of fat in the diet could also be an explanation. Indeed, in our study, the dietary fat was derived from cocoa butter (diets rich in stearic acid), which could not have any effect on
Apoe-/- body weight gain or glucose tolerance, compared to chow diet [
65]. However, the fat diet used in the study mentioned above is very obesogenic, as the body weight gain of
Apoe-/- mice nearly reached 100% compared to their initial body weight after only 14 weeks of feeding [
23]. This overweightness of mice was also accompanied by high fasting plasma glucose. Yet, diabetes hindered plaque regression in atherosclerotic mice, even after reduction of h
yperlipidemia [
66,
67]. These results suggest that diabetes induced by an obesogenic diet is not suitable for assessment of the effect of IF on atherosclerosis development.
Figure 1.
Intermittent fasting reduces body weight gain in Apoe-/- male mice fed chow diet. (A), Schematic outline of the experimental design: male or female Apoe-/- mice were divided into four groups that were assigned standard chow diet ad libitum (CD-AL group), intermittent fasting treatment of chow diet, by fasting every other day (CD-IF group) for 16 weeks, high-fat high cholesterol diet ad libitum (HFCD-AL group), or intermittent fasting of HFCD for 16 weeks (HFCD-IF group). Metabolic tests were performed at the indicated time and blood and organ samples were collected from euthanized mice at the end of the treatment. (B-E), The calorie intake was determined and calculated as the weekly average of Kcal intake per mouse (left panel) and the cumulative calorie intake after 16 weeks of treatment (right panel). Calorie intake in CD-fed males (B), CD-fed females (C), HFCD-fed males (D), and HFCD-fed females, n = 12–14 per group. (F-I) Change in body weight per week (left panel) and body weight gain after 16 weeks of treatment (right panel) in CD-fed males (F), CD-fed females (G), HFCD-fed males (H), and HFCD-fed females (I), n = 12–14 per group. Values are expressed as means ± SEM; ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001. IPGTT: intraperitoneal glucose tolerance test, ITT: insulin tolerance test, VLDL: very low-density lipoprotein.
Figure 1.
Intermittent fasting reduces body weight gain in Apoe-/- male mice fed chow diet. (A), Schematic outline of the experimental design: male or female Apoe-/- mice were divided into four groups that were assigned standard chow diet ad libitum (CD-AL group), intermittent fasting treatment of chow diet, by fasting every other day (CD-IF group) for 16 weeks, high-fat high cholesterol diet ad libitum (HFCD-AL group), or intermittent fasting of HFCD for 16 weeks (HFCD-IF group). Metabolic tests were performed at the indicated time and blood and organ samples were collected from euthanized mice at the end of the treatment. (B-E), The calorie intake was determined and calculated as the weekly average of Kcal intake per mouse (left panel) and the cumulative calorie intake after 16 weeks of treatment (right panel). Calorie intake in CD-fed males (B), CD-fed females (C), HFCD-fed males (D), and HFCD-fed females, n = 12–14 per group. (F-I) Change in body weight per week (left panel) and body weight gain after 16 weeks of treatment (right panel) in CD-fed males (F), CD-fed females (G), HFCD-fed males (H), and HFCD-fed females (I), n = 12–14 per group. Values are expressed as means ± SEM; ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001. IPGTT: intraperitoneal glucose tolerance test, ITT: insulin tolerance test, VLDL: very low-density lipoprotein.
Figure 2.
Intermittent fasting reduces hypertriglyceridemia in Apoe-/- mice fed CD while it exacerbates HFCD-induced dyslipidemia. Plasma levels of TG (A), NEFAs (B), total cholesterol (C), HDL-C (D) and non-HDL-C (D), and (E) in ad libitum (AL) or intermittent fasting (IF) Apoe-/- mice fed chow diet (CD) or high-fat high-cholesterol diet (HFCD). Values are expressed as means ± SEM (n = 6–9 per group); ns; not significant, *p < 0.05, **p < 0.01, ***p < 0.001. HDL-C, high-density lipoprotein cholesterol; TG, triglycerides; NEFAs, non-esterified fatty acids.
Figure 2.
Intermittent fasting reduces hypertriglyceridemia in Apoe-/- mice fed CD while it exacerbates HFCD-induced dyslipidemia. Plasma levels of TG (A), NEFAs (B), total cholesterol (C), HDL-C (D) and non-HDL-C (D), and (E) in ad libitum (AL) or intermittent fasting (IF) Apoe-/- mice fed chow diet (CD) or high-fat high-cholesterol diet (HFCD). Values are expressed as means ± SEM (n = 6–9 per group); ns; not significant, *p < 0.05, **p < 0.01, ***p < 0.001. HDL-C, high-density lipoprotein cholesterol; TG, triglycerides; NEFAs, non-esterified fatty acids.
Figure 3.
Intermittent fasting in Apoe-/- mice improves glucose tolerance. (A-D) Intraperitoneal glucose tolerance test (IPGTT). At 12 weeks of intervention, ad libitum (AL) and intermittent fasting (IF) Apoe-/- mice were fasted for 16 hours and received an intraperitoneal injection of glucose (1 g/kg). Glycemia was measured at the indicated times (left panel), and the respective area under the curve (AUC) was calculated (right panel) for CD-fed males (A), CD-fed females (B), HFCD-fed males (C), and HFCD-fed females (D). (E-H) Intraperitoneal insulin tolerance test (ITT). 2 weeks after IPGTT; the same mice were fasted for 5 hours and received an intraperitoneal injection of insulin (0.5 U/kg). Glycemia was measured at the indicated times (left panel), and the respective area under the curve (AUC) was calculated (right panel) for CD-fed males (E), CD-fed females (F), HFCD-fed males (G), and HFCD-fed females (H). Values are expressed as means ± SEM (n = 5–9 per group); ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3.
Intermittent fasting in Apoe-/- mice improves glucose tolerance. (A-D) Intraperitoneal glucose tolerance test (IPGTT). At 12 weeks of intervention, ad libitum (AL) and intermittent fasting (IF) Apoe-/- mice were fasted for 16 hours and received an intraperitoneal injection of glucose (1 g/kg). Glycemia was measured at the indicated times (left panel), and the respective area under the curve (AUC) was calculated (right panel) for CD-fed males (A), CD-fed females (B), HFCD-fed males (C), and HFCD-fed females (D). (E-H) Intraperitoneal insulin tolerance test (ITT). 2 weeks after IPGTT; the same mice were fasted for 5 hours and received an intraperitoneal injection of insulin (0.5 U/kg). Glycemia was measured at the indicated times (left panel), and the respective area under the curve (AUC) was calculated (right panel) for CD-fed males (E), CD-fed females (F), HFCD-fed males (G), and HFCD-fed females (H). Values are expressed as means ± SEM (n = 5–9 per group); ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 4.
Intermittent fasting in Apoe-/- mice regulates adipose tissue phenotypes depending on the diet.(A,B) Representative images of H&E staining of WAT tissue sections of ad libitum (AL) or intermittent fasting (IF) CD-fed males (A), CD-fed females (B), HFCD-fed males (C), and HFCD-fed females (D). Scale bar: 100 μm, n = 4 per group. (E,F) Expression of genes related to browning and lipid metabolism in ad libitum (AL) or intermittent fasting (IF) CD-fed males (E) and HFCD-fed males (F). (G-J) Representative images of H&E staining of BAT-tissue sections of ad libitum (AL) or intermittent fasting (IF) CD-fed males (G), CD-fed females (H), HFCD-fed males (I), and HFCD-fed females (J). Scale bar: 100 μm, n = 3 per group. (K,L) Expression of genes related to BAT activation in ad libitum (AL) or intermittent fasting (IF) CD-fed males (K) and HFCD-fed males (L). Values are expressed as means ± SEM (n = 6–7 per group); ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 4.
Intermittent fasting in Apoe-/- mice regulates adipose tissue phenotypes depending on the diet.(A,B) Representative images of H&E staining of WAT tissue sections of ad libitum (AL) or intermittent fasting (IF) CD-fed males (A), CD-fed females (B), HFCD-fed males (C), and HFCD-fed females (D). Scale bar: 100 μm, n = 4 per group. (E,F) Expression of genes related to browning and lipid metabolism in ad libitum (AL) or intermittent fasting (IF) CD-fed males (E) and HFCD-fed males (F). (G-J) Representative images of H&E staining of BAT-tissue sections of ad libitum (AL) or intermittent fasting (IF) CD-fed males (G), CD-fed females (H), HFCD-fed males (I), and HFCD-fed females (J). Scale bar: 100 μm, n = 3 per group. (K,L) Expression of genes related to BAT activation in ad libitum (AL) or intermittent fasting (IF) CD-fed males (K) and HFCD-fed males (L). Values are expressed as means ± SEM (n = 6–7 per group); ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 5.
Intermittent fasting reduced hepatic triglyceride content in Apoe-/- male mice fed chow diet while it exacerbated HFCD-induced steatosis. (A-D) Representative images of H&E staining of sections of liver tissue of ad libitum (AL) and intermittent fasting (IF) CD-fed males (A), CD-fed females (B), HFCD-fed males (C), and HFCD-fed females (D). Scale bars: 250 μm (E) Liver triglycerides content in AL and IF CD-fed mice (left panel) and HFCD-fed mice (right panel). (F) Liver cholesterol content in AL and IF CD-fed mice (left panel) and HFCD-fed mice (right panel). (G) Liver reactive oxygen species (ROS) content in AL and IF CD-fed mice (left panel) and HFCD-fed mice (right panel). (H) Liver thiobarbituric acid response substrates (TBARs) level in AL and IF CD-fed mice (left panel) and HFCD-fed mice (right panel). Values are expressed as means ± SEM (n = 6–9 per group); ns; not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 5.
Intermittent fasting reduced hepatic triglyceride content in Apoe-/- male mice fed chow diet while it exacerbated HFCD-induced steatosis. (A-D) Representative images of H&E staining of sections of liver tissue of ad libitum (AL) and intermittent fasting (IF) CD-fed males (A), CD-fed females (B), HFCD-fed males (C), and HFCD-fed females (D). Scale bars: 250 μm (E) Liver triglycerides content in AL and IF CD-fed mice (left panel) and HFCD-fed mice (right panel). (F) Liver cholesterol content in AL and IF CD-fed mice (left panel) and HFCD-fed mice (right panel). (G) Liver reactive oxygen species (ROS) content in AL and IF CD-fed mice (left panel) and HFCD-fed mice (right panel). (H) Liver thiobarbituric acid response substrates (TBARs) level in AL and IF CD-fed mice (left panel) and HFCD-fed mice (right panel). Values are expressed as means ± SEM (n = 6–9 per group); ns; not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 6.
Intermittent fasting increased biliary lipid secretion in Apoe-/- mice fed CD. The gallbladder was cannulated and bile was collected for 30 min, after a stabilization time of 30 min. Biliary secretions of cholesterol (A), bile acids (B) and phospholipids (C) were determined in ad libitum (AL) or intermittent fasting (IF) Apoe-/- mice fed chow-diet (CD) or high-fat high-cholesterol diet (HFCD). Values are expressed as means ± SEM (n = 6–7 per group); ns; not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 6.
Intermittent fasting increased biliary lipid secretion in Apoe-/- mice fed CD. The gallbladder was cannulated and bile was collected for 30 min, after a stabilization time of 30 min. Biliary secretions of cholesterol (A), bile acids (B) and phospholipids (C) were determined in ad libitum (AL) or intermittent fasting (IF) Apoe-/- mice fed chow-diet (CD) or high-fat high-cholesterol diet (HFCD). Values are expressed as means ± SEM (n = 6–7 per group); ns; not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 7.
Intermittent fasting induced significant changes in the hepatic expression of genes involved in lipid and cholesterol metabolism only in males fed CD. (A-B) Expression of genes related to lipolysis in ad libitum (AL) or intermittent fasting (IF) CD-fed males (A) and HFCD-fed males (B). (C-D) Expression of genes related to lipogenesis in ad libitum (AL) or intermittent fasting (IF) CD-fed males (C) and HFCD-fed males (D). (E-F) Expression of genes related to cholesterol synthesis in ad libitum (AL) or intermittent fasting (IF) CD-fed males (E) and HFCD-fed males (F). (G-H) Expression of genes related to cholesterol transport in ad libitum (AL) or intermittent fasting (IF) CD-fed males (G) and HFCD-fed males (H). Values are expressed as means ± SEM (n = 5–8 per group); ns; not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 7.
Intermittent fasting induced significant changes in the hepatic expression of genes involved in lipid and cholesterol metabolism only in males fed CD. (A-B) Expression of genes related to lipolysis in ad libitum (AL) or intermittent fasting (IF) CD-fed males (A) and HFCD-fed males (B). (C-D) Expression of genes related to lipogenesis in ad libitum (AL) or intermittent fasting (IF) CD-fed males (C) and HFCD-fed males (D). (E-F) Expression of genes related to cholesterol synthesis in ad libitum (AL) or intermittent fasting (IF) CD-fed males (E) and HFCD-fed males (F). (G-H) Expression of genes related to cholesterol transport in ad libitum (AL) or intermittent fasting (IF) CD-fed males (G) and HFCD-fed males (H). Values are expressed as means ± SEM (n = 5–8 per group); ns; not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 8.
Intermittent fasting reduced atherosclerotic lesions only when Apoe-/- mice were fed CD. (A,D,G,J) Quantification of the Oil Red O-stained aortic root at the indicated distances from the heart of ad libitum (AL) or intermittent fasting (IF) males fed CD (A), females fed CD (D), males fed HFCD (G), and females fed HFCD (J). (B,E,H,K) Calculation of the AUC regarding the quantification of atherosclerotic lesions of ad libitum (AL) or intermittent fasting (IF) males fed CD (B), females fed CD (E), males fed HFCD (H), and females fed HFCD (K). (C,F,I,L) Representative images of Oil Red O-stained sections of aortic valve of ad libitum (AL) or intermittent fasting (IF) males fed CD (C), females fed CD (F), males fed HFCD (I), and females fed HFCD (L). Scale bars: 1 mm. Values are expressed as means ± SEM (n = 5–7/group); ns; not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 8.
Intermittent fasting reduced atherosclerotic lesions only when Apoe-/- mice were fed CD. (A,D,G,J) Quantification of the Oil Red O-stained aortic root at the indicated distances from the heart of ad libitum (AL) or intermittent fasting (IF) males fed CD (A), females fed CD (D), males fed HFCD (G), and females fed HFCD (J). (B,E,H,K) Calculation of the AUC regarding the quantification of atherosclerotic lesions of ad libitum (AL) or intermittent fasting (IF) males fed CD (B), females fed CD (E), males fed HFCD (H), and females fed HFCD (K). (C,F,I,L) Representative images of Oil Red O-stained sections of aortic valve of ad libitum (AL) or intermittent fasting (IF) males fed CD (C), females fed CD (F), males fed HFCD (I), and females fed HFCD (L). Scale bars: 1 mm. Values are expressed as means ± SEM (n = 5–7/group); ns; not significant, *p < 0.05, **p < 0.01, ***p < 0.001.