Intermittent Fasting Resolves Dyslipidemia and Atherogenesis in Apolipoprotein E-Deficient Mice in a Diet-Dependent Manner, Irrespective of Sex

Abstract

In humans and animal models, intermittent fasting (IF) interventions promote body weight loss, improve metabolic health, and are thought to lower cardiovascular disease risk. However, there is a paucity of reports on the relevance of such nutritional interventions in the context of dyslipidemia and atherosclerotic cardiovascular diseases. The present study assessed the metabolic and atheroprotective effects of intermittent fasting intervention (IF) in atherosclerosis-prone apolipoprotein E-deficient (Apoe^-/-) mice. Groups of male and female Apoe^-/- mice were fed a regular (chow) or atherogenic (high-fat, high-cholesterol, HFCD) diet for 4 months, either ad libitum or in an alternate-day fasting manner. The results show that IF intervention improved glucose and lipid metabolism independently of sex. However, IF only decreased body weight gain in males fed chow diet and differentially modulated adipose tissue parameters and liver steatosis in a diet composition-dependent manner. Finally, IF prevented spontaneous aortic atherosclerotic lesion formation in mice fed chow diet, irrespective of sex, but failed to reduce HFCD-diet-induced atherosclerosis. Overall, the current work indicates that IF interventions can efficiently improve glucose homeostasis and treat atherogenic dyslipidemia, but a degree of caution is warranted with regard to the individual sex and the composition of the dietary regimen.

Keywords: adipose tissue; atherosclerosis; glucose tolerance; hepatic steatosis; intermittent fasting; reverse cholesterol transport.

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 (HFCD-IF group) for 16 weeks. 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 per mouse was calculated from the weekly calorie intake values per cage divided by the number of co-housed mice and expressed per week (left panel) and cumulatively over the 16 weeks of follow-up (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 (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 h 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). Two weeks after IPGTT, the same mice were fasted for 5 h 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–D) 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) WAT 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) BAT expression of genes related to BAT activation in ad libitum (AL) or intermittent fasting (IF) CD-fed males (K) and HFCD-fed males (L). Upper right insets (I,J): zoom-in on white squares. Values are expressed as means ± SEM (n = 6–7 per group); * p < 0.05, ** p < 0.01.

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 triglyceride 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). Upper right insets (A–D): zoom-in on white squares. Values are expressed as means ± SEM (n = 6–9 per group); ns: not significant; * p < 0.05, ** p < 0.01.

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 < 0.01, *** 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); * 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). Arrows indicate the atherosclerotic lesions. Scale bars: 1 mm. Values are expressed as means ± SEM (n = 5–7/group); ns: not significant; ** p < 0.01, *** p < 0.001.