Krill Oil Supplementation Reduces Exacerbated Hepatic Steatosis Induced by Thermoneutral Housing in Mice with Diet-Induced Obesity

Abstract

Preclinical evidence suggests that n-3 fatty acids EPA and DHA (Omega-3) supplemented as phospholipids (PLs) may be more effective than triacylglycerols (TAGs) in reducing hepatic steatosis. To further test the ability of Omega-3 PLs to alleviate liver steatosis, we used a model of exacerbated non-alcoholic fatty liver disease based on high-fat feeding at thermoneutral temperature. Male C57BL/6N mice were fed for 24 weeks a lard-based diet given either alone (LHF) or supplemented with Omega-3 (30 mg/g diet) as PLs (krill oil; ω3PL) or TAGs (Epax 3000TG concentrate; ω3TG), which had a similar total content of EPA and DHA and their ratio. Substantial levels of TAG accumulation (~250 mg/g) but relatively low inflammation/fibrosis levels were achieved in the livers of control LHF mice. Liver steatosis was reduced by >40% in the ω3PL but not ω3TG group, and plasma ALT levels were markedly reduced (by 68%) in ω3PL mice as well. Krill oil administration also improved hepatic insulin sensitivity, and its effects were associated with high plasma adiponectin levels (150% of LHF mice) along with superior bioavailability of EPA, increased content of alkaloids stachydrine and trigonelline, suppression of lipogenic gene expression, and decreased diacylglycerol levels in the liver. This study reveals that in addition to Omega-3 PLs, other constituents of krill oil, such as alkaloids, may contribute to its strong antisteatotic effects in the liver.

Keywords: C57BL/6N mice; NAFLD; high-fat diet; krill oil; obesity; omega-3; phospholipids; thermoneutral temperature.

Figure 1

Overview of the experimental setup. (A) Four groups of mice (n = 8) housed in a thermoneutral environment (~30 °C) were used: (i) the control LHF group, which was fed a lard-based high-fat diet (i.e., LHF diet) for 24 weeks; (ii) ω3PL group fed a LHF-based diet supplemented with omega-3 PUFAs as PLs in the form of krill oil (i.e., ω3PL diet) for the duration of the experiment (i.e., “preventive” approach); (iii) ω3PL-R group fed the LHF diet for the first eight weeks and then from the ninth week on the ω3PL diet until the end of the experiment (i.e., “reverse” approach; marked with the letter “R” at the end of the group name); and (iv) ω3TG-R group fed the LHF diet for the first 8 weeks and then from the ninth week on the LHF-based diet supplemented with omega-3 PUFAs in the form of a concentrate of re-esterified TAGs (i.e., ω3TG diet) until the end of the experiment. (B) Three groups of mice (n = 8) housed in a thermoneutral environment (~30 °C) were used: (i) Chow group, in which mice were fed a standard low-fat diet and served as lean controls; (ii) the control LHF group, which was fed a lard-based high-fat diet (i.e., LHF diet) for 24 weeks; and (iii) ω3PL group fed the ω3PL diet for the duration of the experiment. Further details in Section 2.2. PTT, pyruvate tolerance test; VLDL, liver VLDL-TAGs secretion test; HEC, hyperinsulinemic-euglycemic clamp.

Figure 2

The effect of omega-3 PUFA supplementation on parameters related to energy balance, adipose tissue health and insulin sensitivity: changes in body weight during the study (A), average daily food intake (B), feeding efficiency (C), average size of adipocytes (D) and macrophage accumulation in epididymal WAT (E), plasma adiponectin levels (F), and insulin resistance based on the HOMA-IR index (G). Data are means ± SEM (n = 7–8). *, significant effect of omega-3 PUFAs (vs. LHF); #, significant difference from ω3TG-R (One Way ANOVA or Kruskal–Wallis).

Figure 3

Representative histological sections of liver stained with hematoxylin and eosin. Bars = 200 µm.

Figure 4

The effect of omega-3 PUFA supplementation on NAFLD-related parameters: liver weight (A), liver TAG content (B), steatosis (C), lobular inflammation (D), NAFLD activity score—NAS (E), fibrosis (F), and plasma AST and ALT levels (G). The results presented in panels C–F are based on histological analysis of liver sections. Data are means ± SEM (n = 7–8). *, significant effect of omega-3 PUFAs (vs. LHF); #, significant difference from ω3TG-R (one-way ANOVA or Kruskal–Wallis).

Figure 5

Selected parameters determining the effects of omega-3 PUFAs in the liver: bioavailability of FAs such as arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) measured in the neutral (A) and polar (B) fraction of liver lipids; correlation between plasma adiponectin levels and the degree of TAG accumulation in the liver (C); hepatic expression of genes related to DNL (D), cholesterol biosynthesis (E), inflammation (F), and tissue remodeling (G). Data are means ± SEM (n = 7–8). *, significant effect of omega-3 PUFAs (vs. LHF); #, significant difference from ω3TG-R (one-way ANOVA or Kruskal–Wallis).

Figure 6

Effect of krill oil supplementation on hepatic VLDL-TAG production (A), fasting plasma TAG levels (B), glycemia during the pyruvate tolerance test (C), the level of pyruvate-driven gluconeogenesis (D), as well as glycemia during the last hour of hyperinsulinemic-euglycemic clamp (E) and clamp-related parameters including glucose infusion rate (GIR), glucose turnover (GTO) and endogenous glucose production (EGP; F). Data are means ± SEM (n = 6–7). *, significant difference between LHF and Chow; #, significant difference between LHF and ω3PL; $, significant difference between ω3PL and Chow (one-way ANOVA or Kruskal–Wallis).

Figure 7

Four-class PLS-DA score plots of complex lipids (A; n = 507) and polar metabolites (B; n = 157) in the liver in response to dietary challenges, and the most discriminating complex lipids (C) and polar metabolites (D) based on VIP scores from PLS-DA.

Figure 8

Box plots for stachydrine (A) and trigonelline (B) levels in the liver (arbitrary units; left panel) and in experimental diets (mg/kg; right panel).

Figure 9

Box plots and correlation analysis for DAGs and TAGs lipid classes in the liver in response to dietary challenges: the sum of all DAG species (A; n = 37), the sum of DAGs with at least 1 SFA (B; n = 20), and representative species of DAGs containing either SFA (i.e., DAG 32:0; DAG 16:0_16:0; C) or omega-3 PUFAs (i.e., DAG 44:12; DAG 22:6_22:6; D). Correlation between the sum of all DAG species (n = 37) and the sum of DAGs with at least 1 SFA (n = 20; E). The sum of TAG species with short/medium-chain FAs (F; n = 9), the sum of TAG species with only SFAs (G; n = 3), and a representative SFA-containing TAG species TAG 48:0; TAG 16:0_16:0_16:0 (H). Lipid intensities are in arbitrary units (A.U.).

Figure 10

The potential mechanisms involved in the effects of krill oil supplementation on liver fat accumulation and insulin sensitivity in a mouse model of exacerbated hepatic steatosis induced in C57BL/6N mice fed a high-fat (lard) diet in a thermoneutral environment. The effects of krill oil are determined not only by omega-3 PUFA-containing PLs (ω3 PLs) in this marine oil, but also by its other bioactive constituents, including palmitoleic acid (POA) and the alkaloids stachydrine and trigonelline, and may involve direct or indirect mechanisms. The livers of mice fed a high-fat diet supplemented with krill oil have markedly reduced TAG accumulation and improved insulin sensitivity, which is associated with increased bioavailability of omega-3 PUFAs, suppressed DNL, decreased tissue DAG levels, and stimulated FA oxidation (FAOX). While many of these changes may be due to indirect mechanisms based on the beneficial effect of krill oil on WAT functionality associated with markedly elevated plasma adiponectin levels, direct mechanisms may include the effect of stachydrine and/or trigonelline, i.e., alkaloids contained in krill oil, which have previously been shown to have positive effects on NAFLD, presumably by restoring hepatic autophagy.