Male apoE*3-Leiden.CETP mice on high-fat high-cholesterol diet exhibit a biphasic dyslipidemic response, mimicking the changes in plasma lipids observed through life in men

Abstract

Physiological adaptations resulting in the development of the metabolic syndrome in man occur over a time span of several decades. This combined with the prohibitive financial cost and ethical concerns to measure key metabolic parameters repeatedly in subjects for the major part of their life span makes that comprehensive longitudinal human data sets are virtually nonexistent. While experimental mice are often used, little is known whether this species is in fact an adequate model to better understand the mechanisms that drive the metabolic syndrome in man. We took up the challenge to study the response of male apoE*3-Leiden.CETP mice (with a humanized lipid profile) to a high-fat high-cholesterol diet for 6 months. Study parameters include body weight, food intake, plasma and liver lipids, hepatic transcriptome, VLDL - triglyceride production and importantly the use of stable isotopes to measure hepatic de novo lipogenesis, gluconeogenesis, and biliary/fecal sterol secretion to assess metabolic fluxes. The key observations include (1) high inter-individual variation; (2) a largely unaffected hepatic transcriptome at 2, 3, and 6 months; (3) a biphasic response curve of the main metabolic features over time; and (4) maximum insulin resistance preceding dyslipidemia. The biphasic response in plasma triglyceride and total cholesterol appears to mimic that of men in cross-sectional studies. Combined, these observations suggest that studies such as these can help to delineate the causes of metabolic derangements in patients suffering from metabolic syndrome.

Keywords: Biphasic; dyslipidemia; insulin resistance; metabolic syndrome.

Figure 1

Body Weights of male E3L.CETP mice on HFCD over time. Note that body weight plateaus around 12–15 weeks. Blue‐dotted lines represents the group mean response of the mixed model. Red lines connect measurements performed on the same individual animal. Mixed modeling suggests a maximum at 18 weeks HFCD.

Figure 2

Correlations between individual median food intake over time with the maximally attained body weight of the respective cohorts. Red points indicate animals that dropped out before the end of the experimental period. Pearson correlations shown are with dropouts excluded. When including dropouts correlations are ρ = 0.62 (P = 0.0037) and ρ = 0.24 (P = 0.10) for the 13 and 28 weeks cohort, respectively.

Figure 3

Plasma triglyceride (A), total cholesterol (B), HDL‐C (C) and non‐HDLC (D), for male apoE*3L.CETP mice as measured at the indicated time points. Data for the respective cohorts was pooled when measured on the same time point. Blue‐dotted lines represents the group mean response of the mixed model. Red lines connect measurements performed on the same individual animal.

Figure 4

Whole blood glucose (A) and plasma insulin (B) of male apoE*3L.CETP mice as measured at the indicated time points. Data for the respective cohorts was pooled when measured on the same time point. Blue‐dotted lines represents the group mean response of the mixed model. Red lines connect measurements performed on the same individual animal. Note the biphasic response and that glucose peaks earlier than plasma insulin levels.

Figure 5

Liver weights (A), liver triglyceride (B), total cholesterol (C), free cholesterol (D) and phospholipid (E) concentrations, and NASH scores (F) and fibrotic area (G) of livers in male apoE*3L.CETP cohorts sacrificed after 4, 9, 13, and 28 weeks HFCD, respectively. NASH scores show a statistically significant difference both between 4 weeks and 13 (P = 0.047) and 28 weeks (P < 0.01) and between 9 weeks and 28 weeks (P < 0.01), using the one‐way Kruskal–Wallis test. Fibrotic area was remarkably similar for the cohorts sacrificed up to 13 weeks while fibrotic area after 28 weeks HFCD diet was notably increased (P < 0.01). N = 10, 10, 9 and 21 for 4, 9, 13, and 28 weeks HFCD diet, respectively. Significant differences are denoted with brackets and asterisks: P < 0.05 (*), P < 0.01 (**), P < 0.001 (***).

Figure 6

Heatmap of selected gene expressions over time.

Figure 7

VLDL‐TG production in the respective cohorts does not convey a clear up or downward trend. One‐way ANOVA shows no significant differences between the time points. N = 10, 9, 9 and 16 for 4, 9, 13, and 28 weeks HFCD diet, respectively.

Figure 8

Fractional de novo lipogenesis for palmitate (C16:0) (A), stearate (C18:0) (B), oleate (C18:1) (C), and chain elongation of palmitate to stearate (C18:0) (E) and to oleate (C18:1) (F), across the respective cohorts. N = 10, 9, 10 and 17 for 4, 9, 13 and 28 weeks HFCD diet, respectively. Significant differences, according to one‐way ANOVA, are denoted with brackets and asterisks: P < 0.05 (*), P < 0.01 (**), P < 0.001 (***).

Figure 9

Steady state glucose (A), post‐test plasma insulin (B), endogenous glucose production rate (C) of male E3L.CETP mice on HFCD. From these values we can calculate insulin sensitivity (D), peripheral insulin sensitivity (E) and hepatic insulin sensitivity (F). Note that peripheral insulin resistance decreases and increases over time while hepatic insulin resistance does not. Blue‐dotted lines represents the group mean response of the mixed model. Red lines connect measurements performed on the same individual animal. n = 16, 16, 15 and 14 for 3, 9, 15 and 27 weeks, respectively.

Figure 10

Biliary bile acids (A), biliary neutral sterol (B) and biliary phospholipid secretion rate (C) in male E3L.CETP mice on HFCD diet for 4 weeks (n = 10), 9 weeks (n = 10), 13 weeks, (n = 9), or 28 weeks (n = 16), respectively.

Figure 11

Fecal neutral sterol (NS) and bile acid excretion in male E3L.CETP mice on HFCD diet at 3 weeks (n = 30), 6 weeks (n = 16), 9 weeks (n = 30), 10 weeks (n = 16), 13 weeks (n = 30), 16 weeks (n = 15), 17 weeks (n = 29), 21 weeks (n = 29), 22 weeks (n = 14), 27 weeks (n = 24) and 28 weeks (n = 14). The blue‐dotted line represents the group mean response of the mixed model. Red lines connect measurements performed on the same individual animal.