Oral Fat Tolerance Test in Male and Female C57BL/6 Mice

Background

The amount of triglycerides (TG) produced in lipid metabolism has an important correlation with the onset and development of cardiometabolic diseases. Postprandial lipemia is an important cardiovascular risk biomarker since, among the usual blood lipid fractions analyzed, TG is the first to change after food intake (Nordestgaard, 2016). Recently, international associations recommended that fasting is no longer required for blood lipid analysis. It is justified firstly by the fact that individuals remain most of their time in a postprandial state, and secondly because there is no significant variation in TG between fed or fasted individuals (Nordestgaard et al., 2016). The oral fat tolerance test (OFTT) is a reliable method for assessing postprandial lipemia because it represents the body’s capacity to normalize hyperlipidemia after lipid intake, allowing a more profound analysis of lipid metabolism. The OFTT consists of giving a particular food and monitoring its metabolic response in the body (Langsted and Nordestgaard, 2011).

Animal models are essential for studying metabolism and cardiometabolic diseases; however, there is currently no consensus regarding how to perform the OFTT in rodents, and as a consequence, there is a wide range of variation among published OFTT protocols. A recent study demonstrated that OFTT results are influenced by mouse strain, dietary lipid source, fasting duration, and sex (Ochiai, 2020). Although Ochiai et al. indicated that other strains may have a better response in OFTT (Ochiai, 2020), C57BL/6 mice continue to be the most studied strain for cardiometabolic diseases (Fazio and Linton, 2001); therefore, it has become crucial to establish a protocol to assess postprandial lipemia in these animals.

The vast majority of OFTT studies performed in C57BL/6 mice use olive oil (Kimura et al., 2011 and 2013; Toyoda-Ono et al., 2007; Hiel et al., 2018; Sairyo et al., 2018); although, there are some reports with coconut (enriched with oleic acid) (Hernandez Vallejo et al., 2009), safflower (Yamazaki et al., 2012), and corn (King et al., 2010) oils. Ochiai et al. demonstrated that olive and soybean oils present similar OFTT results in ddY mice (Ochiai, 2020). It is also important to consider the amount of fat administered, since the dose reported in the literature ranges from 5 ml/kg to 17 ml/kg (Toyoda-Ono et al., 2007; King et al., 2010; Sairyo et al., 2018) and the volume ranges from 150 μl to 400 μl (Hernandez Vallejo et al., 2009; Kimura et al., 2011 and 2013; Yamazaki et al., 2012). In the face of this evidence, we propose an OFTT meal protocol of 200 μl soybean oil, which corresponds to approximately 7 ml/kg. We did not correct the oil volume by body weight because we noticed that body mass fluctuations within the range expected for this mouse strain did not significantly alter the volume of oil administered, and it is also difficult to measure precisely in the syringe.

The fasting duration can range from 4 h to 24 h (Toyoda-Ono et al., 2007; Hernandez Vallejo et al., 2009; King et al., 2010; Kimura et al., 2011 and 2013; Yamazaki et al., 2012; Sairyo et al., 2018). Considering that fasting is a stressful event for rodents (Choi et al., 2005) and current guidelines for humans suggest that fasting is no longer required for TG analysis (Nordestgaard et al., 2016), we propose a short fasting protocol of 2 h. Lastly, sex should also be considered, since most studies are conducted in male mice (Toyoda-Ono et al., 2007; Hernandez Vallejo et al., 2009; King et al., 2010; Kimura et al., 2011 and 2013; Yamazaki et al., 2012; Hiel et al., 2018; Sairyo et al., 2018). Ochiai et al. showed that females are less prone to lipid-induced hypertriglyceridemia; however, these results were reported only in ddY mice (Ochiai, 2020), thus, we chose to propose a protocol for male and female mice.

Another two important factors to consider are the frequency and timing of blood sampling and mouse metabolic background. Concerning the former, the TG peak would appear between 2 h and 3 h after lipid administration (Yamazaki et al., 2012; Sairyo et al., 2018; Ochiai, 2020). Regarding the latter, some studies conducted the OFTT in high fat-fed mice, while others were performed in healthy mice. According to Ochiai et al., high-fat and high-sucrose feeding for one week is sufficient to promote lipid-induced hypertriglyceridemia during the OFTT (Ochiai, 2020).

Based on these data, we aimed to demonstrate a protocol for OFTT in male and female C57BL/6 mice using 200 μl soybean oil after a 2-h fasting period.