Adipose tissue is a major site for cholesterol storage
Adipose tissue is a major site for cholesterol storage. Over half of total body cholesterol resides within the adipose tissues in obesity [, , ]. In EAT and SAT from high cholesterol diet (Western or HFD + Cho)-fed mice, we detected about 300–400 mg of cholesterol per gram of tissue, whereas in the same tissue from HFD-fed mice, we detected about 100 mg cholesterol less (Figs. 1F, 2F, and 3F). Dietary cholesterol also affects adipocyte cholesterol uptake [15,39]. Although not tested in this study, adipocytes from high cholesterol diet (Western or HFD + Cho)-fed mice may contain much more intracellular cholesterol and become more potent in activating MCs than those from cholesterol-free HFD-fed mice. In addition to this possibility, dietary cholesterol may also aggravate adipose tissue macrophage accumulation in obese mice  or directly activate MCs . Here, we showed that MCs produced similar amounts of β-hexosaminidase and serotonin in response to 1000 μg/mL LDL, HDL, and VLDL, but the same dose of LDL produced five-fold more serotonin than HDL and VLDL did (Figs. 6D–F and 7B). IgE is one of the best-known endogenous MC activators [42,43]. LDL at 1000 μg/mL produced similar levels of β-hexosaminidase from BMMCs to those treated with 50 μg/mL of IgE antibody (Figs. 6D and 7A), although IgE induced 4μ8C production from BMMCs twice as much as those from LDL (Figs. 6E and 7A). However, 1000 μg/mL LDL produced nearly ten-fold levels of serotonin than 50 μg/mL IgE did (Figs. 6F and 7A). In Western diet- and HFD + Cho diet-fed mice, the plasma LDL levels reached about 1500 μg/mL (Figs. 2G and 3G), whereas plasma IgE levels reached about 300 ng/mL after following a Western diet [25,26], more than 150-fold lower than what we used here. Together, our observations from this study and those from earlier work indicate that, in high cholesterol diet-fed mice, cholesterol may be the major MC activator. Relative to the high plasma cholesterol, the IgE activity in MC activation may become negligible. Both DSCG and ketotifen are MC inhibitors. From mice fed with all three types of diets, ketotifen showed much stronger inhibitory activities than DSCG in bodyweight gain, glucose tolerance, insulin insensitivity, and all other tested variables (Fig. 1, Fig. 2, Fig. 3). Yet, in cultured BMMCs, both DSCG and ketotifen showed comparable inhibitory activities on IgE- and LDL-induced releases of all tested MC mediators (β-hexosaminidase, histamine, and serotonin) (Figs. 6G–I, 7A, and C). These observations suggest that ketotifen acts as more than just MC inhibitor. It may target other components in HFD-fed mice (Fig. 1). For example, ketotifen is a non-competitive histamine antagonist (H1-receptor)  and it may also target monocytes  and other untested inflammatory cells. The recent development of Kit-independent MC-deficient models may advance the studies of MC pathobiology because these mice showed no significant impact of most other immune cells , unlike the Kit-dependent MC-deficient mice . Based on our observations presented in this study, it appears that many studies from Kit-dependent models may remain valid. It is possible that Kit-independent MC-deficient Cpa3 mice  and Mcpt5-Cre R-DTA mice  will be protected from cholesterol-rich, diet-induced obesity and diabetes, a hypothesis that merits careful investigation using different diets described in this study. Therefore, same experimental conditions remain essential to fairly evaluate results from different experimental models . Together, this study revealed an essential role of dietary cholesterol in MC activation. High cholesterol diet increases the cholesterol level in adipose tissues, plasma, and possibly adipocytes and other cell types, and turns them into potent MC activators that may obscure the activities from other endogenous MC activators. Demonstrated MC activation is required in order to evaluate MC functions in disease models.