Our current understanding of oxysterol metabolism during different disease states such as obesity and dyslipidemia is limited. Therefore, the aim of this study was to determine the effect of diet-induced obesity on the tissue distribution of various oxysterols and the mRNA expression of key enzymes involved in oxysterol metabolism. To induce obesity, male C57BL/6J mice were fed a high fat-cholesterol diet for 24 weeks. Following diet-induced obesity, plasma levels of 4
During obesity and insulin resistance, increased adipocyte lipolysis paired with an inability of the fat cell to expand further to store lipid promotes excess ectopic lipid deposition (i.e., lipotoxicity), a major contributor to the development of cardiometabolic diseases [
During hepatic lipotoxicity, the hepatic pools of FFA and cholesterol increase significantly, thereby promoting a cytotoxic environment [
Cholesterol is essential for proper cell function as a key component of cellular membranes and precursor of steroid hormones [
Data from a growing number of research studies suggest that oxysterols play an important role in the regulation of lipid metabolism [
It remains to be elucidated to what extent oxysterol metabolism is altered during diet-induced obesity. Therefore, the purpose of this study was to determine the effects of obesity induced by a high fat-cholesterol diet on oxysterol levels in plasma, liver, and adipose tissue. In addition, we measured the expression of oxysterol synthesizing enzymes CYP7A1, CYP3A11, and CYP27A1 mRNA in liver and adipose tissue. Furthermore, we examined the expression of SULT2B1b mRNA, a cytosolic sulfotransferase that is believed to sulfonate oxysterols to prevent their cytotoxic effects. Tissue lipid composition and circulating lipoprotein composition were quantified to describe the severity of the dyslipidemia in these mice after 24 weeks of a high fat-cholesterol (HFC) diet.
Male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were used and divided into two dietary groups (normal control diet or HFC diet) at 8 weeks of age. The normal control diet (ND) consisted of 5.0% w/w fat (12.0% of kcal from fat) and 0.03% w/w cholesterol (LabDiet #5053, Brentwood, MO) and the HFC contained 21.0% w/w fat (41% of kcal from fat) and 0.15% w/w cholesterol (Dyets Inc., Bethlehem, PA). Mice were fed
Lipoproteins were isolated from 12 hr fasted plasma (500
Tissues were weighed (30–50 mg), finely ground to a powder under liquid N2, and incubated overnight at room temperature in 1 mL of hexane: isopropanol (3 : 2, v/v) with 0.1% (w/v) butylated hydroxytoluene (BHT). Samples are centrifuged (15 min at 4000 g) and the supernatant was transferred to a clean tube and evaporated under N2. The dried lipid extract was weighed to determine the total lipid content of the tissues [
Total cholesterol and triglyceride assays were performed on the liver and adipose tissue lipid extracts using standard colorimetric assay kits (Wako Chemicals, Richmond, VA). From the protein extract, protein concentrations were measured using a standard Lowry assay (Bio-Rad, Hercules, CA).
Oxysterol measurements were performed on diet, plasma, liver, and adipose tissue by gas chromatography-mass spectrometry (GC-MS) using a modified isotope dilution method described by Dzeletovic et al. [
Dried plasma, tissue, and diet lipid extracts were subjected to hydrolysis for 2 hr following addition of 1 mL PBS and 2 mL 0.5 M KOH. To terminate hydrolysis, 1 mL of 1 M HCl was added followed by 2 mL of hexane with 0.1% BHT. Following centrifugation, the upper organic phase was extracted and evaporated under N2. Prior to solid-phase separation of cholesterol and oxysterols, 1 mL of toluene was added to each sample. Solid-phase extraction was performed using 200 mg solute silica cartridges (Agilent Technologies, Inc., Santa Clara, CA). Cholesterol was eluted from the cartridges using 12–18 mL of 0.5% (v/v) isopropanol in hexane followed by elution of oxysterols with 8 mL 60% (v/v) isopropanol in hexane. The oxysterol fraction was evaporated under N2 and converted to trimethysiyl ether by adding 1 : 1 (v/v) pyridine : bis(trimethylsilyl)trifluoroacetamide and heated for 30 min at 60°C. Following evaporation under N2, the residue was dissolved in 100
Oxysterol concentration was measured by GC-MS on an Agilent GC6890N equipped with a DB-5MS capillary column, connected to an Agilent 5973 inert mass selective detector (Agilent Technologies, Inc., Santa Clara, CA). The mass spectrometer was operated in the selected ion monitoring mode, and the ions used for analysis (
mRNA of CYP7A1, CYP27A1, CYP3A11, and SULT2B1b in mouse liver and adipose tissue was examined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) using predesigned primers and probes (Applied Biosystems, Carlsbad, CA) as previously described [
Data are presented as mean ± SD. Independent sample
The total body weight of the HFC group was significantly (
The percentage of lipid by weight recovered from the adipose tissue and liver is shown in Figure
Post-diet lipids in liver tissue and adipose tissue.
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Percentage of lipid recovered from the adipose and liver tissue. No significant (
The mean lipoprotein-FPLC profile for each diet group is presented in Figure
(a) FPLC lipoprotein profiles from mice after normal chow diet (solid line) and HFC diet (dashed line). Total lipoproteins were isolated by ultracentrifugation and lipoproteins were separated over two Superose 6 columns. Fractions 15–19 contain VLDL, 21–24 contain LDL, 25–27 contain LDL/HDL1, and HDL is found in fractions 29–34. (b) Western blot analysis of apo B, apo E, and apo AI in the pooled (six animals per group) FPLC fractions 25–27 (LDL/HDL1).
Lipid and protein composition of the pooled lipoprotein-FPLC fractions are presented in Table
The effects of the high fat/high cholesterol diet on fasting lipoprotein lipid composition.
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Cholesterol |
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No significant difference was observed for the majority of the measured dietary oxysterols between the ND and HFC chow (Table
Oxysterol levels in control and high fat-cholesterol diets.
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Plasma, liver, and adipose tissue oxysterol concentrations are presented in Table
Post-diet oxysterols in plasma, liver tissue, and adipose tissue.
Variables | Plasma (ng/mL) | Liver tissue (ng/mg protein) | Adipose tissue (ng/mg protein) | |||
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ND | HFC | ND | HFC | ND | HFC | |
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Tissue specific CYP mRNA expression is illustrated in Figure
Cytochrome P450 27A1, 7A1, and 3A11 and SULT2B1b mRNA levels quantified by qPCR in adipose tissue and liver tissue of C57BL/6J mice (
In the present study, the HFC diet induced obesity in the mice as witnessed by the presence of increased total body mass, perigonadal fat pad mass, and liver mass. The adipose tissue was characterized by higher intracellular concentrations of cholesterol, as well as triglycerides. In the liver, the 3.3-fold increase in lipid mass was more pronounced and was matched by a 2.3-fold increase in cholesterol and 3.0-fold increase in triglyceride concentrations, characteristic of nonalcoholic fatty liver disease (NAFLD) [
The lipoprotein profile and lipid composition of circulating lipoproteins were significantly modified by the 24-week HFC diet. We observed a significant increase in total cholesterol concentration of all lipoprotein fractions in the obese mice. The most substantial change was in the VLDL fraction where we observed an 11.5-fold increase in cholesterol. This change in VLDL lipid composition in C57BL/6 mice appears to be typical following a HFC feeding [
Mice on the HFC diet had markedly increased (2- to 10-fold) plasma oxysterol levels, with the exception of plasma 7-ketocholesterol and 25-hydroxycholesterol. The elevated circulating oxysterol levels are most likely a result of the diet-induced hypercholesterolemia rather than increased dietary oxysterol intake, since the oxysterol content of the HFC diet was extremely low and did not differ substantially from the control diet with exception of 27-hydroxycholesterol. Previous studies in humans have reported that the major oxysterols present in the circulation are from high-to-low concentration, 27-hydroxycholesterol, 24-hydroxycholesterol, 7
In the obese mice, 4
We did not find any evidence of altered hepatic CYP7A1 mRNA expression in the mice on the HFC diet. This seems in contradiction with a previous report by Gnerre and coworkers who showed that hepatic CYP7A1 mRNA expression was significantly elevated in C57BL/6 mice fed a low-fat standard chow diet supplemented with 2% cholesterol for 1 week [
Diet-induced obesity in C57BL/6 mice has been shown to cause an altered immune response observed by increased proliferation of splenic lymphocytes and the release of cytokines interferon- (IFN-)
27-Hydroxycholesterol has been demonstrated to be an alternative pathway for bile acid synthesis and efflux of cholesterol from macrophages [
In the mice on the HFC diet elevated plasma 27-hydroxycholesterol levels coincided with increased hepatic and adipose levels. The elevated 27-hydroxycholesterol tissue levels were not due to increased CYP27A1 mRNA expression, since both liver and adipose tissue CYP27A1 mRNA appeared to be slightly downregulated in the HFC group.
7
In contrast to 7-ketocholesterol, 7
Compared to the other oxysterols measured in this study, less is known about the function and metabolism of epoxycholesterol oxysterols. In patients with hypercholesterolemia, increased serum 5,6
In summary, we have demonstrated that obesity induced by a HFC diet resulted in markedly increased concentrations of both enzymatically and nonenzymatically derived oxysterols in the circulation as well as hepatic and adipose tissues. The increased circulating oxysterols were associated with increased cholesterol, triglyceride, and phospholipid in all lipoprotein fractions and the presence of the LDL/HDL1 phenotype typically observed in obese mice. Despite the increase in hepatic and adipose tissue oxysterols, we did not observe a change in the expression of hepatic CYP7A1, 3A11, or 27A1 mRNA and CYP27A1 mRNA in adipose tissue. We did detect the presence of SULT2B1b mRNA in the adipose tissue and an increase in hepatic SULT2B1b mRNA expression in the obese mice, possibly as a compensatory mechanism to prevent the accumulation of these cytotoxic oxysterols in these tissues. To our knowledge, this study is first to characterize the metabolism of oxysterols in diet-induced obesity. Future studies using this diet-induced obesity mouse model are needed to elucidate the role of increased oxidative stress on the modulation of oxysterol metabolism.
The authors declare no conflict of interests.
The authors wish to thank Drs. Henry Pownall and Corina Rosales for their technical assistance. This work was supported by a NIH Grants T32-HL07812 (J. S. Wooten), R01-HL098839 (H. Wu), and R01-DK078847 (C. M. Ballantyne).