Two Weeks of Western Diet Disrupts Liver Molecular Markers of Cholesterol Metabolism in Rats

doi:10.21203/rs.3.rs-26176/v1

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Two Weeks of Western Diet Disrupts Liver Molecular Markers of Cholesterol Metabolism in Rats

https://doi.org/10.21203/rs.3.rs-26176/v1

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published 21 Aug, 2020

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Background

The present study was designed to test the hypothesis that excessive fat accumulations impair cholesterol metabolism mainly through alterations in the LDL-receptor (LDL-R) pathway in liver.

Method:

Rats were either submitted to standard (SD), high fat (HFD; 60% kcal) or western (WD; 40% fat + 35% sucrose (17.5% fructose)) diets for 2 or 6 weeks.

Results

Weight gain (~ 40 g) was observed only following 6 weeks of the obesogenic diets (P < 0.01). Compared to the 2-week treatment, obesogenic diets tripled fat pad weight (~ 20 vs 7 g) after 6 weeks. Hepatic triglyceride (TG) levels were greater in response to both the WD and HFD compared to the SD (P < 0.01) at 2 and 6 weeks and their concentrations were greater (P < 0.05) in WD than HFD at 2 weeks. Plasma cholesterol levels were higher (P < 0.05) in animals submitted to WD. After 2 and 6 weeks, liver expression of LDL-R, PCSKk9 and SREBP2, involved in LDL-cholesterol uptake, was lower in animals submitted to WD than in others treated with HFD or SD (P < 0.01). Similarly, LRP1 and ACAT2 mRNA levels were lower (P < 0.01) among WD compared to SD-fed rats. Expression of the gene coding the main regulator of endogenous cholesterol synthesis, HMGCoAR was reduced in response to WD compared to SD and HFD at 2 (P < 0.001) and 6 (P < 0.05) weeks. Being enriched in fructose, the WD strongly promoted the expression of ChREBP and ACC, two key regulators of de novo lipogenesis.

Conclusion

These results show that the WD promptly increased TG levels in the liver by potentiating dietary fat storage and de novo lipogenesis. This impaired the pathway of hepatic cholesterol uptake via the LDL-R axis, promoting a rapid increase in plasma cholesterol levels.

Cardiac & Cardiovascular Systems

Endocrinology & Metabolism

fructose diet

LDL-R

PCSK9

liver cholesterol metabolism

Non-alcoholic fatty liver disease (NAFLD), also referred to as hepatic steatosis is characterized by excessive fat accumulation (5 to 10% of liver weight) in absence of significant alcohol consumption (1). This state is associated with several clinical and pathological manifestations. NAFLD is considered a risk factor for diabetes and cardiovascular diseases (CVD) independently of other usual risk factors (2, 3). Hepatic steatosis by itself is also associated with a proatherogenic lipid profile and production of proinflammatory markers (4, 5). Some authors (6) have even claimed that with regards to body fat accumulation, hepatocytes are probably the first cells to indicate that something is wrong.

Although the association between liver fat accumulation, CVD, and a proatherogenic lipid profile is well substantiated, hepatic cholesterol metabolism is one important factor linking these health conditions that has been overlooked in the past. The liver is the master regulator of several pathways of cholesterol metabolism including synthesis, uptake from chylomicron remnants, re-uptake from LDL and HDL lipoproteins as well as release of cholesterol under VLDL, and biliary acids production. All of these pathways may be involved in changes in plasma cholesterol levels and the subsequent proatherogenic profile associated with fat accumulation in liver.

The association between hepatic steatosis and cholesterol metabolism is most often viewed as how cholesterol metabolism dysregulation contributes to NAFLD in animal models (7). To our knowledge, there is only one study indicating that NAFLD may in turn, dysregulate cholesterol metabolism in humans (8). Long-term studies do not provide any clear indication that accumulation of fat in the liver is at the origin of alterations of cholesterol metabolism. Our group recently addressed this possibility by showing that several molecular markers of cholesterol metabolism in the liver were disturbed by a short-term period (2 weeks) HFD in young rats (9). These results support the concept that excessive accumulation of fat in the liver may contribute to increased circulating cholesterol levels (9). A short-term 2-weeks HFD was used in that study because it has been shown to result in a rapid fat accumulation in the liver while minimizing the overall metabolic disorders normally observed with longer periods of obesogenic diets (10, 11).

The notion that disturbances in lipid metabolism such as TG accumulation in liver play a crucial role in the etiology of plasma hypercholesterolemia is supported by the knowledge that TG and cholesterol metabolic pathways are closely interrelated in liver. Cholesterol and fatty acid synthesis both use acetyl CoA in their initiation, and the reducing equivalent NADPH as their source of energy for their synthesis. Therefore, there is potential competition between fatty acid synthesis stored as TG and cholesterol synthesis in liver. This raises the possibility that de novo lipogenesis resulting in liver fat accumulation may impair hepatic cholesterol metabolism. Elements supporting this hypothesis were recently provided by Ichigo et al. (12) who reported that compared to high glucose (65%) diet, 12 days to a high fructose diet (65%) increased ACAT2 while decreasing ABCG5/G8 gene expression in the liver. In the present study, we tested the hypothesis that a short period (2 weeks) of a WD (40% fat and 35% sucrose (17.5% fructose)) known to stimulate de novo lipogenesis disrupts molecular pathways of cholesterol metabolism in the liver. In line with our previous study (9), we mainly targeted the important receptor LDL-R involved in cholesterol uptake of LDL particles in plasma to the liver and its posttranscriptional regulator PCSK9 along with their transcription factor SREBP2.

Animals care and treatments

Four-week old male Wistar rats (Charles River, St-Constant, Québec, Canada) weighing, 190 to 200 g, were housed individually and had ad libitum access to food and tap water after they were received in our laboratory. Their environment was controlled in terms of light (12 h light-dark cycling starting at 06:00 AM), humidity, and room temperature (20–23 °C). After a 3-day acclimatisation period, rats were randomly submitted either to a SD chow diet, a 60% HFD, or a WD (40% fat-35% sucrose) diet for 2 or 6 weeks for a total of 6 experimental groups (n = 10/group). Food intake and body weight were measured every 2 and 3 days, respectively. The protocol was approved by the Comité Institutionnel de Protection des Animaux (CIPA; 1218-956-1219) of UQAM and developed in agreement with the Canadian Council on Animal Care’s rules (CCAC-CCPA).

At the end of their respective nutritional treatment, rats were euthanized between 09:00 and 11:00 AM after a 3-h fast. Immediately after complete anaesthesia by inhalation of isofluorane (3% introduction and 1.5% maintenance) blood samples drawn from the heart were collected in tubes containing 0.5 ml EDTA. Blood was centrifuged (1,500 RPM for 10 min) and the plasma kept for the measurement of triacylglycerol (TG), cholesterol, and PCSK9. The abdominal cavity was opened and the median lobe of the liver was freeze-clamped, removed and used for TG, cholesterol and gene expression of several key markers of cholesterol metabolism. The mesenteric, retroperitoneal, epididymal, and subcutaneous (sc) fat deposits were, thereafter, rapidly excised and weighed. The mesenteric fat pad consisted of adipose tissue surrounding the gastrointestinal tract from the gastroesophageal sphincter to the end of the rectum with special care taken in distinguishing and removing pancreatic cells. The epididymal fat pad included adipose tissue surrounding the ureters and bladder. The retroperitoneal fat pad represented the distinct deposit behind each kidney, along the lumbar muscles. For sc fat deposit measurement, a rectangular piece of skin was taken on the right side of each animal from the median line of the abdomen to the spine and the right hip to the first rib (13). All tissue and plasma samples were stored at -78 °C until analyses.

Diets

The SD (14) consisted of 13% lipid, 65.6% CHO and 21.4% protein (kcal; Charles River Rodent Diet 5075, Cargill Animal Nutrition, MN, USA). The HFD and the WD were obtained from Research Diets (NJ, USA). The HFD consisted of 60% fat, 20% CHO and 20% protein (kcal) while the WD consisted of 40% lipid, 43% CHO (35% sucrose), and 17% protein (kcal). The caloric equivalent of 2.89, 5.21 and 4.67 kcal/g for the SD, HFD, and WD, respectively was used to report food intake in kcal. Details of the HFD and WD are presented in Table 1.

Table 1

Description of the diets
Ingredients (g)	High Fat diet D12492	Western diet D12079B
Casein, Lactic, 30 Mesh	200	195
Methionine, DL	0	3
Cystine, L	3	0
Sucrose, Fine Granulated	72.8	350
Startch, Corn	0	50
Lodex 10	125	100
Solka Floc, FCC200 (Fiber)	50	50
Butter, Anhydrous	0	200
Corn oil	0	10
Lard	245	0
Soybean Oil, USP	25	0
S10026B (Mineral)	50	0
S10001A (Mineral)	0	17.5
Calcium Phosphate, Dibasic (Mineral)	0	17.5
Calcium Carbonate, Light, USP (Mineral)	0	4
Choline Bitartrate (Vitamin)	2	2
V10001C (Vitamin)	1	1
Ethoxyquin (Anti-oxydant)	0	0.04
Cholesterol, NF	0	1.5
Dye, Blue FD&C #1	0.05	0

Biochemical analyses

Hepatic TG and cholesterol levels were quantified using approximately 100 mg of tissue. Lipids were extracted by overnight 2:1 chloroform/methanol solution. After centrifugation, the lower phase was dried up under nitrogen flux, and resuspended with Floch and Thesit solution 20%. Quantification of total cholesterol and TG from the liver and plasma was performed using Cholesterol E kits (Wako Diagnostics,Mountain View, CA, USA), and TG assay (Randox Laboratories Ltd., Crumlin, UK). respectively. Plasma PCSK9 concentrations were measured using Mouse/Rat PCSK9 ELISA kits from CircuLex (Cat# CY-8078).

Real-time polymerase chain reaction

Quick-frozen tissue samples of liver were powdered with a cold pestle and mortar, and ~ 100 mg was used for the isolation of RNA. Total RNA was extracted using RNeasy Micro (Qiagen). RNA integrity was validated using a Bioanalyzer 2100 (Agilent). Total RNA was treated with DNase and reverse transcribed using the Maxima First Strand cDNA synthesis kit with ds DNase (Thermo Fisher Scientific). A BioDrop spectrophotometer was used to determine RNA concentrations and the ratio of absorbance at 260 and 280 nm was used to assess purity. RNA integrity was evaluated by visualization of intact 18S and 28S RNA bands following agarose gel electrophoresis. SuperScript VILO Master Mix (Invitrogen) was used to synthesize cDNA with 1 µg of RNA per 20 µL reaction.

Real-time qPCR reactions were performed in triplicate using 384‐well plates in the QuantStudio6 system (Life Technologies) with SYBR Select Master Mix kit (Applied Biosystems). Reaction conditions consisted of 2 µL of a 1:10 cDNA dilution and 0.3 µM primers in a final volume of 10 µL. The following cycling protocol was used: 2 minutes at 50 °C, 2 minutes at 95 °C, followed by 40 cycles of 15 seconds at 95 °C, 15 seconds at 58 °C, and 1 minute at 72 °C. The reaction's specificity was verified by melting curve analysis. Negative controls (RT) and no template controls (NTC) were included in the PCR runs. Reference genes were identified by testing 10 different genes (picked from literature) and running TaqMan Array Rat Endogenous Control Plates (Applied Biosystems) on all samples. The 3 reference (housekeeping) genes with the best gene expression stability measure (Expression Suite, Applied Biosystems) were selected for normalization: Hprt, Actb, and Gapdh. Primers used (Table 2) were designed using Primer‐BLAST (NCBI). Whenever possible, intron‐spanning primers were selected to avoid amplification of genomic DNA. A standard curve obtained by scalar dilution of a cDNA pool was generated to verify PCR efficiency. The triplicate average values of cycle threshold (Ct) were used for quantification, and target genes were determined by 2ΔΔCt calculation method.

Table 2

Oligonucleotide primers used for quantitative real-time polymerase chain reaction
Gene name NCBI	Alias	Oligo FWD	Oligo REV	Sonde UPL
Abcg5	ABCG5	cggagagttggtgttctgtg	caccgatgtcaagtccatgt	82
Abcg8	ABCG8	cagatgctggctatcataggg	ctgatttcatcttgccacca	13
Acat2	ACAT-2	agaagtccttcagagagccaaa	cactggcttgtcgagtagga	50
Acaca	ACC	acagagatggtggctgatgtc	gatccccatggcaatctg	4
Actb	Actb (control)	cccgcgagtacaaccttct	cgtcatccatggcgaact	17
Mlxipl	ChREBP	aatcccagcccctacacc	ctgggaggagccaatgtg	10
Gapdh	Gapdh (control)	ccctcaagattgtcagcaatg	agttgtcatggatgaccttgg	73
Hmgcr	HMGCoAR	caaccttctacctcagcaagc	acagtgccacacacaattcg	80
Hprt	Hprt (control)	gaccggttctgtcatgtcg	acctggttcatcatcactaatcac	95
Ldlr	LDL-R	tgctactggccaaggacat	ctgggtggtcggtacagtg	16
Lrp1	LRP-1	aatcgagggcaagatgacac	ccagtctgtccagtacatccac	81
Pcsk9	PCSK9	ccagtgtccacgcttcct	gatgccatgctccttgattt	1
Scarb1	SR-B1	ggtgcccatcatttaccaac	gcgagccctttttactacca	71
Srebf2	SREBP2	gtgcagacagtcgctacacc	aatctgaggctgaaccagga	62

Statistical analyses

All data are expressed as means ± SD. Statistical comparisons were performed using a two-way analysis of variance (ANOVA) for non-repeated measures, using diet and time as main effects. Fisher’s post-hoc test was used in the event of a significant (P < 0.05) F ratio.

Anthropometric parameters and food intake

Body weight was similar for all dietary groups at the beginning of the experimental period (Table 3). Body weight was not statistically modified by the obesogenic diets after 2 weeks of treatment. However, it was significantly higher (P < 0.01) at 6 weeks. Hence, body weight was greater (P < 0.01) in rats fed with the HFD and WD than those submitted to the SD. Average food intake in g/d was lower (P < 0.01) in rats fed the HFD and WD as compared to rats fed the SD. Food intake in g/d was also lower (P < 0.05) in rats fed the HFD as compared to rats fed the WD (Table 3). When food intake was calculated in kcal/d, animals treated with the HFD displayed slightly lower values (P < 0.05) than those of other dietary conditions. The sum of 3 visceral fat pad weights as well as the sc fat depot were significantly (P < 0.05) higher in rats fed the HFD and WD compared to rats fed the SD after 2 (P < 0.05) and 6 weeks (P < 0.001; Table 3). HFD and WD resulted in similar body fat accumulations.

Liver and plasma triglycerides and cholesterol

Liver TG accumulation was significantly higher (P < 0.001) in rats fed both the HFD and the WD after 2 as well as after 6 weeks (Fig. 1a). Liver TG levels were even slightly higher (P < 0.05) in WD than in HFD fed animals after the 2-week feeding period. Liver TG levels were higher (P < 0.01) after 6 weeks than after 2 weeks in the SD fed group. On the other hand, HFD and WD did not affect plasma TG levels after 2 weeks of feeding but resulted in a slight but significant (P < 0.05) decrease after 6 weeks of feeding (Fig. 1b).

Although some tendencies (P < 0.1) can be observed, liver cholesterol concentrations were not changed significantly (P > 0.05) by the diets with the exception of the group of animals fed the WD for 6 weeks that showed significantly (P < 0.01) higher levels compared to SD and HFD fed rats (Fig. 1c). On the other hand, plasma cholesterol levels were higher (P < 0.05) in WD compared to SD and HFD fed animals after 2 and 6 weeks (Fig. 1d). No significant difference in plasma cholesterol levels was found between rats fed the SD and the HFD. Overall, plasma cholesterol levels were higher (P < 0.05) with the longer diet duration (Fig. 1d).

Liver molecular markers

As presented in Fig. 2a, rats submitted to the WD for 2 weeks depicted lower LDL-R gene expression levels as compared to SD (P < 0.05) and HFD (P < 0.05) animals. LDL-R mRNA levels were also lower (P < 0.05) in rats fed the WD compared to those fed the SD after 6 weeks. The same pattern of response was observed for the transcription factor SREBP2 (P < 0.001) that regulates both LDL-R and PCSK9 (Fig. 2b). Similarly, PCSK9 mRNA expression was lower (P < 0.01) in rats fed the WD as compared to animals fed the SD and HFD after 2 and 6 weeks (Fig. 2c). This pattern of response was similar for plasma PCSK9 concentrations after 2 weeks while WD fed animals depicted lower levels as compared to HFD fed rats after 6 weeks (Fig. 2d). The transcription of LRP-1, a member of the LDL-R gene family, was also significantly (P < 0.05) lower after 2 and 6 weeks in animals submitted to the WD when compared to animals fed the SD (Fig. 2e). Gene expression of ACAT-2 involved in cholesterol esterification was also lower (P < 0.05) following 2 and 6 weeks of WD compared to the SD with the 6-week treatment showing even lower levels that the 2-week (Fig. 2f).

Transcripts of HMGCoAR, the main enzyme of cholesterol synthesis, were decreased (P < 0.001) after 2 weeks in rats fed the HFD and the WD while after 6 weeks lower levels (P < 0.05) were found only in rats fed the WD (Fig. 3a). Animals fed the SD depicted lower gene expression of HMGCoAR after 6 weeks than after 2 weeks. Gene expression of SR-B1, the transporter responsible to exchange cholesterol from HDL particles to the liver, was significantly (P < 0.001) lower in WD-fed animals compared to SD- and HFD-fed rats in both feeding durations (Fig. 3b). SR-B1 mRNA levels was higher (P < 0.001) in HFD compared to SD fed animals after 6 weeks. A small but significant (P < 0.05) effect of overall diet duration was also found with higher values observed after 6 weeks. Gene expressions of ABCG5 and ABCG8 both involved in cholesterol export into bile ducts, were significantly (P < 0.01) higher in rats fed the HFD as compared to those submitted 2 and 6 weeks of the other diets (Fig. 3c and d). Gene expression of ACC, an important enzyme regulating the first steps of de novo lipogenesis, was higher in rats fed the WD compared to rats fed the two other diets for 2 weeks (Fig. 3e). After 6 weeks of treatment, ACC transcription was also higher (P < 0.001) in rats fed the WD than those treated with the HFD. ACC mRNA levels were also lower (P < 0.001) in HFD than in SD fed rats after 2 and 6 weeks. Gene transcription of ChREBP, the transcription factor involved in regulating mediators of de novo lipogenesis, was highly stimulated (P < 0.01) in rats fed with the WD compared to those treated for 2 weeks of the other dietary regimens (Fig. 3f). This response was not observed after 6 weeks of treatment.

The main finding of the present study is that animals fed with a WD (high fat + high sucrose) for only 2 weeks depicted lower gene expression of several key markers of liver cholesterol metabolism including LDL-R, PCSK9, SREBP2, and HMGCoAR. The same response was also observed for PCSK9 measured in plasma. These findings were not observed when rats were fed the HFD without fructose. The 2-week WD also resulted in higher plasma cholesterol levels. The HFD and even more so the WD promptly (2 weeks) induced excessive TG accumulation in liver. These results indicate that the WD, a diet that does not contain higher levels of cholesterol, disturbs the mechanisms involved in the regulation of cholesterol metabolism in liver. The higher level of fructose together with higher fat content of the WD that promptly and potently resulted in liver TG accumulation may be put forward as responsible for the cholesterol pathway disturbances. As a clinical point of view, these results suggest that high plasma cholesterol levels may be linked to lipid metabolism in liver.

The first observation of the present study is that a short period (2 weeks) of an HFD (60%) or a WD (40% fat + 35% sucrose (17.5% fructose)) induced an important increase in the liver TG levels that remained stable after 6 weeks of treatment. An early accumulation of lipids in the liver in response to high-fat feeding has been previously observed (11, 15, 16). This response has been interpreted as if the liver was acting as a buffer to protect the other organs from a potentially deleterious surge of lipids (11, 17). Accordingly, plasma TG levels were not changed after 2 weeks of HFD or WD. An additional finding of the present study is that liver TG accumulation after 2 weeks was even higher when the dietary fat was reduced from 60 to 40% and replaced by fructose (17.5%). Fructose is a strong inducer of hepatic lipogenic enzymes (18). Hence, the present increases in gene expression of ACC and ChREBP measured in the liver of animals submitted to 2 weeks of WD is an indication that de novo lipogenesis promoted the excessive accumulation of fat in this organ. These data, therefore, indicate that liver fat accumulation in rats fed the WD involved two mechanisms, which are dietary fat storage and de novo lipid synthesis.

In addition to resulting in a rapid increase in liver TG accumulation, the short 2-week period of feeding applied in the present study has the advantage of minimizing the overall metabolic effects of an obesogenic diet. Body weight in animals fed the HFD and WD was similar to that found in rats fed the SD diet after 2 weeks while it was approximately 50 g higher after 6 weeks. Moreover, the sum of abdominal and subcutaneous fat accumulation was approximately 3 times lower after 2 weeks than after 6 weeks of the obesogenic diets (7 vs 20 g), while liver TG levels were similar after 2 and 6 weeks. It is, therefore, our view that the present 2-week feeding experimental design is appropriate to isolate the metabolic effects of an increase in liver fat. For this reason, the present discussion is centered on results derived from the 2-week feeding condition.

Using this experimental design, the present study aimed to gather evidence for the existence of an association between a rapid increase in liver TG in rats submitted to short-term (2 weeks) obesogenic diets and disturbances in gene expression of key regulators of cholesterol metabolism in the liver. The main finding of the present study was that feeding animals with a WD for 2 or 6 weeks resulted in a decrease in gene expression of three important and related markers of liver cholesterol metabolism: LDL-R, PCSK9, and the transcription factor SREBP2. These 3 molecules combine their action to regulate transport of highly atherogenic LDL particles. With approximately 70% of its expression carried out in the liver, it is clear that LDL-R is crucial for the clearance of LDL-cholesterol particles from circulation (19). Therefore, the increase in plasma cholesterol observed in rats fed with WD at 2 and 6 weeks could be the result of the decrease in gene expression of liver LDL-R.

Our results also showed that the levels of mRNA and plasma PCSK9 (a protease that down-regulate LDL-R protein expression (20, 21, 22)) are reduced at 2 and 6 weeks after WD feeding. This could have normally contributed to promote the clearance of plasma cholesterol by increasing LDL-R protein levels. However, because of the decrease in upstream gene expression of SREBP2, a transcription factor that regulates the expression of both LDL-R and PCSK9 (23, 24), the reduction in PCSK9 levels did not positively affect the expression of LDL-R. Consequently, plasma cholesterol remained high in rats fed the WD at 2 and 6 weeks. Similarly, gene expression of LRP1, responsible for the removal of circulating lipoprotein remnants enriched in cholesterol (25), was reduced in rats fed the WD, thus potentially contributing to the increased plasma cholesterol levels. In addition, cholesterol transport from HDL to the liver is carried out through its interaction with its receptor SR-B1 (26) whose gene expression was also lowered by the WD. All of these responses may have contributed to the decrease in cholesterol uptake by the liver and, in turn, promoted its increased concentration in the circulation.

Along with LDL-R and PCSK9, the expression of HMGCoAR, the key marker of cholesterol synthesis in the liver, is regulated by the transcription factor SREBP2 (23, 24). Accordingly, SREBP2 and HMGCoAR mRNA levels were highly reduced in rats fed with the WD. At the same time, mRNA expression of ACAT-2 that is involved in cholesterol esterification was reduced in rats fed the WD compared to rats submitted to the HFD. Taken together, these results concourse to the interpretation that in the liver, a short-term WD yielding a large increase in liver TG content promotes disruption of pathways involved in cholesterol metabolism.

If liver cholesterol uptake from the circulation is reduced under the present WD, the next question is what can trigger this response? The accumulation of cholesterol in the liver may be an important factor inducing the reduction in gene expression of key markers of the LDL-R pathway and, in turn, the increase in plasma cholesterol level (23). Liver cholesterol concentrations show a tendency (P < 0. 1) to be higher after 2 weeks of both the HFD and WD, a tendency that became highly significant for the WD after 6 weeks. In this regard, it is noticeable that gene expression of HMGCoAR was lowered in rats fed the HFD and even more so in rats fed the WD for 2 weeks. This response may have been stimulated by a transient rise of cholesterol levels in the liver. In addition, the low mRNA levels of ABCG5/G8 transporters (responsible for the exportation of cholesterol in the bile ducts) in rats fed the WD might have contributed to transient higher liver cholesterol levels in these animals. However, this association does not hold for the HFD-fed animals since ABCG5/G8 mRNA levels were increased in these groups of rats. It is likely that other mechanisms such as fecal excretion of biliary acids (27) or increased synthesis and secretion of VLDL are involved

Another important question emerging from the present results is what factors alter the expression of key metabolic markers of hepatic cholesterol homeostasis in response to 2 weeks of WD and why does the response in animals submitted to the HFD not reach the same extent? It must be taken into account that after 2 weeks, hepatic TG levels were higher in WD than in HFD fed rats and this might have interfered with cholesterol metabolism. A key element of the WD diet is its content of fructose. Fructose that has been reported to increase protein levels of all de novo lipogenesis enzymes during its conversion into triglycerides (28, 29). The WD-induced lipogenesis necessitates the synthesis of malonyl-CoA, which is not the case for dietary fat. Malonyl-CoA is a potent inhibitor of mitochondrial oxidation (30). By doing so, increased malonyl-CoA levels may have indirectly contributed to the higher level of liver fat accumulation in animals submitted to the WD. A second interaction between fructose and cholesterol metabolism is that lipogenesis and cholesterol synthesis pathways both use acetyl CoA as a molecular precursor, and both use the reducing equivalent NADPH as a sole source of energy. Although other factors may be involved, this may have contributed to the lower gene expression of HMGCoAR found in rats treated with the WD as compared to rats fed with the HFD for 2 weeks. In one of the rare studies evoking the potential link between fructose and hepatic cholesterol metabolism, Ichigo et al. (12) reported a decrease in ABCG5/G8 gene expression in rats submitted to 12 days of high fructose as compared to rats fed a high glucose diet. Although comparisons between this study and ours remain difficult, these authors did hypothesize that a fructose diet might affect genes involved in regulation of cholesterol metabolism.

Although the precise mechanism linking liver fat accumulation to disruption of liver cholesterol metabolism is beyond the scope of the present study, our results do indicate that a short-term WD disturbs hepatic cholesterol metabolism while promoting a rapid increase in circulating cholesterol levels. Results derived from previous reports show that cholesterol homeostasis in the liver may be perturbed by activities of other metabolic pathways. For instance, Kemper et al. (31) reported that the ingestion of an ester of B-hydroxybutyrate in rats promoted an increase in LDL-R and SREBP2 protein levels. More recently, Rana et al. (32) reported that the cell surface protease-activated receptor PAR2 is a factor linking gene expression of several markers of cholesterol metabolism (i.e. LDL-R, SR-B1) to increased TG levels in the liver. This effect could potentially be mediated through the action of the de novo lipogenesis pathway. On a long-term basis, fructose has been highly associated with the development of liver steatosis in human (33) while several metabolic pathways modulating hepatic cholesterol are dysregulated in NAFLD (7). Notwithstanding these observations, NAFLD is associated with several metabolic disorders (i.e. inflammation) making it difficult the direct association between liver fat accumulation and hepatic cholesterol dysregulation. The present study indicates that a 2-week WD used as a model of isolated hepatic steatosis, resulted in the disruption of several cholesterol metabolic pathways most likely leading to a rapid increase in circulating cholesterol levels. The presence of fructose in the WD suggests that de novo lipogenesis pathway interferes with the proper regulation of hepatic cholesterol metabolism. From a clinical point of view, the present findings suggest that an increase in plasma cholesterol may result from an increase in dietary fat and fructose.

ACKNOWLEDGEMENTS

FINCIAL SUPPORT

This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC; 7594).

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

AUTHORSHIP

This work was performed at Université du Québec à Montréal (UQAM).

RS-A (conception, design, acquisition of data), ENS and SQ (acquisition of data) JML and DS-P (conception, design).

All authors contributed to data interpretation and drafting/revising the article for important intellectual content. All authors approved the final version to be published.

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Two Weeks of Western Diet Disrupts Liver Molecular Markers of Cholesterol Metabolism in Rats

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Abstract

Background

Method:

Results

Conclusion

Figures

Introduction

Material And Methods

Animals care and treatments

Diets

Biochemical analyses

Real-time polymerase chain reaction

Statistical analyses

Results

Anthropometric parameters and food intake

Liver and plasma triglycerides and cholesterol

Liver molecular markers

Discussion

Declarations

ACKNOWLEDGEMENTS

FINCIAL SUPPORT

CONFLICT OF INTEREST

AUTHORSHIP

References

Status:

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