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© 2007 American Society for Nutrition J. Nutr. 137:2219-2223, October 2007

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Carbohydrate Restriction Alters Hepatic Cholesterol Metabolism in Guinea Pigs Fed a Hypercholesterolemic Diet1

Moises Torres-Gonzalez2, Sudeep Shrestha2, Mathew Sharman3, Hedley C. Freake2, Jeff S. Volek3 and Maria Luz Fernandez2,*

2 Department of Nutritional Sciences and 3 Department of Kinesiology, University of Connecticut, Storrs, CT 06269

* To whom correspondence should be addressed. E-mail: maria-luz.fernandez{at}uconn.edu.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
The current study was undertaken to evaluate the effect of carbohydrate restriction on hepatic cholesterol metabolism in guinea pigs fed a hypercholesterolemic diet. Hartley male guinea pigs (n = 10 per group) were fed 1 of 3 diets: a diet with a percent energy distribution of 42:23:35 carbohydrate:protein:fat and 0.04% cholesterol (control), a diet with the same macronutrient distribution but with 0.25% cholesterol (HChol), or a carbohydrate-restricted (CR) diet with a percent energy distribution of 11:30:59 carbohydrate:protein:fat and 0.25% cholesterol for 12 wk. There was more accumulation of hepatic cholesterol and triglycerides as well as lower 3-hydroxy-3-methyl glutaryl-CoA reductase messenger RNA abundance in guinea pigs fed the high-cholesterol diets (HChol and CR) (P < 0.01). Guinea pigs fed the CR diet had lower concentrations of hepatic total cholesterol and cholesteryl ester than those fed the HChol diet (P < 0.05). There was no diet effect on hepatic LDL receptor expression. Hepatic acyl CoA cholesteryl acyltransferase (ACAT) activity was lowest in guinea pigs fed the low-cholesterol diet (9.7 ± 4.8 pmol·min–1·mg–1), intermediate in those fed the CR diet (37.3 ± 12.4 pmol·min–1·mg protein–1), and highest in guinea pigs fed the HChol diet (55.9 ± 11.2 pmol·min–1·mg–1). ACAT activity was significantly correlated with hepatic cholesterol (r = 0.715; P < 0.01) and LDL cholesterol (r = 0.59; P < 0.01) for all dietary groups, suggesting a major role of this enzyme in hepatic cholesterol homeostasis and in lipoprotein concentrations. These results indicate that dietary cholesterol increases hepatic lipid accumulation and affects hepatic cholesterol homeostasis. Carbohydrate restriction in the presence of high cholesterol is associated with lower hepatic ACAT activity and an attenuation of hepatic cholesterol accumulation.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
The liver is essential for controlling plasma glucose, protein, and lipid levels as well as for producing bile acids for fat digestion and absorption, thereby contributing to the processing, distribution, and maintenance of the body's fuel supply (1). Although it is not the main organ that synthesizes cholesterol de novo, the liver is responsible for controlling cholesterol homeostasis in the body (2). The liver can regulate plasma cholesterol levels through several mechanisms. One of these mechanisms, perhaps the most important one, relies on the regulation of HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis (3). 3-Hydroxy-3-methyl glutaryl (HMG)3-CoA reductase is found in virtually all tissues, but the liver expresses the highest level of this enzyme and the feedback regulation by dietary cholesterol occurs in this organ (4). Acyl CoA cholesteryl acyltransferase (ACAT)4 is another enzyme utilized by the liver and together with HMG-CoA reductase, they are the 2 key enzymes involved in controlling the intracellular and extracellular levels of cholesterol (4). ACAT esterifies free cholesterol (FC) with fatty acyl CoA, thereby reducing the toxic effects of excessive cholesterol in cell membranes (5). The liver is also the organ that expresses the highest concentrations of the LDL receptor (LDL-r), which is a key mechanism used to remove LDL cholesterol (LDL-C) from circulation (5).

Recently, much attention has been paid to carbohydrate-restricted (CR) diets, because they lead to favorable changes in plasma lipids, which may reduce the risk of cardiovascular disease. They have been effective in reducing body weight and plasma triglycerides (TG) and in increasing HDL cholesterol (6,7). However, little is known about their effects on hepatic lipid metabolism and their overall effect on the main regulators utilized by the liver to control cholesterol homeostasis. It is difficult to investigate these effects in human liver, mainly due to the lack of adequate techniques to explore the possible in vivo mechanisms. The selection of a suitable animal model that resembles humans in regards to lipid metabolism is helpful. Our laboratory group has extensively demonstrated that guinea pigs are an excellent animal model to study lipid metabolism due to their many similarities with humans. Most importantly, in their normal state, guinea pigs carry the majority of their cholesterol in LDL and have high LDL:HDL ratios (8,9). In addition, studies have shown that guinea pigs, like humans, have higher levels of hepatic FC than hepatic cholesterol-ester as well as similar activities of hepatic ACAT and HMG-CoA reductase (10). Another important aspect is that they respond similarly to dietary interventions such as CR diets (11).

Given that the liver is the main organ that controls macronutrient metabolism and cholesterol levels in the body and due to the current lack of knowledge on the effects of carbohydrate restriction on this organ, this study was undertaken to evaluate the effects of CR diets during a cholesterol challenge on hepatic lipid metabolism in guinea pigs. The first objective of this study was to assess the effects of dietary cholesterol and CR diets on hepatic accumulation of FC, cholesterol ester (CE), and TG. The other objective was to evaluate the effects of these diets on the main hepatic cholesterol regulators, HMG-CoA reductase, ACAT activity, and LDL-r. We hypothesized that dietary cholesterol would result in alterations in hepatic cholesterol metabolism and that carbohydrate restriction would attenuate hepatic cholesterol accumulation.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Materials. Kits to measure plasma TG and cholesterol were purchased from Roche Diagnostics. Ketone kits were from Waco Diagnostics. DL-Hydroxy- [3-14C] methyl glutaryl CoA (1.81 GBq/mmol), DL- [5-3H] mevalonic acid (370 GBq/mmol), cholesteryl- [1,2,6,7-3H] oleate (370 GBq/mmol), Aquasol, Liquiflor (toluene concentrate), and [14C] cholesterol were purchased from DuPont NEN. Oleoyl- [1-14C] CoA (1.8 GBq/mmol) and DL-3-hydroxy-3-methyl glutaryl CoA were obtained from Amersham. Cholesteryl oleate, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, NADP, sodium fluoride, Triton, bovine serum albumin, and sucrose were obtained from Sigma Chemical. Glass silica gel plates were purchased from EM Science.

    Diets. Isocaloric diets were designed to meet the nutritional requirements of guinea pigs. The 3 diets differed in their cholesterol, carbohydrate, and fat content (diet composition shown in Table 1). Briefly, diet 1 (control) was high in carbohydrate (42% energy) and low in cholesterol (0.04%). Diet 2 was high in cholesterol (0.25%) and had the same amount of carbohydrate (HChol). Diet 3 was high in cholesterol (0.25%) and low in carbohydrate (11% of total energy) and was defined as the CR diet. The level of cholesterol in diets 2 and 3 is known to cause hypercholesterolemia in guinea pigs. Dietary cholesterol at 0.25% in this model corresponds to an absorbed amount equal to the daily cholesterol synthesis rates (9) and is equivalent to 1800 mg/d for a human diet. The fat mix was rich in lauric and myristic acids, known to cause endogenous hypercholesterolemia in guinea pigs (10).


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  		TABLE 1 Composition of control, HChol, and CR diets

 
    Guinea pigs. Male Hartley guinea pigs weighing between 250 and 300 g were purchased from Harlan Sprague-Dawley. The guinea pigs (n = 10 per group), were randomly allocated to 1 of 3 treatments for 12 wk: control, HChol, or CR diet. Two guinea pigs were housed per cage in a light cycle room (light from 0700 to 1900) at 23°C. Guinea pigs consumed diet and water ad libitum. During this time, the diets were weighed every 2 d to measure the amount of food consumed. All animal experiments were conducted in accordance with U.S. Public Health Service/USDA guidelines. Experimental protocols were approved by the University of Connecticut Institutional Animal Care and Use Committee.

After 12 wk of dietary treatment, guinea pigs were anesthetized under isofluorane vapors and blood was obtained via heart puncture. The livers were harvested and divided to isolate microsomes, to determine hepatic lipids, and to extract RNA for the measurement of the LDL-r and HMG-CoA reductase. The liver used for RNA extraction was frozen in liquid nitrogen.

    Plasma lipids and ketones. Plasma total cholesterol and LDL-C were measured as previously reported (11). Total plasma ketones were measured using cyclic enzymatic methodology in commercially available kits, which measure both the concentrations of acetoacetate and 3-hydroxy-butyrate as previously reported (12).

    Hepatic lipids. Hepatic total and FC (13) and TG (14) were determined as previously described. Briefly, 1 g of liver was sliced into small pieces and combined with 10 mL chloroform:methanol (2:1) overnight. Lipid extraction was accomplished by mixing with acidified water and separating the 2 phases with a separatory funnel. An aliquot of 0.2 mL, taken from the lower phase, was completely evaporated and suspended in 0.2 mL ethanol for enzymatic determination of free and total cholesterol utilizing cholesterol oxidase and cholesterol esterase, respectively. Cholesteryl ester concentrations were calculated by subtracting FC from total cholesterol. Then 2 mL were recovered from the lower phase for TG determination using enzymatic methods as previously reported (9).

    Hepatic microsome isolation. The hepatic microsomal fraction was isolated by 2 25-min centrifugations at 10,000 x g (JA-20 rotor, J221) followed by ultracentrifugation at 100,000 x g; 1 h at 4°C in a Ti-50 rotor for as previously described (9). Microsomes were resuspended in the homogenization buffer and centrifuged for an additional hour at 100,000 x g. After centrifugation, microsomal pellets were homogenized and stored at –70°C.

    Hepatic ACAT activity. Hepatic ACAT activity was measured by the incorporation of [14C] oleoyl CoA in cholesteryl ester in hepatic microsomes by preincubating 0.8–1 mg of microsomal protein per assay with 84 g/L albumin and buffer for microsomal isolation (15). Recoveries of [C–14] cholesteryl oleate were ~90%.

    RNA extraction. Total RNA was extracted from liver by the Trizol method (16). To check the integrity of the extracted RNA, samples were electrophoresed through a 1% agarose gel at 125 V for 45 min. Two clear sharp bands representing 28S and 18S ribosomal fractions of RNA were obtained (data not shown).

    RNA quantification. First, cDNA was synthesized for HMG-CoA reductase and LDL-r using an iScript cDNA synthesis kit following the manufacturer's instructions. cDNA was amplified using real time PCR. This procedure was conducted in duplicate using the LightCycler FastStart DNA Masterplus SYBR Green I (Roche Diagnostics), also following the manufacturer's instructions. The oligonucleotide primers were those used for amplification for HMG-CoA reductase and the LDL-r in humans (17). The primers used for ß-actin, a house-keeping gene, were taken from guinea pig sequences and have been previously reported (18). The size of each reaction product is as follows: HMG-CoA reductase, 70 bp; LDL-r, 74 bp; and ß-actin, 220 bp. ß-Actin was used as a control in all reactions. The reactions were conducted under the following conditions: polymerase activation at 95°C for 5 min followed 45 cycles of denaturing at 95°C for 10 s, annealing at 57°C for 10 s, and extension at 72°C for 10 s. Quantification was conducted by analyzing the fluorescence curves detecting the crossing point of samples using LightCycler Software 4.0 (Roche Diagnostics).

    Statistical analysis. One-way ANOVA was used to determine differences between groups in hepatic lipids, ACAT activity, and HMG-CoA reductase and LDL-r messenger RNA (mRNA) abundance. The least significant difference test was used to evaluate differences in means. P < 0.05 was considered significant. Pearson correlations were calculated for hepatic lipids, enzyme activity, and LDL-C. Values in the text are means ± SD.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
Plasma lipids of the guinea pigs have been previously reported (11). Total plasma cholesterol and TG did not differ between the HChol (10.7 ± 3.5 mmol/L) and CR (14.0 ± 3.8 mmol/L) groups and they were higher than the controls (3.4 ± 1.4 mmol/L) (P < 0.01). These changes in total cholesterol were associated with increased plasma LDL-C concentrations, which were 2.3 ± 1.2 mmol/L in the controls, 7.9 ± 2.3 mmol/L in the HChol group, and 10.0 ± 3.3 mmol/L in the CR group. The plasma TG concentrations were 0.32 ± 0.14, 0.62 ± 0.27, and 0.83 ± 0.33 mmol/L in the control, HChol, and CR groups, respectively.

The guinea pigs fed the CR diet had higher concentrations of plasma ketones (P < 0.01) (Table 2), which indicated that these animals develop ketosis in a similar way to humans. Hepatic total cholesterol and CE concentrations were higher in the Hchol group than in the control and the CR groups (P < 0.01). In contrast, FC and TG concentrations in the liver did not differ between the HChol and CR groups and were both higher than the controls (P < 0.05) (Table 2). Hepatic ACAT activity was correlated with total cholesterol (r = 0.715; P < 0.001) (Fig. 1A) as well as free and esterified cholesterol (data not shown). Guinea pigs fed the control diet had the lowest hepatic ACAT activity, the CR group had an intermediate value, and the HChol had the highest activity (Table 2). Plasma LDL-C concentrations were positively correlated with hepatic ACAT activity (r = 0.59; P < 0.01) for all groups of guinea pigs.


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  		TABLE 2 Hepatic lipids, ACAT activity, and plasma ketones of guinea pigs fed control, HChol, and CR diets for 12 wk1

 

Figure 1
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  		FIGURE 1  Correlations between hepatic ACAT activity and hepatic cholesterol (A) and plasma LDL-C (B) concentrations in 30 guinea pigs fed control, HChol, or CR diets for 12 wk.

 
HMG-CoA reductase mRNA levels were affected by dietary cholesterol (Fig. 2). The mRNA abundance for this enzyme was lower for the HChol (16.0 ± 6.7 arbitrary units) and CR (12.9 ± 4.2) groups than for the control group (26.0 ± 12.6) (P < 0.025). These results indicate that high-cholesterol diets suppress the expression of this enzyme and show that HMG-CoA reductase is an important regulator of cholesterol homeostasis in guinea pigs, as it is in humans. CR did not affect HMG-CoA reductase mRNA expression.


Figure 2
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  		FIGURE 2  Hepatic mRNA abundance of the LDL-r and HMG-CoA reductase for guinea pigs fed control, Hchol, or CR diets for 12 wk. Values are means ± SD, n = 10. Bars with unlike letters differ, P < 0.025.

 
Dietary cholesterol and dietary carbohydrate did not affect the expression of LDL-r, because mRNA abundance did not differ in the 3 groups (Fig. 2).


   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
    Dietary cholesterol, carbohydrate restriction, and hepatic lipid accumulation. This study investigated the effects of carbohydrate restriction during a cholesterol challenge on hepatic lipid metabolism in guinea pigs. The cholesterol derived from the diet is delivered mainly as CE to the liver through chylomicron remnants. Inside the liver, CE are hydrolyzed, generating FC and fatty acids (4). Therefore, high-cholesterol diets increase the levels of FC accumulated in this organ, as we observed in the current study. Several studies have reported the same effect in mice (19), hamsters (20), opossums (21), and rabbits (22). The liver processes FC toxicity by transforming it into CE, which can be stored or used for secretion into VLDL particles (5). Therefore, as the FC in the liver increases, a rise in CE ester is also expected. However, in this study, high cholesterol in combination with a CR diet did not increase hepatic CE concentrations. Regardless of the high cholesterol in the diet, the CR group had similar hepatic CE concentrations as the low cholesterol group. Several animal studies have demonstrated that cholesterol induces the expression of lipogenic enzymes such as steroyl CoA desaturase (SCD) (23). SCD synthesizes monounsaturated fatty acids, mainly palmitoleic and oleic fatty acids, which are the preferred fatty acids utilized for the formation of CE (24). In contrast, it has been demonstrated that high-carbohydrate diets also induce the expression of SCD (25). In this study, we speculate that high carbohydrate in combination with high dietary cholesterol may act synergistically, stimulating the synthesis of CE and this may explain why the HChol group had higher levels of CE than the CR group.

The high-cholesterol diets resulted in higher levels of hepatic cholesterol than the control group. As discussed above, cholesterol not only stimulates the expression of SCD, but it also induces the expression of diacyl glycerol acyltransferase, an enzyme involved in the synthesis of TG (26); hence, higher levels of dietary cholesterol result in higher concentrations of TG stored in the liver. In addition, high-carbohydrate diets stimulate de novo synthesis of fatty acids if they are mainly composed of simple sugars (25), as was the case in the current study. When glucose is delivered into the portal vein in large quantities, it can be converted into TG through de novo lipogenesis (27). Enzymes of the lipogenic pathway that have been reported to be regulated by carbohydrates include acetyl-CoA carboxylase, fatty acid synthase, and SCD (28). Thus, the high hepatic TG levels in the HChol group were due to the combined effect of high cholesterol and high carbohydrate in the diet, which stimulated de novo fatty acids synthesis, producing similar TG levels as the CR group.

    Dietary cholesterol, carbohydrate restriction, and ACAT activity. Several authors have demonstrated that substrate availability (e.g. FC and fatty acids) is the major determinant for ACAT activity (29). In the present investigation, high cholesterol in the diets increased ACAT activity. However, CR had lower ACAT activity than the HChol group, suggesting that CR attenuates ACAT activity, explaining why this group also had lower CE levels than the HChol group. Again, these findings indicate that carbohydrate in combination with cholesterol produces a stronger effect on the synthesis of CE through ACAT induction than the combination of fat and cholesterol.

As in other studies (30,31), this study found positive correlations between hepatic free, total, esterified cholesterol, and ACAT, which clearly indicates 2 important aspects: 1) the substrate availability is the major determinant for ACAT activity; and 2) as ACAT activity increased, so did levels of CE, suggesting that cholesterol esterification is one of the major mechanisms utilized by the liver to control the toxic effects that can be produced by FC.

High ACAT activity also induced the secretion of more VLDL (21,32,33), because CE is one of the components carried by these particles. VLDL in plasma can lead to the formation of intermediate density lipoprotein and LDL and the final outcome is high plasma LDL-C as a result of high ACAT activity in the liver. Previously, we reported that a high-cholesterol diet increased VLDL cholesterol as well as LDL-C (11). In this study, as expected, we observed a positive correlation between ACAT activity and LDL-C levels. This result again confirms that ACAT plays an important role in determining plasma LDL-C concentrations, which is why ACAT inhibition has lately been tested as a target to reduce plasma cholesterol.

    Dietary cholesterol, carbohydrate restriction, and HMG-CoA reductase and LDL-r mRNA levels. When animals face a cholesterol challenge, they respond by markedly downregulating the expression of hepatic HMG-CoA reductase, the rate-limiting enzyme in de novo synthesis of cholesterol (34). In this study, we found that high cholesterol in the diet (CR and HChol diets) reduced hepatic HMG-CoA reductase mRNA levels. In other studies using different animal models such as mice, hamsters, and Golden Syrian hamsters fed diets containing 5, 2, and 0.5% cholesterol for 10, 14, and 12 d, respectively, decreased mRNA levels of this enzyme were also found (35).

LDL-r is another important regulator used by the liver to control cholesterol levels in the body (2). However, the findings relating to LDL-r expression have been contradictory, because some investigators have reported an upregulation (36), a downregulation (37), or no effect (38) when animals were fed high-cholesterol diets. Some authors have found that animals challenged with high-cholesterol diets increased the expression of hepatic LDL-r and they have suggested that this response might be utilized by the liver to help maintain cholesterol homeostasis (36). Other studies, however, have found the opposite. During a cholesterol challenge, animals have downregulated both the mRNA and the protein levels of LDL-r (37). In other animal models, LDL-r mRNA levels have not been affected during a cholesterol challenge (1% of cholesterol in the diet), although they did find a downregulation in HMG-CoA reductase mRNA levels with the same levels of dietary cholesterol (38). In the current study, we found the same effect: a downregulation in the HMG-CoA reductase mRNA and no effect in the LDL-r mRNA levels by high-cholesterol diets. The results from our work, together with those of other studies, may indicate that the control in the transcription of HMG-CoA reductase is a more important mechanism used by the liver to regulate the body's cholesterol levels, because LDL-r mRNA levels were not affected. However, we did not measure LDL-r protein levels, and other studies in guinea pigs fed with hypercholesterolemic diets reported decreased LDL binding to hepatic membranes (a measurement of the LDL-r) as the level of cholesterol in the diet increased (39,40). Thus, we cannot exclude a posttranscriptional effect on LDL-r.

In conclusion, a challenge with dietary cholesterol has a profound effect on hepatic cholesterol homeostasis, which results in elevated plasma LDL-C concentrations. However, the CR diet attenuated hepatic cholesterol accumulation induced by high concentrations of dietary cholesterol and decreased the concentration of small dense LDL as previously reported (11).


   FOOTNOTES
 
1 Author disclosures: M. Torres-Gonzalez, S. Shrestha, M. Sharman, H. C. Freake, J. S. Volek, and M. L. Fernandez, no conflicts of interest. Back

4 Abbreviations used: ACAT, acyl CoA cholesterol acyltransferase; CE, cholesteryl ester; CR, carbohydrate restricted; FC, free cholesterol; HChol, high cholesterol diet; HMG, 3-hydroxy-3-methyl glutaryl; LDL-C, LDL cholesterol; LDL-r, LDL receptor; mRNA, messenger RNA; SCD, steroyl CoA desaturase; TG, triglyceride. Back

Manuscript received 12 June 2007. Initial review completed 13 July 2007. Revision accepted 19 July 2007.


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 Materials and Methods
 Results
 Discussion
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