Comparison of free fructose and glucose to sucrose in the ability to cause fatty liver
- First Online:
- Cite this article as:
- Sánchez-Lozada, L.G., Mu, W., Roncal, C. et al. Eur J Nutr (2010) 49: 1. doi:10.1007/s00394-009-0042-x
- 476 Downloads
There is evidence that disaccharide sucrose produce a greater increase in serum fructose and triglycerides (TGs) than the effect produced by their equivalent monosaccharides, suggesting that long-term exposure to sucrose or fructose + glucose could potentially result in different effects.
Aim of the study
We studied the chronic effects of a combination of free fructose and glucose relative to sucrose on rat liver.
Rats were fed either a combination of 30% fructose and 30% glucose (FG) or 60% sucrose (S). Control rats were fed normal rat chow (C). All rats were pair fed and were followed for 4 months. After killing, blood chemistries and liver tissue were examined.
Both FG-fed- and S-fed rats developed early features of metabolic syndrome when compared with C. In addition, both diets induced hepatic alterations, including variable increases in hepatic TG accumulation and fatty liver, an increase in uric acid content in the liver, as well as an increase in hepatic levels of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-α) measured in liver homogenates.
Diets containing 30% of fructose either as free fructose and glucose, or as sucrose, induce metabolic syndrome, intrahepatic accumulation of uric acid and TGs, increased MCP-1 and TNF-α as well as fatty liver in rats. It will be relevant to determine clinically whether pharmacological reduction in uric acid levels might have a therapeutic advantage in the treatment of non-alcoholic fatty liver disease.
KeywordsNon-alcoholic steatosis Metabolic syndrome Sucrose Fructose
Fructose intake and/or sugar-sweetened beverages have recently received attention as a potential risk factor for metabolic syndrome and diabetes [15, 16, 33, 38]. The intake of fructose, as sucrose (disaccharide consisting of glucose and fructose) or as high-fructose corn syrup (HFCS), has increased markedly in the last 2 decades in association with increasing rates of obesity, diabetes, hypertension, and kidney disease . Moreover, experimental evidence has shown that fructose can induce metabolic syndrome in animals, whereas this is not observed in controls fed equal amounts of glucose . Studies in humans have also shown that high doses of fructose can result in insulin resistance, postprandial hypertriglyceridemia, intra-abdominal fat accumulation, and elevated blood pressure [3, 5, 13, 14, 40, 41, 43]. In this regard, Swarbrick et al. showed that prolonged fructose consumption can increase fasting glucose, postprandial triglycerides (TGs), and apolipoprotein-B in overweight women .
The mechanism by which fructose causes metabolic syndrome is due to its unique metabolism which facilitates the synthesis of TGs and uric acid (reviewed in ref. [15, 29]). Furthermore, the incubation of human endothelial cells, renal tubular cells and hepatocytes with postprandial concentrations of fructose induced the overexpression and increased activity of fructokinase, reduced intracellular ATP, increased intracellular uric acid levels and activated proinflammatory and pro-oxidative responses [7, 12, 32]. Of note is that allopurinol blocked fructose-induced monocyte chemoattractant protein-1 (MCP-1) production in renal tubular cells and stimulation with 15 mg/dl uric acid mimicked the effect of fructose, suggesting that xanthine oxidase pathway might mediate the proinflammatory effects of fructose .
Long-term fructose intake is also associated with non-alcoholic fatty liver disease (NAFLD) which is another manifestation of the metabolic syndrome . Fructose rapidly increases hepatic TG synthesis and deposition, and can cause fatty liver in rats . Chronic sucrose ingestion has also been reported to raise TG and liver transaminase enzymes in humans . In one study, the amount of fructose ingested from soft drinks was over twofold higher in subjects with NAFLD than that in the control group (89 vs. 42 g/day) . These individuals also displayed twofold higher levels of fructokinase mRNA, consistent with a history of increased fructose intake .
The hallmark feature of the pathogenesis of NAFLD, both histologically and metabolically, is the accumulation of TGs in the liver . Regulation of fatty acid metabolism is highly different in fasting and fed states. Recent studies showed that 59% of TG within hepatocytes arose from the plasma-free fatty acid pool in the fed state . Interestingly, three studies have shown that postprandial hypertriglyceridemia correlates with fat liver content [25, 30, 35]. In one study of patients with type-II diabetes mellitus and normal fasting TG, the increase in postprandial TG levels was directly predicted by the amount of hepatic fat . In this regard, the administration of fructose-sweetened beverages providing 25% of the daily caloric intake for 10 weeks was shown to increase hepatic de novo lipogenesis and reduce postprandial lipoprotein lipase activity suggesting that both mechanisms may contribute to fructose-induced postprandial hypertriglyceridemia .
If fructose is a mediator of metabolic syndrome, a key question is whether sucrose is different from their individual components, fructose and glucose, in its metabolic effects. Sucrose is degraded by intestinal sucrase (EC 220.127.116.11) prior to the release of fructose for absorption. Acute studies have shown that serum fructose and TGs increase more after a sucrose drink than when an equivalent amount of a mixture of fructose and glucose is administered to healthy individuals (disaccharide effect) [22, 44]. Moreover, there are a few short-term studies that have compared the effects of sucrose with HFCS, which contains roughly the same amount of fructose and glucose as free sugars. In general, those studies have not found significantly different effects on appetite and satiety between both sweeteners [26, 27]. Interestingly, in male humans, a short term study showed that consuming HFCS- and sucrose-sweetened beverages increased postprandial TGs concentrations to the same extent as fructose alone . However, it is unknown whether the effects of sucrose (disaccharide) vs. free monosaccharides (fructose + glucose) on liver adaptation differ in a chronic setting.
We, therefore, studied the long-term effects of a combination of free fructose and free glucose compared with equivalent amounts of fructose and glucose supplied as sucrose on the development of fatty liver in rats.
Relevant nutrient content of the rodent experimental diets
Normal diet (n = 6)
Sucrose diet (n = 6)
Fructose + glucose diet (n = 6)
Serum glucose, TGs, total cholesterol, and uric acid levels were measured using the VetAce autoanalyzer (Alpha Wasserman, West Caldwell, NJ, USA). Rat serum insulin, MCP-1 and tumor necrosis factor (TNF) were measured by ELISA using commercial kits (Insulin, Crystal Chem, Chicago, IL, USA; MCP-1 and TNF, BD Biosciences Pharmingen, San Diego, CA, USA).
Tissue was fixed in methyl-Carnoy’s solution, dehydrated in alcohols, and embedded in paraffin (Sigma-Aldrich, St Louis, MO, USA), and 4-μm sections were stained with periodic-acid Schiff (PAS) stain. The histologic analysis was performed blinded regarding treatment groups. Steatosis attributable to NAFLD is typically macrovesicular rather than microvesicular . In the present study, macrovesicular steatosis was defined as a single vacuole that is larger than the nucleus and usually displaces it to the periphery of the cell . The scoring grade as the percentage of macrovesicles on PAS-stained liver tissue (20×) was set as follows; <25% = 0, 25% = 1+, 50% = 2+, 75% = 3+, and 100% = 4+. In addition, indirect immunoperoxidase for macrophages and for CD8-positive lymphocytes was performed using buffered paraformaldehyde-fixed tissue that had been frozen in liquid nitrogen using ED-1 and antiCD8 antibodies from BD Pharmingen (San Diego, CA, USA) as previously described. Twenty fields (1,100 μm × 1,400 μm) of each biopsy were measured at 200× magnification, and the mean number of macrophages per field was calculated. We also stained type-III collagen in methyl-Carnoy’s fixed tissue with a goat anti-collagen III antibody (Southern Biotech, Birmingham, AL, USA) as a measure of fibrosis and determined the percent area of collagen deposition using an Axioplan 2 imaging microscope (Carl Zeiss, Munich, Germany) with Zeiss AutoMeasure software (Axiovision 4.1).
Measurements of liver tissue homogenates
Liver tissue homogenates were prepared by mechanical homogenization after the addition of fresh tissue to RIPA buffer (Sigma-Aldrich) to which Compete (Roche, Mannheim, Germany) protease inhibitors were added. Samples were incubated at 4°C for 1 h and then centrifuged at 4°C at 16,000g for 60 min. The supernatant was removed, and the protein concentration was standardized at 8 mg/ml after total protein measurement (Pierce, Rockford, IL, USA). Rat MCP-1 and TNF-alpha (TNF-α) protein was measured in the homogenates by ELISA with a kit (BD Biosciences Pharmingen, San Diego, CA, USA) according to the manufacturer’s directions. Both the TG and uric acid levels in the liver tissue homogenates were determined using the VetAce autoanalyzer (Alpha Wasserman, West Caldwell, NJ, USA).
Statistical analysis was performed using Prism 5 for Windows (Graph Pad Software, Inc., San Diego, CA, USA). Values are expressed as mean ± SD. All studied parameters were analyzed by one-way ANOVA. When the ANOVA P value was <0.05, post-test comparisons were made using a Bonferroni multiple-comparison test. Correlation analysis was used to measure the degree to which two variables were related.
General characteristics and systemic chemistries
Mean caloric intake, and body weight (BW), abdominal, retroperitoneal and total fat (per 100 g of BW) after 4 months of a control, sucrose, or fructose–glucose diet
Daily intake (kcal)
Abdominal fat (g)
Retroperitoneal fat (g)
Total fat (g)
C (n = 6)
202 ± 24
634 ± 63
1.53 ± 0.62
3.1 ± 0.79
4.6 ± 1.35
S (n = 6)
201 ± 24
662 ± 48
2.37 ± 0.41
4.2 ± 0.92
6.58 ± 1.23
FG (n = 6)
203 ± 6
647 ± 53
2.33 ± 0.70
4.2 ± 0.7
6.48 ± 1.36
Laboratory tests obtained at 8 and 16 weeks in rats fed control, sucrose, or fructose–glucose diet in postprandial state
C (n = 6)
160 ± 97
81 ± 13
3.0 ± 0.2
164 ± 25
S (n = 6)
284 ± 32
139 ± 26**
3.4 ± 0.4
174 ± 18
FG (n = 6)
422 ± 277*
140 ± 34**
3.0 ± 0.2
179 ± 26
C (n = 6)
190 ± 103
85 ± 13
2.7 ± 0.4
193 ± 39
2.5 ± 0.7
S (n = 6)
294 ± 63
121 ± 13*
3.4 ± 0.3
194 ± 19
4.1 ± 1.2*
FG (n = 6)
486 ± 220*
132 ± 21**
3.8 ± 1.0*
189 ± 32
3.6 ± 0.9
Effect of the different diets on the liver
Induction of MCP-1 and TNF-α in liver tissue
Most of the fat-laden hepatic parenchyma showed minimal inflammation and fibrosis. Although no difference in hepatic macrophage infiltration was observed among all groups (data not shown), there was focal inflammation consisting of CD8-positive T cells and ED-1-positive macrophages in the periportal regions in the FG and S groups. Periportal areas also stained for alpha actin pericytes (Ito cells) and type-III collagen, but no differences were noted in any of the three groups (data not shown).
In the present study we tested the hypothesis whether sucrose is different from their individual components, fructose and glucose, in its metabolic effects, particularly as it relates to the development of fatty liver in rats. Rats were pair fed to assure equivalent energy intake, and the study was conducted over 4 months so that the chronic effects could be determined. Our hypothesis was that if there were to be observable differences between the two diets it would most likely be noted in the liver because this is the primary site of metabolism of fructose.
As expected, both groups showed systemic features consistent with metabolic syndrome, with elevations in serum TGs and with the development of insulin resistance. Serum uric acid levels were also increased, particularly in the FG group, consistent with the known effect of fructose to stimulate uric acid production . Furthermore, the serum uric acid correlated with various markers of metabolic syndrome such as serum TGs (r = 0.81, P < 0.0001), cholesterol (r = 0.81, P < 0.0001), and insulin (r = 0.65, P = 0.004) as well as with abdominal (r = 0.72, P = 0.0007) and total fat (r = 0.72, P = 0.0008). These observations are consistent with our previous studies [28, 36]. In the present studies, we did not include a control group receiving a 60% glucose diet. In this regard, we have previously reported that rats consuming a 60% glucose diet had clinical chemistry values similar to rats receiving control regular diet [11, 28]. The use of diets containing 60% of energy derived from simple sugars has been questioned. However, it is a common maneuver in experimentation to use high doses as a way to induce disease conditions in a short period of time. For example, if rats are administered 60% of fructose, metabolic syndrome is induced within 4 weeks [28, 36], whereas if doses of 14% of total energy as fructose are administered, a period of 9 months is required for rats to develop insulin resistance .
The principal finding in this study was related to the effect of the diets on the development of fatty liver. Both diets induced fatty liver and increased hepatic TG content, although this tended to be worse in the FG group. We also documented that postprandial levels of TGs positively correlated with fatty liver score and intrahepatic TGs; similar findings have been observed in human studies [10, 30, 35]. In this regard, it is known that the maximal effects of fructose on TGs are best observed in the postprandial state, and recent studies by Stanhope et al.  have shown that this is particularly true in overweight humans fed fructose. In addition, postprandial hypertriglyceridemia is strongly associated with fat liver content [25, 30, 35], which was one of the main aims of the study.
Interestingly, sucrose- and glucose + fructose-fed animals also were found to have increased intrahepatic levels of uric acid. It has been established that the effects of uric acid on various cell types are mediated by intracellular uric acid [2, 4, 12, 16]. Importantly, we observed a parallel increase in uric acid and intrahepatic TGs as well as fatty liver. In addition, a strong positive correlation between liver uric acid and TG was observed (r = 0.92, P < 0.0001), despite the fact that we had a limited number of animals per group.
The role of uric acid as a biomarker of fatty liver has been recently underscored and there are epidemiological and clinical studies that support that concept [20, 21, 24, 37]. For instance, a recent large population study from China , reported that the prevalence rate of NAFLD was significantly higher in subjects with hyperuricemia than in those without hyperuricemia (24.75 vs. 9.54%; P < 0.001) and the prevalence increased with progressively higher serum uric acid levels (P value for trend <0.001) . Moreover, the multiple regression analysis showed that hyperuricemia was associated with an increased risk of NAFLD (P < 0.001) . There is also experimental evidence that chronic hyperuricemia may be able to cause hypertriglyceridemia and fatty liver in rats. In those studies, hyperuricemia was induced by the inhibition of uricase with allantoxanamide  or oxonic acid  and after 4 weeks the animals had developed elevated serum TGs and fatty liver. The mechanism by which uric acid might be able to cause fatty liver is unknown.
In the present studies, we also documented that both diets induced mild inflammation in the liver tissues which occurred in the periportal areas, and there was no fibrosis. However, subtle inflammatory processes could be discerned as noted by the higher hepatic and serum TNF-α levels in the FG- and S-fed rats and the higher MCP-1 levels in the S-fed rats. In this regard, data from animal and clinical studies indicate that TNF-α mediates not only the early stages of fatty liver disease but also the transition to more advanced stages of liver damage [8, 9, 23]; moreover serum levels of TNF-α strongly correlates with hepatic injury score in pediatric NAFLD . Others have also reported that fructose feeding of rats can induce signaling pathways associated with inflammation in the liver . Whether we would have observed more inflammatory and fibrotic changes if the diets had been continued for a longer period can only be speculated. These findings may be relevant to NAFLD, a condition commonly observed in subjects with metabolic syndrome. Whereas NAFLD was once rare, it has dramatically increased over the last several decades. Cirrhosis secondary to NAFLD has also increased and is now one of the more common reasons for liver transplantation in the United States. There is increasing evidence that this condition is due to increased fructose intake. In this regard, Ouyang et al. recently reported that subjects with NAFLD are ingesting more than twofold greater fructose from soft drinks than controls, amounting for 90 g of fructose per day from this source alone . Recently Abdelmalek et al. have also reported a correlation between fructose intake and liver injury in subjects with NAFLD, as noted by the presence of ballooning degeneration, inflammation, and fibrosis in subjects with NAFLD .
In conclusion, we demonstrated that diets containing 30% fructose either as free fructose and glucose, or as sucrose, induce metabolic syndrome, intrahepatic accumulation of uric acid and TGs, increased MCP-1 and TNF-α as well as fatty liver in rats. Since uric acid can induce proinflammatory changes [17, 18], including in response to fructose , it will be important in future studies to determine if lowering uric acid can prevent the development of fatty liver or the proinflammatory changes that can accompany chronic fatty liver disease.
Supported by NIH grants HL-68607 and generous funds from Gatorade. Laura G. Sánchez-Lozada is supported by grant 081054 from CONACyT, Mexico.