Nutrition Research 27 (2007) 423 – 431 www.elsevier.com/locate/nutres
Long-term fructose intake reduces oxidative defense and alters mitochondrial performance in mice Tali A. Kizhner ⁎, Orit Shilovizki, Moshe J. Werman Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel Received 9 October 2006; revised 16 April 2007; accepted 25 April 2007
Abstract Mitochondria are involved in the production of reactive oxygen species and subsequently are very susceptible to oxidative stress. Fructose, a reducing monosaccharide, is widely used as a food ingredient and has a high potential to intensify oxidative stress through the Maillard reaction or autooxidation processes. This study presents a new insight into the long-term effects of fructose consumption on the mouse mitochondria. Examination of carbonyl levels, as a marker of protein oxidative modification, in 17-month-old ICR mice introduced to fructose solutions vs water, showed a significant decrease in hepatic carbonyl levels of fructose-treated animals although no changes in brain and skeletal mitochondria. The activity of manganese superoxide dismutase (MnSOD), the main mitochondrial antioxidative enzyme, was significantly decreased in all the tested tissues after fructose consumption. No correlation was found between the expression levels of MnSOD and its specific activity. These findings suggest that a defense mechanism is activated in hepatic mitochondria of fructose-treated mice, excluding MnSOD as an option. Electron microscopy examination indicated changes in mitochondrial morphology caused by prolonged drinking of fructose solutions. These findings support the concern directed at the extensive use of fructose in the food industry. © 2007 Elsevier Inc. All rights reserved. Keywords:
Fructose; Glycoxidation; Mitochondria; Carbonyls; Manganese superoxide dismutase; Mouse
1. Introduction The monosaccharide, fructose, has long been a component of the human diet . Current interest in fructose metabolism and its chemical properties has arisen because of the increased fructose use by the food industry in its crystalline form and as high-fructose corn syrup . Digestion, absorption, and metabolism of fructose differ from that of glucose. Hepatic metabolism of fructose favors de novo lipogenesis. In addition, unlike glucose, fructose does not stimulate insulin secretion or enhance leptin production, thus suggesting that dietary fructose may ⁎ Corresponding author. Technion-Israel Institute of Technology, The Bruce Rappaport Faculty of Medicine, P.O. Box 9649, Bat-Galim, Haifa 31096, Israel. Tel.: +972 4 8295391; fax: +972 4 8295403. E-mail address: [email protected]
(T.A. Kizhner). 0271-5317/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2007.04.018
contribute to increased energy intake and weight gain . From a chemical point of view, the reducing free carbonyl group of fructose may react with free amino groups of biological molecules, initiating a cascade of complex reactions, named glycoxidation process, or the Maillard reaction . Concern has arisen because of the realization that a high concentration of fructose can promote potentially deleterious metabolic changes such as hyperlipidemia, hyperuricemia, nonenzymic fructosylation of macromolecules, lactacidemia, and disturbance in copper metabolism . Previous studies in our laboratory showed the detrimental effects of long-term fructose intake on protein and lipid oxidation . In addition, excessive exposure of plasmid and genomic DNA to fructose resulted in molecular damages [6,7].
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During the last decades, the involvement of the mitochondria, as a major contributor to various types of diseases and natural aging, has become apparent. These include myopathies, encephalomyopathies, heart diseases, late-onset diabetes, and other age-related degenerative diseases such as Parkinson, Huntington, and Alzheimer . Mitochondria are involved in the production of reactive oxygen species through one-electron carriers in the respiratory chain. Mitochondrial structures are also very susceptible to oxidative stress [9-12] as reported in many studies on lipid peroxidation [13,14], protein oxidation , and mitochondrial DNA mutations [8,16]. Because fructose is a potent agent in the glycoxidation process, it may intensify oxidative stress [17,18]. This study aimed at exploring the connection between the harmful effects caused by increased fructose consumption and mitochondrial performance. We determined the activity and expression of manganese superoxide dismutase (MnSOD), the major enzymatic antioxidant in the mitochondria, together with protein carbonyl content and the morphology of skeletal muscle of murine mitochondria after continuous intake of fructose solutions vs tap water for 17 months.
26.8 mmol/L KCl (pH 7.4). Tissues were kept frozen in liquid nitrogen until use. RNA extraction was preformed by grinding 50 to 100 mg of the tissue in liquid nitrogen, with subsequent homogenizing and lysis in 1 mL of commercial TRI Reagent (Sigma, Rehovot, Israel). The isolation procedure was carried out according to the manufacturer's instructions. RNA extracts were then transferred to a “spin basket” assembly of SV Total RNA Isolation System (Promega, Madison, Wis) following the manufacturer's instructions. RNA concentrations were determined by UV absorbance, and extracts were stored at −70°C. 2.3. Reverse transcription
2. Methods and materials
First-strand complementary DNA (cDNA) was synthesized from 2 μg of total RNA in a 25-μL reaction mixture containing 0.5 mmol/L dNTPs (Pharmacia, Piscataway, NJ), 0.5 μg random hexamers (Pharmacia) per 1 μg RNA, 200 U of Moloney murine leukemia virus reverse transcriptase (Promega), 5μL of 5× reverse transcription buffer (250 mmol/L Tris-HCl, pH 8.3, 375 mmol/L KCl, 15 mmol/L MgCl2, 50 mmol/L DTT), and 25 U of recombinant RNasin ribonuclease inhibitor (Promega). The mixture was incubated at 39°C for 60 minutes and then at 95°C for 5 minutes to inactivate the enzyme.
2.4. Quantitative real-time polymerase chain reaction
Male ICR mice, weighing 15 to 20 g each, were obtained from the animal colony of the Department of Biotechnology and Food Engineering, Technion, Haifa, Israel. The mice were randomly divided into 3 groups (9 mice per group) and housed in polycarbonate cages (3 per cage) containing wood shavings and fitted with stainless steel wire mesh tops. The animal room was maintained at 21°C with 12-hour light/dark periods. The animals were treated according to the ethics committee of the Technion for experimentation in animals. All mice were fed a balanced commercial diet (no. 19510, Koffolk, Tel Aviv, Israel). The composition of the diet was as follows: energy, 16 527 kJ/kg [3950 kcal/kg]; total protein, 21%; total fat, 4%; cellulose, 3.75%; carbohydrates, 53.5%; ash, 6.7%; moisture, 13.0%. The control group received tap water, while the rest were provided with fructose solutions (11% or 25%). Mice were allowed free access to the diet and their respective drinking solutions for 17 months. One week before the termination of the study, mice were placed in metabolic cages for 24 hours to measure daily feed, liquid, and energy expenditure. At the end of the experiment, animals were killed by cervical dislocation and immediately preceded for mitochondrial isolation.
The cDNA concentrations were determined by TaqManbased real-time polymerase chain reaction (PCR) in duplicate. The assay was performed in a 96-well optical tray with a 10 μL final reaction volume containing 5 μL TaqMan Universal Master Mix (Applied Biosystems, Foster City, Calif). The expression of the target genes was detected with the Assays-on-Demand primer and probe sets, MnSOD Mm00449726_m1 and 18S Hs99999901_s1 (Applied Biosystems). Gene-specific PCR products were measured continuously by an ABI PRISM 7000 Sequence Detection System (Applied Biosystems) during 40 cycles. The quantity of the specific cDNA was normalized by the amount of 18S ribosomal RNA. Relative amounts of cDNA were calculated by the comparative CT method (Applied Biosystems user bulletin).
2.2. RNA extraction Total RNA was purified from skeletal muscles, brain, and liver. Tissues were excised and thoroughly washed with ice-cold phosphate-buffered saline (PBS) containing 8.1 mmol/L disodium hydrogen phosphate, 1.47 mmol/L potassium dihydrogen phosphate, 140 mmol/L NaCl, and
2.5. Isolation of mitochondrial fraction Tissues (liver, skeletal muscles, and brain) were removed and immediately minced in ice-cold mitochondrial isolation buffer (MSHE) containing 0.21 mol/L mannitol, 0.07 mol/L sucrose, 10 mmol/L HEPES, pH = 7.4, 1 mmol/L EDTA, 1 mmol/L EGTA, 0.15 mmol/L spermine, and 0.75 mmol/L spermidine. The MSHE was replaced with fresh buffer (10% wt/vol), and tissues were homogenized with glass and Teflon homogenizers. Unbroken cells and nuclei were precipitated by centrifugation at 500g for 7 minutes and discarded. The supernatant, containing the mitochondria, was centrifuged for 7 minutes at 9500g. The mitochondrial pellet was washed twice with MSHE buffer, followed by
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centrifugation for 7 minutes at 9500g, and finally suspended in MSHE buffer. Total protein content in fresh mitochondrial extracts was determined by the Micro BCA kit (Pierce Chemical Company, Rockford, IL). 2.6. Activity of mitochondrial citrate synthase Activity of citrate synthase, as a protein marker of mitochondrial fraction, was assayed in a reaction mixture composed of 810 μL water, 0.1 mmol/L 5′,5′-dithiobis-2nitrobenzoate solution (0.4 mg/mL in 1 mol/L Tris-HCl, pH = 8.1), 30 μmol/L acetyl coenzyme A, and 0.05% Triton X-100, according to Robinson et al . Mitochondrial suspension (10 μL) was added, and 1.5 minutes later, 50 μL of sodium oxaloacetate fresh solution (1.32 mg/mL) was added to give a total volume of 1 mL. Activity was determined by following the increase in absorbance at 412 nm. Citrate synthase activity was calculated by subtraction in the change in absorbance occurring during the last minute before the addition of oxaloacetate, from the change in absorbance of the first minute of the linear range. 2.7. Activity of MnSOD Manganese superoxide dismutase activity was measured as described by Del Maestro and McDonald . The ability of MnSOD to scavenge superoxide radicals in a mitochondrial protein sample was monitored by pyrogallol autoxidation. A 1-mL aliquot of freshly prepared reaction buffer (0.05 mol/L Tris-HCl buffer, 0.1 mol/L NaCN, and 1 mmol/L diethylenetriamine pentaacetic acid; pH 8.2) was added to 20 μg of mitochondrial extract. To enable the inhibition of copper-zinc superoxide dismutase by sodium cyanide, the reaction mixture was kept for 15 minutes in a chemical hood. The reaction was initiated by the addition of 0.2 mmol/L pyrogallol, and the change in optical density at 420 nm was recorded for 3 minutes. Manganese superoxide dismutase activity was calculated as units per milligram of protein, and 1 U of MnSOD was defined as the amount that inhibited the rate of pyrogallol autoxidation by 50%. 2.8. Carbonyl content in mitochondrial protein The extent of carbonyls in mitochondrial proteins of different tissues was measured as described by Buss et al . Mitochondrial extracts were diluted in PBS (pH 7.4) to give a protein concentration of 4 mg/mL. Low-protein samples were first concentrated by mixing an aliquot containing 60 μg protein with 0.8 volumes of 28% trichloroacetic acid, centrifuged at 10 000g, and the supernatant was discarded. Then, protein precipitate was suspended in PBS (15 μL). Protein derivatization was carried out in a 1.5-mL reaction tube, containing a 15-μL sample (4 mg protein per milliliter) and 45 μL dinitrophenylhydrazine (DNP) reagent (10 mmol/L DNP in 6 mol/L guanidine hydrochloride, pH 2.5) to give a final protein concentration of 1 mg/mL. Samples were incubated at room temperature for 45 minutes and vortex-mixed after
10, 13, and 15 minutes. Subsequently, 5 μL was added to 1 mL coating buffer (10 mmol/L disodium phosphate and 140 mmol/L NaCl), pH 7.0. Triplicates of 200-μL aliquots (containing 1 μg protein) were transferred to wells on a NUNC Immuno Maxisorp plate (NUNC, Roskilde, Denmark) and incubated overnight at 4°C. Wells were washed 5 times with PBS between each of the following steps: (a) blocking with 250 μL/well of 0.1% BSA (previously reduced with sodium borohydride ) in PBS for 1.5 hours at room temperature; (b) addition of 200 μL/well of biotinylated anti-DNP antibody (1:10000 dilution; Molecular Probes, Eugene, OR) and incubation for 1 hour at 37°C; and (c) incubation for an additional hour at room temperature with 200 μL of streptavidin-biotinylated horseradish peroxidase (1:3000 dilution; Amersham Pharmacia Biotech, Piscataway, NJ). Then, color was developed for 25 minutes after the addition of 200 μL/well substrate made of o-phenylenediamine (0.6 mg/mL), 24 mmol/L citric acid, 6 mmol/L hydrogen peroxide, and 50 mmol/L disodium hydrogen phosphate (pH 5.6). Color developing was stopped by adding 100 μL/well of sulfuric acid (2.5 mol/L), and intensity was determined at 490 nm using a Versa Max Tunable Microplate Reader. A 6-point standard curve of reduced and oxidized BSA, prepared according to Buss et al , was included in each plate and a blank for DNP reagent in PBS without protein was subtracted. 2.9. Electron microscopy of skeletal muscle Samples were retrieved, pinned at their extremities onto a cork sheet, and immediately immersed in buffered glutaraldehyde (2.5%, pH 7.00) for at least 24 hours. Subsequently, samples were postfixed in osmium tetroxide solution, dehydrated in ethanol series, and embedded in epoxy. After polymerization, semithin sections were cut with a glass knife, stained with 5% toluidine solution, and examined with an optic microscope for orientation. Sections, considered informative, were thereafter cut with a diamond knife producing ultrathin sections. These were spread on copper grids, stained with uranyl acetate and lead citrate, and examined in a JEOL 100B electron microscope (Japan Electron Optics Laboratory, Tokyo, Japan) operated at 60 and 80 kV. For each muscle sample, 2 blocks were available, and of each block, 2 grids were examined, that is, 4 grids per sample. Electron micrographs were taken at various enlargements, on Kodak special films SO 163 (Eastman Kodak Co, Rochester, NY), developed, and printed with 2.5 enlargement factor of the original magnification. 2.10. Statistical analysis Data are presented as means ± SEM. Group contrasts were tested by either 1- or 2-way analysis of variance and then subjected to a multicomparison Tukey-Kramer honestly significant difference test [22,23] using the Matlab software for Windows (MathWorks, Inc, Natick, MA). Differences with P b .05 were considered significant.
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Fig. 1. Daily feed, liquid, and energy expenditure of 17-month-old ICR male mice. Six animals from each experimental group (water control, 11% fructose solution, or 25% fructose solution) were placed in metabolic cages for 24 hours of monitoring. Values are expressed as means ± SEM. Asterisk indicates P b .05 compared to the water group.
Fig. 2. Carbonyl content in mitochondrial proteins. Mitochondrial fraction was isolated from the brain, skeletal muscles, and liver of 17-month-old ICR male mice treated with water (control), 11% fructose solution, or 25% fructose solution. Carbonyl content was measured by a method based on recognition of carbonyl-DNP adduct with an anti-DNP antibody. The results were normalized to citrate synthase activity. Values are expressed as means ± SEM for the number of animals indicated in parentheses. Asterisk indicates P b .05 compared to the water group.
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Fig. 3. The activity of mitochondrial MnSOD. Mitochondrial fraction was isolated from the brain, skeletal muscles, and liver of 17-month-old ICR male mice supplied with water (control), a 11% fructose solution, or a 25% fructose solution. Manganese superoxide dismutase activity was calculated as unit per milligram of protein, with 1 U of activity defined as the amount that inhibits the rate of pyrogallol authorization by 50%. The results were normalized to citrate syntheses (CS) activity. Values are expressed as means ± SEM for the number of animals indicated in parentheses. Bars with different symbols (*) differ significantly (P b .05).
Fig. 4. Relative quantification (RQ) of mitochondrial MnSOD expression. Total RNA was isolated, using a commercial kit, from the brain, skeletal muscles, and liver of 17-month-old ICR male mice supplied with water (control), a 11% fructose solution, or a 25% fructose solution. An aliquot of 2 μg of total RNA was subjected to reverse transcription followed by quantification of the obtained cDNA with TaqMan-based real-time PCR. Results were normalized to the amount of 18S ribosomal RNA. Values are expressed as means ± SEM for the number of animals indicated in parentheses. Bars with different symbols (*,▲) differ significantly (P b .05).
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3. Results 3.1. Feed, liquid, and energy expenditure Daily feed and liquid intake was measured 1 week before the end of the study (Fig. 1). Mice consumed more fructose solutions compared with those given tap water with no significant difference between the 11% and 25% fructose solution treatments. When daily energy expenditure was calculated, we observed a 160% and 250% increase in animals treated with 11% and 25% fructose solution, respectively, compared with those given water. 3.2. Mitochondrial protein carbonyl content Carbonyl content in mitochondrial proteins was measured as an index of oxidative injury that occurred in the liver, brain, and skeletal muscles of 17-month-old mice (Fig. 2). No changes in the level of carbonyl adduct in the mitochondria of the brain and muscles were detected. However, carbonyl content was significantly decreased in the liver mitochondria of the experimental animals compared with the controls. Tissue differences in carbonyl levels after fructose intake indicate that the liver is more susceptible to fructose-related modification than the brain or skeletal muscles. 3.3. Manganese superoxide dismutase activities Fig. 3 shows the activities of MnSOD in skeletal muscles, brain, and liver of 17-month-old male ICR mice. Long-term exposure to fructose caused a significant decrease in MnSOD activity in all the tested tissues as compared with water. In the liver, MnSOD activity was decreased in a concentration-dependent manner. The lowest activity was observed in mice treated with the 25% fructose solution. 3.4. Expression of MnSOD messenger RNA The expression of MnSOD messenger RNA was detected by a quantitative TaqMan-based real-time PCR and presented in Fig. 4. Brain and skeletal muscles demonstrated a significant reduction in MnSOD expression in animals treated with 25% fructose solution as compared with the control animals. Tissues from animals treated with 11% fructose solution revealed a similar but not significant effect. Results are in agreement with data acquired for MnSOD activities for these tissues (Fig. 3). In contrast, hepatic MnSOD expression did not correlate with MnSOD activity, as animals treated with fructose showed an elevation in enzyme expression although being significant only in animals treated with the 25% fructose solution. 3.5. Mitochondrial morphology We examined the potential of fructose to perturb mitochondrial ultrastructure. As shown in representative photographs (Fig. 5), mitochondria from muscles of control animals have a more clearly defined internal membrane structure, including wider cristae. There were no drastic
Fig. 5. Electron micrographs of skeletal muscles. Samples were retrieved, pinned at their extremities on a cork sheet, and immediately immersed in 2.5% buffered glutaraldehyde (pH 7.00) for at least 24 hours. Subsequently, samples were postfixed in osmium tetroxide solution, dehydrated in ethanol, embedded in epoxy, coarsely sliced, stained with 5% toluidine solution, and examined with an optic microscope for orientation. Sections considered informative were thereafter ultrathin sliced with a diamond knife, spread on copper grids, stained with uranyl acetate and lead citrate, and examined in a JEOL 100B electron microscope operated at 60 and 80 kV. (A) A normal sample of the skeletal muscle from control mouse. Arrow indicates the mitochondrion with normal cristae. (B) A sample of the skeletal muscle from fructose-fed (25%) mice. Arrow indicates the mitochondrion with absent cristae; m, mitochondria. Bar = 1 μm.
changes in the number or size of the mitochondria in the samples tested. However, profound alterations were observed in cristae structure and matrix density in the organelles of the fructose-fed animals. Incidence counting revealed that 5 of 12 fructose-treated mice demonstrated various negative morphological changes. 4. Discussion It is well accepted that oxygen free radicals catalyze oxidative modifications of proteins . Protein oxidation consequently results in the introduction of carbonyl groups, which can be easily detected . Proteins bearing carbonyl adducts are generally dysfunctional because these moieties may alter both structure and functional properties of the protein. Carbonyl levels have been shown to be elevated in some pathologies  and to increase with age .
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Cumulative oxidative damage is often proposed as the major cause of mitochondrial dysfunction associated with aging [28,29]. Because fructose is a potent agent in the glycoxidation processes, it may intensify oxidative stress. Brain and skeletal muscle tissues excised from fructose-treated mice demonstrated similar carbonyl profiles compared with the control group. This could be explained by a high preservation of these organs or, alternatively, the absence of fructose toxicity. In comparison, hepatic mitochondria proteins of fructose-treated mice demonstrated a significant decrease in the introduction of carbonyl moieties. Because the liver is the main metabolic site of fructose , it could be assumed that hepatocytes would demonstrate a greater reaction to the sugar than the brain or skeletal muscles. This finding led us to consider the possible defense mechanisms that diminish oxidative damage. Thus, the expression and activity of the key mitochondrial antioxidant enzyme, MnSOD, located in the matrix of the mitochondria and responsible for converting superoxide radical into hydrogen peroxide  were examined. The results showed a significant decrease in the activity of MnSOD in all 3 tested tissues after fructose intake as compared to tap water. This finding is very important when considering public health issues, as Van Remmen et al  demonstrated that the primary phenotype resulting from reduced MnSOD activity, in Sod2+/− mice, is the increase incidence of cancer. The major alteration in MnSOD expression in all tested tissues was observed after the consumption of 25% fructose solution. Enzyme transcription was reduced by 35% and 50% in muscles and brain, respectively, whereas a 3-fold elevation was detected in the liver. No correlation was observed between MnSOD expression and its activity in the tested animals. Eukaryotic regulation of MnSOD gene expression remains unclear . MacMillan-Crow et al demonstrated that, in humans, a significant decrease in MnSOD activity occurred in rejected renal allograft in spite of an elevation in the translation rates of the protein. This finding implicates the susceptibility of the enzyme to posttranslational modifications. We observed a similar reverse pattern in the livers of mice maintained on fructose solutions for 17 months, namely, an elevated expression vs a reduction in protein activity. Increased protein expression represents an appropriate response to oxidative injury in an attempt to compensate for the loss of protection against oxidative stress. Manganese superoxide dismutase regulation appears to be a tissue-dependent event. It is unlikely that fructose interacts directly with the enzyme molecule, as the sugar does not enter the mitochondria [33,34]. It was demonstrated earlier that among simple sugars, fructose undergoes a more rapid autooxidation process, generating reactive oxygen species . Apparently, fructose intensifies oxidative stress by increasing free radical production, which in turn causes a posttranslational inactivation of mitochondrial MnSOD. In our study, low hepatic MnSOD activity did not help to clarify the low carbonyl levels. There are other possible
alternative mechanisms for preventing carbonyl accumulation in the mitochondria. (1) Alkaline proteases activity. Alkaline proteases preferentially degrade oxidative modified proteins . Further studies must determine the dynamics of their activity after continuous fructose consumption. (2) Uncoupling protein activity. Uncoupling proteins (UCPs) regulate the dissipation of membrane potential formed through respiration. Instead of being used for adenosine triphosphate synthesis, energy is converted to heat. Heat production not only protects against cold environments but also regulates mitochondrial energy balance . Through the regulation of uncoupling , mitochondria can adjust their metabolism according to the supply of various substrates and cellular ATP requirement, while minimizing ROS production by lowering membrane potential . In addition to enhancing oxidative stress, fructose is known to down-regulate leptin production . Recently, leptin was found to control UCP expression levels in different tissues . Because not all UCP functions and regulation are yet established, further studies are needed to clarify the complicated mechanism of fructose-induced metabolic alterations. Mitochondrial morphology is important because changes in mitochondrial ultrastructure modulate mitochondrial function . Electron microscopy and flow cytometry studies in old animals revealed mitochondrial enlargement, matrix vacuolization, and altered cristae [43-45]. Alterations of mitochondrial cristae in old mitochondria may be responsible for the age-related impairment in mitochondrial membrane potential. Acute oxidative stress is well known to cause mitochondrial swelling . Skeletal muscle is the largest tissue in the body and is responsible for most of the lipid oxidation. Because skeletal muscles strongly rely on oxidative phosphorylation for energy production, they are richly endowed with mitochondria that, along with the smooth endoplasmic reticulum, are located in the cytoplasm between the myofibrils. It is accepted that insulin resistance of skeletal muscle in diabetes mellitus and obesity implies altered regulation of both carbohydrate and lipid oxidation . Thus, we assumed that a similar outcome may occur as a result of long-term fructose intake. Fructose-treated animals contained ballooned mitochondria, in which the cristae were either absent or extremely displaced toward the periphery of the organelles. Enlarged and apparently ballooned mitochondria displaying normal arrangement of cristae were also observed. There were no drastic changes in the number or size of the mitochondria in the tested samples. However, electron microscopy revealed profound alterations in cristae structure and matrix density in the organelles of the fructose-treated animals. Extensive examination of micrographs from fructose-fed mice vs controls indicated that the changes in mitochondrial morphology can be solely related to the fructose treatments. The irregular matrix observed in the mitochondria of fructose-treated mice may have a negative impact on the efficiency of β-oxidation and the tricarboxylic acid cycle, both occur in the mitochondrial matrix.
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In conclusion, our study indicates a significant decrease in the activity of MnSOD in mice, an important mitochondrial antioxidant after 17 months of exposure to fructose solution. Data show that the liver is the main target of fructose metabolic consequences, although there are minor alterations in postmitotic tissues (skeletal muscles and brain). Electron microscopy analysis of skeletal muscles obtained from mice maintained on fructose solutions revealed some pathologic alterations in mitochondrial morphology. We can say that extensive continuous fructose consumption has a negative impact on animal mitochondria, expanding the data on its deleterious influence to mammal metabolism. We assume that these findings cannot be linked only to the excess energy expenditure but mainly to the type of carbohydrate used in the experiment. Future work is necessary to reveal the severity of oxidative stress imposed by fructose consumption in the mitochondria, followed by reconsidering the extensive use of fructose as one the main sweeteners in the beverages and bakery industries. Acknowledgment We thank Abigail Morgenshtern for her extraordinary technical assistance, Theodore C. Iancu for the electron microscopy examination, and Sarah Maurice for her critical review of the manuscript. References  Hanover LM, White JS. Manufacturing, composition, and applications of fructose. Am J Clin Nutr 1993;58:724S-32S.  Mayes PA. Intermediary metabolism of fructose. Am J Clin Nutr 1993;58:754S-65S.  Bray GA, Nielsen SJ, Popkin BM. Consumption of high-fructose corn syrup in beverages may play a role in the epidemic of obesity. Am J Clin Nutr 2004;79:537-43.  Monnier VM. Toward a Maillard reaction theory of aging. Prog Clin Biol Res 1989;304:1-22.  Levi B, Werman MJ. Long-term fructose consumption accelerates glycation and several age-related variables in male rats. J Nutr 1998;128:1442-9.  Levi B, Werman MJ. Fructose triggers DNA modification and damage in an Escherichia coli plasmid. J Nutr Biochem 2001;12:235-41.  Levi B, Werman MJ. Fructose and related phosphate derivatives impose DNA damage and apoptosis in L5178Y mouse lymphoma cells. J Nutr Biochem 2003;14:49-60.  Richter C. Oxidative damage to mitochondrial DNA and its relationship to ageing. Int J Biochem Cell Biol 1995;27:647-53.  Sohal RS, Brunk UT. Mitochondrial production of pro-oxidants and cellular senescence. Mutat Res 1992;275:295-304.  Murakami K, Kondo T, Kawase M, Li Y, Sato S, Chen SF, et al. Mitochondrial susceptibility to oxidative stress exacerbates cerebral infarction that follows permanent focal cerebral ischemia in mutant mice with manganese superoxide dismutase deficiency. J Neurosci 1998;18:205-13.  Cadenas E, Davies KJ. Mitochondrial free radical generation, oxidative stress, and aging. Free Radic Biol Med 2000;29:222-30.  Green K, Brand MD, Murphy MP. Prevention of mitochondrial oxidative damage as a therapeutic strategy in diabetes. Diabetes 2004;53(Suppl 1):S110-8.
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