High-fructose diet elevates myocardial superoxide generation in mice in the absence of cardiac hypertrophy

Nutrition 26 (2010) 842–848

Contents lists available at ScienceDirect

Nutrition journal homepage: www.nutritionjrnl.com

Basic nutritional investigation

High-fructose diet elevates myocardial superoxide generation in mice in the absence of cardiac hypertrophy Kimberley Mellor B.Sc. a, Rebecca H. Ritchie Ph.D. c, Greta Meredith B.Sc. a, Owen L. Woodman Ph.D. b, Margaret J. Morris Ph.D. d, Lea M. D. Delbridge Ph.D. a,* a

Department of Physiology, University of Melbourne, Melbourne, Victoria, Australia School of Medical Sciences, Royal Melbourne Institute of Technology, Melbourne, Victoria, Australia Heart Failure Pharmacology, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia d Department of Pharmacology, University of New South Wales, Sydney, New South Wales, Australia b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 October 2008 Accepted 10 August 2009

Objective: Dietary fructose intake has increased considerably in recent decades and this has been paralleled by an increase in the incidence of insulin resistance, especially in children and adolescents. The impact of a high-fructose diet on the myocardium is not fully understood. The aims of this study were to characterize the murine metabolic and cardiac phenotypes associated with a high-fructose diet and to determine whether this diet imparts differential effects with age. Methods: Juvenile (4 wk) and adult (14 wk) C57Bl/6 mice were fed a 60% fructose diet or isoenergetic control (starch) diet for 6 wk. Results: At completion of the dietary intervention (at ages 10 and 20 wk), fructose-fed mice were normotensive; hyperinsulinemia and cardiac hypertrophy were not evident. Interestingly, fructosefed mice exhibited lower blood glucose levels (10 wk: 4.81  0.28 versus 5.42  0.31 mmol/L; 20 wk: 4.88  0.30 versus 5.96  0.42 mmol/L, P < 0.05) compared with controls. Nicotinamide adenosine dinucleotide phosphate–driven myocardial superoxide production was significantly increased in fructose-fed mice at both ages (by approximately 29% of control at 10 wk of age and 16% at 20 wk, P < 0.01). No increase in aortic superoxide production was observed. Fructose feeding did not alter gene expression of the antioxidant thioredoxin-2, suggesting an imbalance between myocardial reactive oxygen species generation and antioxidant induction. Conclusion: These findings indicate that increased myocardial superoxide production may represent an early and primary cardiac pathologic response to the metabolic challenge of excess dietary fructose in juveniles and adults that can be detected in the absence of cardiac hypertrophy and hypertension. Ó 2010 Elsevier Inc. All rights reserved.

Keywords: Cardiac hypertrophy Oxidative stress Insulin resistance Fructose Mice

Introduction The increasing usage of fructose sweeteners in food [1] and the association between high-fructose intake and the development of insulin resistance [2–4] may be contributing factors to the escalating prevalence of metabolic syndrome and type 2 diabetes [5]. In particular, the increased incidence of insulin resistance in children and adolescents has been coincident with increased consumption of the high-fructose content foods that

This study was supported by Diabetes Australia, the National Heart Foundation, and the National Health and Medical Research Council, Australia. * Corresponding author. Tel.: þ61-3-8344-5853; fax: þ61-3-8344-5897. E-mail address: [email protected] (L. M. D. Delbridge). 0899-9007/$ – see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2009.08.017

are commercially targeted to this age cohort. Unlike glucose, fructose bypasses the phosphofructokinase rate-limiting step in glycolysis, potentially resulting in unrestrained production of glycolytic endproducts and systemic metabolic dysregulation [6,7]. In the heart, metabolic dysregulation can lead to pathologies such as oxidative stress and hypertrophy, compromising cellular structure and function [8–11]. Thus, it is important to identify the mechanisms by which insulin resistance contributes to cardiac damage and dysfunction. Although, the fructose-fed rodent is a recognized experimental model of systemic insulin resistance and metabolic dysregulation [2,12–14], the reported phenotype is variable. Some rodent models of fructose-induced insulin resistance exhibit hypertension, but not all [14–20]. Fructose feeding is linked with

K. Mellor et al. / Nutrition 26 (2010) 842–848

dyslipidemia, but hyperinsulinemia and/or hyperglycemia are not always observed [14,16,21–23]. Some of these differences may be species specific (rat versus mouse) and others may arise due to differences in animal age and feeding duration. In addition, inadequately controlled dietary interventions in some previous studies may have contributed to the apparently inconsistent findings and the difficulty in identifying fructosespecific dietary impacts [2,12,14,19,22,24,25]. No studies of the age-dependent effects of fructose dietary intervention have been reported, and the possibility that a maturational window of fructose susceptibility exists has not been explored. The cardiac effects of high-fructose intake have not been investigated in detail, and the few available studies have reported inconsistent findings in relation to cardiac hypertrophy and oxidative stress [12,17]. Cardiac hypertrophy has been observed in response to fructose feeding, but only in the context of elevated blood pressure [17]. Thus, although a cardiac-specific detrimental effect of fructose is indicated, to date this has not been demonstrated to occur independent of pressure loading and may represent a secondary response to dietary intervention. A causal role for oxidative stress in the development of cardiovascular complications in diabetes is increasingly recognized [26,27], although a specific link between oxidative stress and insulin resistance is yet to be established. Myocardial expression of nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase is markedly upregulated in insulin resistance, and antioxidant strategies attenuate cardiac hypertrophy and fibrosis in this setting [28]. Although increased production of reactive oxygen species (ROS) in the vasculature has been reported with fructose feeding [12,20], it has not yet been determined whether evidence of myocardial oxidative stress can be detected with elevated fructose intake. The goal of this study was to examine the age-dependent, cardiac-specific effects of elevated dietary fructose intake. Mice were treated for 6 wk, employing an isoenergetic intervention involving manipulation of only the dietary carbohydrate content. Cardiac growth and parameters relating to ROS generation and oxidative stress were examined. We observed evidence of myocardial metabolic stress associated with fructose feeding in the absence of hypertension and cardiac hypertrophy. Materials and methods Ethical approval All animals were cared for in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and all procedures were approved by the animal ethics committee of the University of Melbourne.

843

Table 1 Diet composition: macro- and micronutrient components (Specialty Feeds, Glen Forrest, WA, Australia) Control (AIN-93 G) Digestible energy (MJ/kg) Components (g/kg) Starch Fructose Sucrose Casein (acid) DL-Methionine Cellulose Canola oil Calcium carbonate Sodium chloride Potassium citrate Potassium dihydrogen phosphate Potassium sulfate Trace minerals Choline chloride (65%) Vitamin mix* Total (100%)

Fructose (SF03-018)

16.1

16.6

536.0 0 100.0 200.0 3.0 50.0 70.0 13.1 2.6 2.5 6.9 1.6 1.4 2.5 10.0 w1000

36.0 600.0 0 200.0 3.0 50.0 70.0 13.1 2.6 2.5 6.9 1.6 1.4 2.5 10.0 w1000

* Vitamin mix includes vitamins A (4000 IU/kg), D3 (1000 IU/kg), E (76 mg/kg), K (1 mg/kg), B1 (6 mg/kg), B2 (6 mg/kg), and B12 (102 mg/kg).

Tissue harvest and plasma analysis At the completion of the 6-wk feeding period (ages 10 and 20 wk), mice were fasted overnight, heparinized (1000 IU, intraperitoneally), and anaesthetized by sodium pentobarbitone (60 mg/kg, intraperitoneally). A blood sample (10 mL) was collected for measurement of blood glucose using a blood glucometer (ACCU-CHEK Advantage, Roche, Mannheim, Germany) and plasma insulin (Rat Insulin RIA kit, Linco Research, St. Charles, MO, USA) concentrations. Hearts were excised (excess tissue was dissected away) and weighed for determination of the cardiac weight index (heart weight in milligrams to body weight in grams). Ventricles were sectioned into tissue portions for gene expression analysis, assay of lipid peroxidation markers, and measurement of cardiac superoxide production as described below. Detection of tissue superoxide production The NADPH-driven superoxide production, an estimate of NADPH oxidase activity, was measured in freshly dissected thoracic aorta and ventricular tissues using lucigenin-enhanced chemiluminescence [28,30]. Small (1–2 mm) fragments of ventricular tissue and aortic rings were incubated with 50 mM NADPH in HEPES-Krebs buffer at 37  C for 45 min. The tissues were transferred to 96-well opti-plates (Perkin Elmer Inc., Boston, MA, USA) containing HEPES-Krebs buffer, 100 mM NADPH, and 5 mM lucigenin (N,N0 -dimethyl-9-90 -biacridianium, Sigma Aldrich, St. Louis, MO, USA). Luminescence generated by the reaction of tissuederived superoxide and lucigenin (5 mM) was counted at 20  C. Background chemiluminescence was measured in the 96-well opti-plate (dark-adapted for 12 h) with HEPES-Krebs buffer, 100 mM NADPH, and 5 mM lucigenin before tissue addition and subtracted from the tissue fragment luminescence. Tissues were dried at 80  C for 48 h and weighed for calculation of normalized superoxide production expressed as counts per second per milligram of dry tissue.

Dietary treatments and in vivo measurements Real-time quantitative reverse transcription–polymerase chain reaction Male C57Bl/6 mice aged 4–5 wk (weaning) and 14–15 wk (adult) were housed in temperature-controlled conditions in a 12-h light/dark cycle. At the commencement of the dietary treatment period, mice were allocated to a control (54% starch, 10% sucrose, 7% total fat, 19.4% protein) or high-fructose (60% fructose, 3.6% starch, 7% total fat, 19.4% protein) diet (n ¼ 12/group). The diets were of equal digestible energy and micronutrient matched (Table 1) and based on the American Institute of Nutrition standard rodent growth diet (Specialty Feeds, Glen Forrest, WA, Australia). Food intake and body weight were monitored throughout the 6-wk treatment period. In the final week of treatment, blood pressure was measured on 3 consecutive days using tail cuff plethysmography [29]. After gentle heating by infrared lamp, the pressure cuff and pulse sensor were placed around the base of the tail and the pressure cuff inflated using a sphygmomanometer (Narco Biosystems, Sydney, NSW, Australia). The pulse was detected using a pneumatic pulse transducer (Narco Biosystems) and recorded using the MacLab data acquisition system (A.D. Instruments, Sydney, NSW, Australia). Determination of mean pressure for each animal was made by averaging three to four concordant measurements for each session and calculating the mean of three session averages [29].

The RNA was extracted from frozen cardiac tissues using the TRIzol reagent (Invitrogen, VIC, Australia) according to the manufacturer’s instructions. Total RNA was reverse transcribed to cDNA with the SuperScript III First-Strand Synthesis System (Invitrogen). Real-time reverse transcription–polymerase chain reaction was used to determine the relative gene expression levels of b-myosin heavy chain (cardiac prohypertrophic gene), the Nox2 subunit of NADPH oxidase (a major source of ROS), and thioredoxin-2 (a mitochondrial antioxidant) in ventricular tissue [28]. Primers and polymerase chain reaction cycling conditions were optimized and primer sequences for each gene were as follows: b-myosin heavy chain: 50 - TCTCCTGCTGTTTCCTTACTTGCTA-30 and 50 - GTACTCCTCTGCTGAGGCTTCCT-30 ; Nox2: 50 -GCTGCCAGTGTGTCGAAATCT-30 and 50 -GCTACCATCTTATGGAAAGTGA GGTT-30 ; thioredoxin-2: 50 -CAGCCTCTGGCACATTTCCT-30 and 50 -GTTCGGCTTCTG GTTTCCTTT-30 ; and 18S: 50 -TCGAGGCCCTGTAATTGGAA-30 and 50 -CCCTCCAATGGATCCTCGTT-30 . The b-tubulin primers were purchased as a preoptimized assay (Entrez Gene ID 22153, QuantiTech Primer Assay, Qiagen Pty. Ltd., Melbourne, VIC, Australia). The comparative DDCt method was utilized in analysis of the genes of interest as described previously [31,32]. This method involves two comparative

844

K. Mellor et al. / Nutrition 26 (2010) 842–848

steps (i.e., two ‘‘delta’’ manipulations using base 2 logarithmic values). First, the threshold cycle number (Ct value) of the gene of interest relative to the Ct value of the housekeeper gene is calculated for each sample (this operation is termed DCt). Second, to reference the expression change in treatment groups to a selected control group, a second manipulation is performed (i.e., DDCt calculation). Gene expression was normalized to the expression level of a housekeeper gene, 18S (b-myosin heavy chain, Nox2) or b-tubulin (thioredoxin-2) and presented relative to the 10-wk-old control group. Markers of lipid peroxidation Ventricular lipid peroxidation was assessed by measurement of thiobarbituric acid-reactive substances. Ground ventricular tissue (15–30 mg) was dissolved in 1.15% KCl (10 mL/mg of tissue). Tissue homogenate (70 mL) was added to 100 mL of 8.1% sodium dodecylsulfate, 100 mL of 20% acetic acid, and 100 mL of 0.82% thiobarbituric acid freshly made on day of experiment. A standard curve was constructed over 0.1–2.5 nmol of 1,1,3,3-tetraethoxypropane. All samples, standards, and blanks were vortex-mixed and incubated at 100  C in a heat block for 45 min. After cooling on ice, absorbance at 532 nm was determined by spectrophotometry (DU800, Beckman Coulter Inc., Fullerton, CA, USA) and the concentration of thiobarbituric acid-reactive substances in the tissue was calculated using the standard curve. Statistical analysis Data are expressed as mean  standard error and were analyzed with SPSS 16 (SPSS Inc., Chicago, IL, USA). Two-way analysis of variance was performed using a univariate, general linear model. The two categorical independent variables were diet and age; interactions between these factors were also analyzed. P < 0.05 was considered statistically significant.

Results Systemic characteristics For each age group, mice were matched for body weight at the commencement of the feeding period. Food intake measured at 2-d intervals throughout the treatment period was not significantly different for the fructose and control diet groups (mean 2.75 g/d). After 6 wk of treatment, the mean body weight in the fructose-fed group was 12% lower than in the control group for 10- and 20-wk-old animals (Table 2). As presented in Table 2, fasted blood glucose concentrations were approximately 11% lower in 10-wk-old fructose-fed mice and 18% lower in 20wk-old mice compared with controls (diet, P < 0.05). Plasma insulin levels after 6 wk of fructose feeding were not different from levels in control-fed animals and did not show a significant age-dependent difference (Table 2). Blood pressure and heart growth index As shown in Figure 1, an age-dependent elevation in blood pressure was apparent, but the fructose diet was not associated with a specific blood pressure effect. An age-dependent Table 2 Systemic characteristics of fructose-fed and control-fed mice at ages 10 and 20 wk*

10 wk Control Fructose 20 wk Control Fructose * y z

Initial body weight (g)

Final body weight (g)

Blood glucose (mmol/L)

Plasma insulin (ng/mL)

(n ¼ 17–18)

(n ¼ 17–18)

(n ¼ 16–17)

(n ¼ 14–17)

15.1  0.7 14.3  0.5

24.9  0.5 21.5  0.6y

5.42  0.31 4.81  0.28y

0.18  0.02 0.20  0.02

26.1  0.7z 25.6  0.6z

28.7  0.5z 25.5  0.7y,z

5.96  0.42 4.88  0.30y

0.23  0.02 0.19  0.02

Values are means  SEMs. P < 0.05 compared with control. P < 0.05 compared with 10 wk.

Fig. 1. Mean systolic blood pressure in the final week of dietary intervention (n ¼ 11–12/group). The horizontal bars depict an age effect. Data are presented as mean  SEM. # Age factor effect, P < 0.05, two-way analysis of variance. cont, control; fruc, fructose.

reduction in the cardiac weight index was evident in both diet groups (age, P < 0.05), due to the proportionally greater increase in body weight relative to heart weight with age (Fig. 2A). No significant difference in cardiac weight index was detected between diets. The 6-wk dietary intervention did not significantly alter gene expression levels of b-myosin heavy chain, the prohypertrophic gene, in either age group (although a nonsignificant trend was evident for increased b-myosin heavy chain gene expression in younger mice). Thus, the high-fructose diet did not induce cardiac hypertrophy in these mice (Fig. 2B).

Parameters of oxidative stress The NADPH-driven superoxide production was measured in ventricular and aortic tissues in freshly harvested tissues using lucigenin-enhanced chemiluminescence. As presented in Figure 3A, the 6-wk period of fructose feeding was associated with a significant elevation in ventricular superoxide production in mice in both age groups, which was increased by approximately 29% in 10-wk-old mice and 16% in 20-wk-old mice. There were no significant differences in aortic superoxide production between fructose-fed and control-fed mice at either age, and no age-dependent differences between groups (10 wk: 2280  320 versus 2840  310 counts $ s1 $ mg1; 20 wk: 2760  390 versus 2840  380 counts $ s1 $ mg1). Average aortic levels of superoxide production were about five-fold greater than the levels measured in cardiac tissue (P < 0.05). Fructose-fed mice at 10 wk showed a non-significant tendency for increased mRNA expression of the NADPH oxidase subunit Nox2 (by 16.8  9.5-fold of control, P ¼ 0.13). This was not observed in the 20-wk-old animals, where Nox2 gene expression was comparable for fructose- and control-fed mice (Fig. 4). A significant correlation was not observed between Nox2 gene expression and myocardial superoxide production (Pearson’s coefficient 0.27, P ¼ 0.15). Gene expression of the mitochondrial isoform of the redox protein thioredoxin-2 was determined using real-time polymerase chain reaction. No effect of fructose feeding on myocardial redox protein thioredoxin-2 expression was observed for either age group (Fig. 5). The extent of myocardial lipid peroxidation (determined using thiobarbituric acid-reactive substances) was not significantly

K. Mellor et al. / Nutrition 26 (2010) 842–848

Fig. 2. (A) Cardiac weight index. Heart weight (milligrams) to body weight (grams) ratio at completion of a 6-wk dietary treatment (n ¼ 17–18/group). The horizontal bars depict an age factor effect. (B) Gene expression level of b-MHC, a prohypertrophic gene in fructose-fed versus control-fed mice at 10 and 20 wk of age (normalized to 18S and determined relative to 10-wk-old control group, n ¼ 11–12/ group). Data are presented as mean  SEM. # Age factor effect, P < 0.05, two-way analysis of variance. b-MHC, b-myosin heavy chain; cont, control; fruc, fructose.

modified by diet at either age (Fig. 6), although age per se tended to elevate lipid peroxidation. Thiobarbituric acid-reactive substances levels increased in control-fed mice (18%) and in fructose-fed mice (11%) from 10 to 20 wk of age (P ¼ 0.09). Discussion The key finding of the present study is that high dietary fructose intake in mice is associated with increased myocardial superoxide generation. In contrast, a fructose-induced increase in ROS generation was not observed in vascular tissue. Fructosefed mice were normotensive, insulin levels were not elevated, and cardiac hypertrophy was not evident. Interestingly, blood glucose levels were lower in fructose-fed mice. Normal maturational differences in blood pressure and cardiac weight index were observed between 10- and 20-wk-old mice, but no agedependent differences in other myocardial measures were detected. Thus, this investigation provides support for the proposition that fructose feeding is associated with a cardiacspecific metabolic stress response. Importantly, this increased capacity for ROS generation in the myocardium was observed with a relatively short dietary intervention of 6 wk and occurred

845

Fig. 3. Nicotinamide adenosine dinucleotide phosphate–stimulated superoxide (O 2 ) production (counts per second per milligram of dry tissue) in fructose-fed and control-fed mice at completion of the 6-wk dietary intervention. Superoxide production in (A) ventricular tissue (n ¼ 6 tissue fragments/animal, n ¼ 15–17/ group) and (B) thoracic aorta (n ¼ 3 aortic rings/animal, n ¼ 16–17/group). Data are presented as mean  SEM. * Diet factor effect, P < 0.05, two-way analysis of variance. cont, control; fruc, fructose.

in the absence of the complicating systemic factors of body mass increase, hyperinsulinemia, and/or hypertension. No evidence of increased susceptibility to juvenile fructose feeding could be detected in this experimental context. This is the first study to investigate the direct effects of highfructose intake on the heart in a mouse model and suggests that the murine myocardium may be compromised by dietary fructoseinduced elevated ROS production. The fructose-induced increase in myocardial NADPH-driven superoxide production was evident in young and older animals, and although not statistically resolvable, the data suggest a more marked effect in the younger animals. A similar trend of increased Nox2 (a catalytic subunit of NADPH oxidase) gene expression in the young but not older mice was also observed. These findings suggest that the fructose diet–induced elevated superoxide production may be mediated in part by NADPH oxidase upregulation, which may be accentuated in younger animals, although in this dataset a significant correlation between these parameters could not be discerned. No changes in the expression of thioredoxin-2, an important mitochondrial antioxidant, were observed in the context of this upregulated ROS production. Thus, an imbalance between ROS generation and antioxidants could potentially render the heart vulnerable to oxidative damage and oxidative stress may represent an early manifestation of fructose-induced cardiac pathology from which further complications emerge. With this 6-wk dietary intervention,

846

K. Mellor et al. / Nutrition 26 (2010) 842–848

Fig. 4. Nox2 gene expression normalized to 18S and determined relative to the 10wk-old control group. Fructose-fed versus control-fed mice at 10 and 20 wk of age (n ¼ 9–10/group). Data are presented as mean  SEM. cont, control; fruc, fructose.

increased myocardial lipid peroxidation damage was not evident in fructose-fed mice. A more prolonged intervention might be expected to confer detectable peroxidation damage, possibly in association with impaired antioxidant defense, as has been reported in the rat [33]. It is notable that this effect of high-fructose intake on ROS generation was specific for the myocardium. The role of oxidative stress in the cardiovascular complications of type 2 diabetes has been previously demonstrated [27,34]. Although the mean level of superoxide production in the aorta was approximately fivefold higher than in the myocardium, possibly reflecting the differential expression of NADPH oxidase subunits in the vasculature relative to the myocardium [35], an effect of fructose feeding was not detected. This finding indicates that, where vascular ROS production has been previously found to be increased with fructose feeding (in rats), coincident hypertension may be the predominant causative factor [12]. In this study cardiac weight indices and expression of the hypertrophic growth marker b-myosin heavy chain were not different in either age cohort after a 6-wk high-fructose feeding regime. A trend for increased b-myosin heavy chain expression was indicated in younger mice, suggesting a heightened

Fig. 5. Thx2 gene expression normalized to b-tubulin and determined relative to the 10-wk-old control group. Fructose-fed versus control-fed mice at 10 and 20 wk of age (n ¼ 5–6/group). Data are presented as mean  SEM. cont, control; fruc, fructose; Thx2, thioredoxin-2.

Fig. 6. Lipid peroxidation (ventricular TBARS) after 6-wk dietary treatment (n ¼ 13–15/group). Data are presented as mean  SEM. cont, control; fruc, fructose; TBARS, thiobarbituric acid-reactive substances.

susceptibility to growth induction. Previous studies with rats have reported cardiac hypertrophic effects in response to fructose feeding [17,36]. Those previous studies reported an occurrence of hypertension coincident with hypertrophy, suggesting that the cardiac growth effects were secondary to the hypertension induced by fructose feeding rather than a direct effect of the diet. Fructose feeding was not associated with hemodynamic volume load (these animals were not obese) or pressure overload. Older mice exhibited a small significant elevation of blood pressure. The capacity to detect this expected normal maturational pressure increase confirmed the sensitivity of the plethysmographic methodology employed. Previous studies in rodents have reported increases in [25,37] and no change in [19] systolic blood pressure with fructose feeding. This discrepancy may reflect difficulties in diet matching in these studies, as discussed below. In the present study, daytime blood pressure measurements within a 2-h time window were taken. Interestingly, a recent telemetric study determined that nocturnal (i.e., active period) blood pressure was significantly elevated in fructose-fed mice after 10 wk of dietary treatment [14]. The possibility that some pressure variation over a 24-h period may occur in mice, particularly with extended duration feeding, remains to be evaluated. In this study, heart rate measurements were not obtained, and the possibility that fructose feeding may influence heart rate cannot be precluded. However, fructose feeding in rats is not associated with heart rate alteration, even when hypertension is evident [38]. Although fructose feeding in rodents is a recognized model of diet-induced insulin resistance, there has been little consistency in the composition of diets utilized. In some studies diet specification was not provided and in others diet caloric content and macronutrient types were not matched [2,12,14,19,22,25]. In the present study, strict matching of diet composition was achieved, and all components of the two diets were matched, with only the type of carbohydrate manipulated. Surprisingly, body weight gain in fructose-fed mice was reduced. The basis for this is unknown and merits further investigation. Most pertinent to the present study is the confirmation that, with these specified wellmatched diets, fructose feeding was not associated with body weight increase and therefore interpretation of the findings is not confounded by consideration of an obesity-related hemodynamic or endocrine effect [39].

K. Mellor et al. / Nutrition 26 (2010) 842–848

Fructose feeding in the present study was not associated with hyperglycemia, but rather, surprisingly, with lower blood glucose levels. Previous studies of fructose feeding in rats and mice have reported no change or elevated blood glucose levels [24,33,40]. In the present study, fasted plasma insulin levels were not different in response to a high-fructose diet, consistent with a recent report in mice [14]. Studies in fructose-fed rats have shown a treatment duration-dependent influence on plasma insulin levels. With 4 wk of fructose feeding in rats, plasma insulin levels were significantly elevated, yet after 7 wk, insulin levels were reportedly not different [12]. The effects of a highfructose diet on plasma insulin are controversial and the limited available evidence is not conclusive. Although the present study suggests an effect of high-fructose intake on systemic glucose homeostasis, there was no evidence of hyperglycemia. Thus, the cardiac-specific effects of high-fructose intake cannot be attributed to elevated plasma glucose or to hyperinsulinemia. In summary, this study provides the first demonstration that high-fructose intake has a direct and selective cardiac effect inducing elevated myocardial ROS production. With a relatively short 6-wk dietary intervention, this indication of cardiac oxidative stress could be observed independent of systemic insulin dysregulation. It is evident that the myocardium responds to the environmental insult of a high-fructose diet in the absence of marked systemic pathology. Importantly, in the present study, the fructose-induced increase in cardiac superoxide production observed was apparent in the absence of hypertrophy and hypertension, demonstrating that the NADPH oxidase upregulation occurs independently of pressure loading and precedes any structural cardiac remodeling event. This investigation also provides suggestive evidence of increased susceptibility of younger hearts to the cardiac effects of highfructose intake, and further work is required to explore this possibility.

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

Acknowledgments The expert technical assistance provided by Bill Meeker, Jill Pavia, and Anh Cao is acknowledged. The contribution of Specialty Feeds (Warren Potts) in managing diet-batch consistency is appreciated.

[24]

[25]

[26]

References [1] Havel PJ. Dietary fructose: implications for dysregulation of energy homeostasis and lipid/carbohydrate metabolism. Nutr Rev 2005;63: 133–57. [2] Hwang IS, Ho H, Hoffman BB, Reaven GM. Fructose-induced insulin resistance and hypertension in rats. Hypertension 1987;10:512–6. [3] Daly M. Sugars, insulin sensitivity, and the postprandial state. Am J Clin Nutr 2003;78:865S–72. [4] Basciano H, Federico L, Adeli K. Fructose, insulin resistance, and metabolic dyslipidemia. Nutr Metab (Lond) 2005;2:5. [5] Steinberger J, Daniels SR. Obesity, insulin resistance, diabetes, and cardiovascular risk in children: an American Heart Association scientific statement from the Atherosclerosis, Hypertension, and Obesity in the Young Committee (Council on Cardiovascular Disease in the Young) and the Diabetes Committee (Council on Nutrition, Physical Activity, and Metabolism). Circulation 2003;107:1448–53. [6] McGuinness OP, Cherrington AD. Effects of fructose on hepatic glucose metabolism. Curr Opin Clin Nutr Metab Care 2003;6:441–8. [7] Mayes PA. Intermediary metabolism of fructose. Am J Clin Nutr 1993;58:754S–65. [8] Huggins CE, Domenighetti AA, Ritchie ME, Khalil N, Favaloro JM, Proietto J, et al. Functional and metabolic remodelling in GLUT4-deficient hearts confers hyper-responsiveness to substrate intervention. J Mol Cell Cardiol 2008;44:270–80. [9] Rutter MK, Parise H, Benjamin EJ, Levy D, Larson MG, Meigs JB, et al. Impact of glucose intolerance and insulin resistance on cardiac structure and

[27]

[28]

[29]

[30] [31] [32]

[33]

[34]

[35]

847

function: sex-related differences in the Framingham Heart Study. Circulation 2003;107:448–54. Witteles RM, Fowler MB. Insulin-resistant cardiomyopathy clinical evidence, mechanisms, and treatment options. J Am Coll Cardiol 2008;51:93–102. Isomaa B, Almgren P, Tuomi T, Forsen B, Lahti K, Nissen M, et al. Cardiovascular morbidity and mortality associated with the metabolic syndrome. Diabetes Care 2001;24:683–9. Delbosc S, Paizanis E, Magous R, Araiz C, Dimo T, Cristol JP, et al. Involvement of oxidative stress and NADPH oxidase activation in the development of cardiovascular complications in a model of insulin resistance, the fructose-fed rat. Atherosclerosis 2005;179:43–9. Oda T, Hirata M, Oshida Y, Han YQ, Koshinaka K, Sato Y. Effect of imidapril, an angiotensin-converting enzyme inhibitor, on fructose-induced insulin resistance in rats. Endocr J 2004;51:69–74. Farah V, Elased KM, Chen Y, Key MP, Cunha TS, Irigoyen MC, Morris M. Nocturnal hypertension in mice consuming a high fructose diet. Auton Neurosci 2006;130:41–50. Girard A, Madani S, El Boustani ES, Belleville J, Prost J. Changes in lipid metabolism and antioxidant defense status in spontaneously hypertensive rats and Wistar rats fed a diet enriched with fructose and saturated fatty acids. Nutrition 2005;21:240–8. Ran J, Hirano T, Fukui T, Saito K, Kageyama H, Okada K, Adachi M. Angiotensin II infusion decreases plasma adiponectin level via its type 1 receptor in rats: an implication for hypertension-related insulin resistance. Metabolism 2006;55:478–88. Kamide K, Rakugi H, Higaki J, Okamura A, Nagai M, Moriguchi K, et al. The renin-angiotensin and adrenergic nervous system in cardiac hypertrophy in fructose-fed rats. Am J Hypertens 2002;15:66–71. Reed MJ, Ho H, Donnelly R, Reaven GM. Salt-sensitive and carbohydratesensitive rodent hypertension: evidence of strain differences. Blood Press 1994;3:197–201. D’Angelo G, Elmarakby AA, Pollock DM, Stepp DW. Fructose feeding increases insulin resistance but not blood pressure in Sprague-Dawley rats. Hypertension 2005;46:806–11. Nyby MD, Abedi K, Smutko V, Eslami P, Tuck ML. Vascular Angiotensin type 1 receptor expression is associated with vascular dysfunction, oxidative stress and inflammation in fructose-fed rats. Hypertens Res 2007;30:451–7. Iyer SN, Raizada MK, Katovich MJ. AT1 receptor density changes during development of hypertension in hyperinsulinemic rats. Clin Exp Hypertens 1996;18:793–810. Hsieh PS, Tai YH, Loh CH, Shih KC, Cheng WT, Chu CH. Functional interaction of AT1 and AT2 receptors in fructose-induced insulin resistance and hypertension in rats. Metabolism 2005;54:157–64. Chess DJ, Lei B, Hoit BD, Azimzadeh AM, Stanley WC. Deleterious effects of sugar and protective effects of starch on cardiac remodeling, contractile dysfunction, and mortality in response to pressure overload. Am J Physiol Heart Circ Physiol 2007;293:H1853–60. Farah V, Elased KM, Morris M. Genetic and dietary interactions: role of angiotensin AT1a receptors in response to a high fructose diet. Am J Physiol Heart Circ Physiol 2007;293:H1083–9. Nyby MD, Matsumoto K, Yamamoto K, Abedi K, Eslami P, Hernandez G, et al. Dietary fish oil prevents vascular dysfunction and oxidative stress in hyperinsulinemic rats. Am J Hypertens 2005;18:213–9. Cai L, Wang J, Li Y, Sun X, Wang L, Zhou Z, Kang YJ. Inhibition of superoxide generation and associated nitrosative damage is involved in metallothionein prevention of diabetic cardiomyopathy. Diabetes 2005;54: 1829–37. Ceriello A, Motz E. Is oxidative stress the pathogenic mechanism underlying insulin resistance, diabetes, and cardiovascular disease? The common soil hypothesis revisited. Arterioscler Thromb Vasc Biol 2004;24:816–23. Ritchie RH, Quinn JM, Cao AH, Drummond GR, Kaye DM, Favaloro JM, et al. The antioxidant Tempol inhibits cardiac hypertrophy in the insulin-resistant GLUT4-deficient mouse in vivo. J Mol Cell Cardiol 2007;42:1119–28. Whitesall SE, Hoff JB, Vollmer AP, D’Alecy LG. Comparison of simultaneous measurement of mouse systolic arterial blood pressure by radiotelemetry and tail-cuff methods. Am J Physiol Heart Circ Physiol 2004;286:H2408–15. Munzel T, Afanas’ev IB, Kleschyov AL, Harrison DG. Detection of superoxide in vascular tissue. Arterioscler Thromb Vasc Biol 2002;22:1761–8. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 2008;3:1101–8. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402–8. Busserolles J, Gueux E, Rock E, Demigne C, Mazur A, Rayssiguier Y. Oligofructose protects against the hypertriglyceridemic and pro-oxidative effects of a high fructose diet in rats. J Nutr 2003;133:1903–8. Choi JS, Song J. Effect of genistein on insulin resistance, renal lipid metabolism, and antioxidative activities in ovariectomized rats. Nutrition 2009;25:676–85. Bengtsson SH, Gulluyan LM, Dusting GJ, Drummond GR. Novel isoforms of NADPH oxidase in vascular physiology and pathophysiology. Clin Exp Pharmacol Physiol 2003;30:849–54.

848

K. Mellor et al. / Nutrition 26 (2010) 842–848

[36] Kobayashi R, Nagano M, Nakamura F, Higaki J, Fujioka Y, Ikegami H, et al. Role of angiotensin II in high fructose-induced left ventricular hypertrophy in rats. Hypertension 1993;21:1051–5. [37] Togashi N, Ura N, Higashiura K, Murakami H, Shimamoto K. The contribution of skeletal muscle tumor necrosis factor-alpha to insulin resistance and hypertension in fructose-fed rats. J Hypertens 2000;18:1605–10. [38] Di Verniero CA, Silberman EA, Mayer MA, Opezzo JA, Taira CA, Hocht C. In vitro and in vivo pharmacodynamic properties of metoprolol in fructosefed hypertensive rats. J Cardiovasc Pharmacol 2008;51:532–41.

[39] Buchanan J, Mazumder PK, Hu P, Chakrabarti G, Roberts MW, Yun UJ, et al. Reduced cardiac efficiency and altered substrate metabolism precedes the onset of hyperglycemia and contractile dysfunction in two mouse models of insulin resistance and obesity. Endocrinology 2005;146:5341–9. [40] Ovide-Bordeaux S, Bescond-Jacquet A, Grynberg A. Cardiac mitochondrial alterations induced by insulin deficiency and hyperinsulinaemia in rats: targeting membrane homeostasis with trimetazidine. Clin Exp Pharmacol Physiol 2005;32:1061–70.