Carbohydrate-enriched diet impairs cardiac performance by decreasing the utilization of fatty acid and glucose

Abstract

Aims: We hypothesized that a high-carbohydrate diet affects the cardiac performance by interfering in the metabolic steps involved in energy transfer in this organ. To verify this, we investigated the myocardial utilization of different substrates and contractile function in rats fed a high-carbohydrate diet, under normal flow and ischemia.

Methods and Results: Male Wistar rats were fed over 9 days with standard (39.5% carbohydrate, 8% fiber) or high-carbohydrate diet (58% carbohydrate) and, afterwards, their cardiac function was examined using isolated heart preparations. The high-carbohydrate diet decreased the activity of the lipoprotein lipase, utilization of fatty acids, expression of the gene of peroxisome proliferator-activated receptor α and its target enzymes. In addition, decreased GLUT4 mass, glucose uptake, glycogen content and glycolytic intermediates were also observed. High-carbohydrate hearts displayed weaker activation of the glycolytic pathway during ischemia, according to minor production of lactate, in relation to control hearts. The functional impairment caused by high-carbohydrate diet shown by the decrease in the ventricular systolic strength, +dT/dt and −dT/dt was, at least in part, due to the low availability of adenosine triphosphate (ATP).

Conclusion: Our data suggest that a high-carbohydrate diet can damage myocardial contractile function by decreasing the cardiac utilization of glucose and fatty acids and, consequently, the ATP pool.

Keywords

cardiac tissue energetic metabolism fatty acid glucose high carbohydrate diet

Introduction

The High-carbohydrate Western diet is not the best choice for reducing cardiovascular disease (CVD). As the population has become increasingly overweight and obese, the emergence of the metabolic syndrome has led to reconsideration of the role of foods containing large quantities of carbohydrate in lipid and glucose metabolism. Carbohydrate-enriched diets, particularly sugars, have been shown to induce atherogenic dyslipidemia, characterized by high concentrations of small dense low-density lipoprotein (LDL) particles, low levels of high-density lipoprotein (HDL) and high triglyceride (TG) concentration, and it may, therefore, increase the risk of CVD [Culling et al. 2009; Diniz et al. 2008; Griel et al. 2006; Fried and Rao, 2003; Krauss et al. 2000]. Recent studies have shown that normal rats fed on a Western diet (high-carbohydrate, high-fat diet) not only became obese, but also presented impairment of global left ventricular function [Wilson et al. 2007], most likely because of enhanced de novo synthesis of saturated long-chain fatty acids (FAs) by the liver [Harmancey et al. 2010].

In contrast to the deleterious effects of refined carbohydrates, dietary fiber has demonstrated consistent beneficial effects on lipid and glucose metabolism. It is well established that diets rich in soluble fiber lower blood cholesterol levels [Griel et al. 2006]. In addition, the lower glycemic index of whole foods may protect against TG-increasing and HDL-lowering associated with traditional high-carbohydrate diets. In this context, glycemic load constitutes an important predictor of the CVD, as it reflects both the quantity and quality of carbohydrates [Chess and Stanley, 2008; Diniz et al. 2008; Sharma et al. 2008; Griel et al. 2006; Halton et al. 2006; Popkin and Nielsen, 2003].

Despite the well-documented increase in the intake of carbohydrates in Western countries and the positive association with metabolic and cardiovascular diseases [Diniz et al. 2008; Griel et al. 2006; Fried and Rao, 2003; Popkin and Nielsen, 2003], there is a paucity of information regarding the role of a carbohydrate-rich diet in cardiac dysfunction. A high intake of carbohydrates, especially sucrose, was strongly associated with an increased risk of coronary heart disease in women [Halton et al. 2006] and accelerated cardiac systolic dysfunction in hypertensive rats [Sharma et al. 2008]. Because functional cardiac performance is closely related to the energetic metabolism of the heart [Lopaschuk et al. 2010], we hypothesized that carbohydrate-rich diet would impair metabolic pathways that generate energy in the heart. This would be clinically relevant to consider since the duration of the cardiac resistance to an episode of ischemia and reperfusion is directly associated with energy reserve of the heart [Institute of Medicine of the National Academies, 2002/2005; Jennings and Steenbergen, 1985].

Thus, the major purpose of this study was to investigate relevant parameters of the metabolism of glucose and FA in hearts of rats fed with a diet rich in refined carbohydrates, under normal flow and ischemic conditions. We simultaneously studied the effect of this diet on the cardiac contractility indices, important indicators of functional integrity of the heart.

Methods and materials

Animals and treatment

Wistar male rats (10 weeks old) were kept in individual cages in an environmentally controlled room with a 14 h/10 h light/dark cycle. The animals received ad libitum a standard rodent chow (C) (Nuvital, Colombo, PR) containing 39.5% carbohydrate, 8% fiber (by weight) and 3.0 kcal/g, or a high-carbohydrate diet (HC), fiberless, containing 58% carbohydrate (29% starch and 29% sucrose) and 4.0 kcal/g [Kettelhut et al. 1980]. The HC diets contained equivalent amounts of fat, protein and micronutrients to C diet, and the groups were fed over the course of 9 days, after 2 days of adapting to this diet. Food intake was carefully monitored by weighing daily the special feeding containers and the weight evolution was obtained by weighing the animals on the first and ninth day of feeding. The animals were killed between 13:00 and 14:00 and the hearts were collected and used immediately or stored at −80°C. The serum was isolated and stored at −20°C until further analysis. The study complied with the standards stated in the Guides for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was approved by the ‘Ethics Committee in Animal Experimentation of the Federal University of Minas Gerais’ (protocol No. 027/05).

Langendorff isolated heart perfusion

After decapitation the thoracic cavities were opened and the hearts quickly excised and perfused to avoid clotting in the coronary arteries. Immediately on excision, the beating hearts were briefly placed in ice-cold (4°C) Krebs–Ringer (K-R) buffer (pH 7.4) containing 11.7 mM glucose. They were then cannulated via the aorta and perfused retrogradely through the coronary arteries in Langendorff mode, using the K-R buffer containing 1% bovine serum albumin. The perfusion fluid was maintained at 37 ± 1°C, with a pressure of 65 mmHg and gassed continuously (5% CO₂; 95% O₂). A force transducer (model FT 03, Grass) was attached through a heart clip to the apex of the ventricles to record the contractile force (tension, g) on a computer, using a data-acquisition system (Codas, Dataq Instruments Inc., Akron, OH, USA). A diastolic tension of 0.5–1.0 g was applied to the hearts. The heart rate and dT/dt were derived from the measurements made. The coronary flow was measured by collecting the perfusate over a period of 1 minute at regular intervals. Of the models submitted to regional ischemia, the left anterior descending coronary artery was ligated beneath the left auricular appendage, together with the adjacent veins [Lubbe et al. 1978]. The ligature was released after 15 min and the reperfusion was performed for an additional 15 min. To measure the activity of the functional LPL, after an initial 20 min stabilization period, 5 IU ml⁻¹ heparin were added to the perfusion solution and the perfusates were then collected at regular intervals.

Measurements of uptake and incorporation of energy substrates

To investigate the utilization of energy substrates, we adapted the method described by Golfman and colleagues [Golfman et al. 2005] and Niu and colleagues [Niu et al. 2004]. After stabilization, the system was switched to a recirculating mode using K-R buffer containing only ³H-triolein (15 μCi/l) or ³H-triolein (15 μCi/l), [U-¹⁴C] glucose (10 μCi/l) and insulin (40 μU/ml). The substrate–emulsion [Faraj and Cianflone, 2004] was added to the K-R solution at a final concentration of 0.4 mM triolein and the glucose concentration was 5.5 mM. Aliquots of 1 ml perfusate were collected at regular intervals for 30 min and the uptake of energetic substrates was determined by measuring the disappearance of these components from the perfusate over the course of 30 min. Incorporation was investigated in fragments of cardiac ventricles. The samples were subjected to lipid extraction with chloroform:methanol (2 : 1v/v) [Folch et al. 1957], so that the uptake and incorporation of FA could be evaluated in the organic phase and the uptake and incorporation of glucose could be evaluated by analyzing the aqueous phase for radioactivity.

Lipoprotein lipase activity

Functional lipoprotein lipase (LPL) (the active LPL released by heparin) was measured in cardiac perfusate after heparin injection in the perfusion solution. The enzymatic activity was measured using a [9,10-³H]triolein-containing substrate emulsified with lecithin [Nilsson-Ehle and Schotz, 1976] and containing 24-h fasted rat serum as a source of apo-CII. The serum was pretreated by heating at 55°C to inactivate nonspecific plasma proteases. The radioactivity in liberated FAs was counted following extraction in methanol–chloroform–heptane [Belfrage and Vaughan, 1969].

Quantitative real-time PCR

Total RNA from hearts (100 mg) was extracted using TRIzol reagent. After quantification, the samples were treated with DNAse and the reverse transcription was carried out using an oligo (dT) primer and MML-V (Moloney murine leukemia virus). cDNA was amplified using specific primers of PPARα (forward: 5′-taccactatggagtccacgcatgt-3′; reverse: 5′-ttgcagcttcgatcacacttgtcg), CPT-1 (forward: 5′-acgtgagtgactggtgggaagaat-3′; reverse: 5′-tctccatggcgtagtagttgctgt-3′), ACO (forward: 5′-atctctgtggttgctgtggagtca-3′, reverse: 5′-tctggatgcttccttctccaaggt-3′), β-actin (forward: 5′-catgaagatcaagatcattgctcct-3′, reverse: 5′-ctgcttgctgatccacatctg-3′) and SYBR® green PCR buffer in an ABI Prism 7000 platform (Applied Biosystems). β-actin was co-amplified as an endogenous normalizing gene. The specific primers sequence was designed from sequences available in geneBank (BLAST, NCBI). The amplification parameters were set at stage 1 (one cycle at 50°C for 2 min), stage 2 (one cycle at 95°C for 10 min), stage 3 (40 cycles of 95°C for 0.15 min and 60°C for 1 min). To analyze the target genes expression, we used the ΔΔCT semiquantitative method [Pfaffl, 2001]. The expression levels were represented as the ratio of signal intensity for each target mRNA relative to β-actin mRNA.

Western blotting analysis

Samples of frozen cardiac ventricles (∼300 mg) were homogenized in lysis buffer containing protease inhibitors. After centrifugation at 1500 g for 20 min at 4°C, the protein content of the supernatant was quantified using a Bradford protein assay and, then, 50 µg was resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; 10%). After transfer, the membranes were blocked in 5% skimmed milk in Tris-buffered saline containing 0.1% Tween 20. The glucose transporter GLUT4 was probed with a polyclonal rabbit anti-GLUT4 antibody (1 : 4,000) and a goat antirabbit IgG antibody conjugated with peroxidase (1 : 2,000) was used as a secondary antibody. The blots were visualized using an ECL® chemiluminescence detection kit.

Biochemical analysis

Ventricular tissues were freeze-dried and the metabolites were extracted with perchloric acid for determination of adenosine triphosphate (ATP), glycolytic intermediates (G-6-P, glucose-6-phosphate; F-6-P, -fructose-6-phosphate) and glycogen. The extracts were then assayed enzymatically using spectrophotometric methods [Trautschold, 1989; Michael, 1988]. The total glycogen content was quantified by the anthrone assay [Hassid and Abraham, 1957]. Serum triglycerides, total cholesterol and glycerol were assayed by conventional enzymatic methods using kits produced by Katal (Belo Horizonte, MG-Brazil). Free fatty acids (FFAs) were estimated using commercial kits from Wako (Pure Chemical Industries, Japan). The lactate present in cardiac perfusate was quantified by using a glucose and lactate oxidase analyzer (Glucose analyzer YSI 300 Plus).

Statistical analysis

Data are expressed as mean ± SE. The statistical significance between the groups was assessed by unpaired Student’s t-test, two-way analysis of variance (ANOVA; with post hoc Bonferroni test) and one-way ANOVA (with post hoc Newman Keuls test) according to viability. All data and statistical analyses were performed using GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, CA). Differences were considered statistically significant at the p < 0.05 level.

Results

Physical and metabolic parameters of control and HC-fed animals

The body weight gain was similar among the analyzed groups and this was associated with lower intake of the HC diet (Table 1), which provided higher metabolizable energy (average 4.0 kcal/g) in relation to control chow (average 3.0 kcal/g). Lipid profile of the animals was affected by intake of refined carbohydrate-enriched diet, showing 83% increase in TG, 42% in total cholesterol and 53% in FFA concentration, in relation to controls. The serum concentration of glycerol was also increased by 56% in the HC group (Table 1).

Table 1.

Weight-related and serum biochemical parameters.

	C	HC
General (n = 5)
Food intake (g/day)	28 ± 1.2	21 ± 0.7**
Food intake (kcal/g)	84 ± 3,6	84 ± 2,8
Final body weight (g)	244 ± 5.3	242 ± 8.6
Body weight gain (g/9 days)	58 ± 6.8	61 ± 8.8
Heart weight (g/100 g bw)	0.38 ± 0.01	0.41 ± 0.01
Serum (n = 7–10)
Triglycerides (mmol/l)	0.85 ± 0.07	1.56 ± 0.12***
Total cholesterol (mmol/l)	2.94 ± 0.07	4.20 ± 0.11****
Free fatty acid (mmol/l)	0.63 ± 0.04	0.97 ± 0.07**
Glycerol (mmol/l)	0.36 ± 0.03	0.56 ± 0.04**

Data are means ± SEM; **p < 0.01, ***p < 0.001, ****p < 0.0001 versus C.

bw, body weight; C, control group; HC, high-carbohydrate group.

LPL activity in cardiac perfusate of control and HC-fed animals

As the lipoproteins constitute the main sources of FA used by the heart muscle and LPL is the enzyme that hydrolyzes the core of triglyceride-rich lipoproteins into FFA and monoglycerides [An et al. 2005], we measured functional LPL activity and found a 41% decrease in the HC hearts compared with controls, during the entire perfusion period (Figure 1A). This may have contributed to lower cardiac uptake of triolein-FA observed in HC animals, in both perfusion with ³H-triolein only (61% lower) and perfusion with ³H-triolein plus [U-¹⁴C] glucose simultaneously (79% lower), compared with control animals (Figure 1B). After perfusion with ³H-triolein alone, cardiac incorporation of triolein-FA by HC hearts was also lower than C hearts (46% lower) (Figure 1C). The addition of glucose to perfusion buffer caused the expected decrease in the cardiac uptake (19% and 55% decrease for C and HC groups, respectively) (Figure 1B) and incorporation (94% and 92% decrease for C and HC groups, respectively) (Figure 1C) of FA, in both groups.

Figure 1.

Lipoprotein lipase activity (A) and ³H-triolein-FA uptake (B) and utilization (C) by hearts from C (control) and HC (high-carbohydrate) treated rats. Activity of heparin-releasable lipoprotein lipase (LPL) was determined in cardiac perfusate, in basal conditions and during occlusion and reperfusion. Hearts were perfused with K-R buffer as described in the text. At the time indicated by the arrow, heparin (5 IU ml⁻¹) was added to the buffer and the LPL activity was measured in the coronary perfusate, which was collected for 1 min at 5 min intervals. Uptake (B) and tissue incorporation (C) of ³H-triolein-FA were measured in hearts perfused with ³H-triolein only or with ³H-triolein plus [U-¹⁴C] glucose and insulin simultaneously. Data are means ± SEM for n = 5–6 animals; ****p < 0.0001 versus C; ***p < 0.001 versus C for triolein alone, ^###p < 0.001 versus C for triolein + glucose, ⁺p < 0.05 versus corresponding value for triolein alone, ^••p < 0.01 versus corresponding value for triolein alone, ^••• ⁺⁺⁺p < 0.001 versus corresponding value for triolein alone.

Gene expression of oxidative enzymes of control and HC-fed animals

We investigated the lipid oxidative pathway by quantifying the gene expression of an important transcriptional regulator, PPARα, and of its target enzymes, CPT-1 and ACO. Real-time PCR quantification was performed to measure mRNA using specific primers for PPARα, CPT-1 or ACO, and the β-actin housekeeping gene was used as internal control after reverse transcription analyses. The results show that HC diet decreased cardiac expression of PPARα (50%), of CPT-1 (85%), and of ACO mRNAs (54%) relative to the expression in the hearts of control animals (Figure 2A–C).

Figure 2.

Cardiac tissue mRNA expression of PPARα (A), CPT-1 (B) and ACO (C) in C (control) and HC (high-carbohydrate) groups. Arbitrary units are relative to values obtained for β-actin. Data are means ± SEM for n = 4–5 animals; *p < 0.05, ****p < 0.0001 versus C.

Glucose uptake by hearts of control and HC-fed animals

The perfusion of the hearts with a mixture of glucose, insulin and triolein showed a 62% reduction in the uptake of glucose (Figure 3A) and consequent decrease (32%) in incorporation of this metabolite (Figure 3B). As GLUT4 is the predominant glucose transporter isoform in the adult heart [Schwenk et al. 2008; Depre et al. 1999], we analyzed GLUT4 expression using Western blotting. Figure 3C shows immunoblots of the 45-kDa GLUT4 band and semiquantitative results of this analysis are shown in Figure 3D. The mass of GLUT4 protein was decreased in the hearts of the rats fed with HC diet, in relation to control diet (Figure 3D). Glycogen content was also decreased, in both baseline state (34%) and postischemia and reperfusion (44%) (Figure 3E).

Figure 3.

Uptake (A) and tissue incorporation (B) of [U-¹⁴C] glucose by C (control) and HC (high-carbohydrate) hearts perfused with ³H-triolein, plus [U-¹⁴C] glucose and insulin. Representative Western blotting of GLUT4 (C), summary data of blots for GLUT4 (D) and glycogen content (E). Data are means ± SEM for n = 3–7 animals; *p < 0.05 versus C, **p < 0.01 versus C.

Glycolytic pathway intermediates of hearts of control and HC-fed animals

The HC hearts showed lower content of G-6-P (59%) and F-6-P (52%) during the basal perfusion and of G-6-P postocclusion and reperfusion (82%) (Figure 4A and B). As expected, coronary occlusion reduced G-6-P (86% and 94% decrease for C and HC groups, respectively) (Figure 4A) and F-6-P (86% and 82% decrease for C and HC groups, respectively) (Figure 4B) contents in both groups, as result of the limited supply of nutrients and intensified glycolytic flow, typical of this phase [Schwenk et al. 2008; Depre et al. 1999; Samaja et al. 1998]. Lactate production in cardiac perfusate was similar between the groups after 20 min of basal perfusion and 15 min of reperfusion, increasing immediately after 15 min of ischemia (360% and 267% increase in C and HC groups, respectively). However, this increase was less intense in the animals undergoing the HC diet, with a lactate production 22% lower than in the C animals (Figure 4C).

Figure 4.

Glycolytic intermediates of basal and ischemic hearts. Concentration of glucose-6-phosphate (A), fructose-6-phosphate (B), and lactate (C) were measured in myocardial tissue from C (control) and HC (high-carbohydrate) groups, after basal perfusion (20 min) and postocclusion and reperfusion (30 min). Data are means ± SEM for n = 7 animals; ***p < 0.001 versus C at basal condition, *p < 0.05 versus C at postocclusion, ^###p < 0.001 versus C at basal condition; ⁺⁺⁺p < 0.01 versus HC at basal condition, ***p < 0.001 versus C at postocclusion, ^###p < 0.001 versus C at basal condition, ^•••p < 0.001 versus C at postocclusion, ⁺⁺⁺p < 0.001 versus HC at basal condition, ⁺ p < 0.05 versus HC at basal condition, ^o p < 0.001 versus HC at postocclusion.

Functional parameters of hearts of control and HC-fed animals

The metabolic changes involving cardiac utilization of glucose and FA affected the energy yield of the hearts of the rats that were fed the HC diet. ATP content of the HC hearts was smaller than that of C hearts in both basal perfusion (60%) and postischemia and reperfusion (76%). After coronary occlusion, the energy store was impaired in both groups, leading to a deficit of 85% and 91% of ATP content of the C and HC hearts, respectively (Figure 5).

Figure 5.

Concentration of ATP in cardiac tissue from C (control) and HC (high-carbohydrate) groups; after basal perfusion (20 min) and postocclusion and reperfusion (30 min). Data are means ± SEM for n = 7 animals; ***p < 0.001 versus C at basal condition, *p < 0.05 versus C at postocclusion, ^###p < 0.001 versus C at basal condition; ⁺⁺ ⁺ p < 0.01 versus OA at basal condition.

When we analyzed the contractility index, no changes were observed in heart rate and diastolic tension according to any of the perfusion conditions. Nevertheless, HC intake decreased ventricular systolic tension (19% lower in both pre-ischemia and ischemia), +dT/dt (20% and 22% lower in pre-ischemia and ischemia, respectively) and –dT/dt (25% lower in both pre-ischemia and ischemia). During ischemia, coronary flow decreased similarly in both groups (Table 2).

Table 2.

Functional parameters from hearts of C (control) and HC (high-carbohydrate) animals.

	C	HC
Basal (n = 15–22)
Heart rate (beats/min)	279 ± 2.9	274 ± 3.3
Systolic tension (g)	7.2 ± 0.2	5.8 ± 0.1**
Diastolic tension (g)	0.8 ± 0.02	0.8 ± 0.01
+dT/dt (g/s)	122 ± 3.8	97 ± 1.4**
−dT/dt (g/s)	123 ± 6.5	92 ± 2.3*
Coronary flow (ml/min)	12.5 ± 0.1	12.6 ± 0.2
Occlusion (n = 9–14)
Heart rate (beats/min)	250 ± 4.4	258 ± 1.1
Systolic tension (g)	5.7 ± 0.1	4.6 ± 0.1***
Diastolic tension (g)	0.9 ± 0.05	1.0 ± 0.1
+dT/dt (g/s)	98 ± 4.0	76 ± 2.4**
−dT/dt (g/s)	76 ± 2.7	57 ± 1.1***
Coronary flow (ml/min)	5.3 ± 0.6	6.5 ± 0.5
Reperfusion (n = 9–11)
Diastolic tension (g)	3.6 ± 0.1	3.2 ± 0.2
Coronary flow (ml/min)	12.1 ± 0.4	11.9 ± 0.1

Data are means ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 versus C.

Discussion

Recent evidence has shown that the large majority of carbohydrates in Western diets, consisting of refined starches and sugars, have adverse metabolic effects and increase risk of CVD [Fried and Rao, 2003; Popkin and Nielsen, 2003; Krauss et al. 2000]; however, there is still very little information linking the macronutrient composition to energy yield in the heart. Under normal aerobic conditions FA and glucose constitute the major sources of energy for the heart, with FA contributing about 70% of this energy supply [Lopaschuk et al. 2010; Schwenk et al. 2008; Sambandam and Lopaschuk, 2003; Depre et al. 1999]. Thus, this study investigated the effect of a diet rich in refined carbohydrates on metabolic pathways generating energy in the heart and the outcome on cardiac performance.

The HC diet used, which contained starch and sucrose (1 : 1 v/v) as source of carbohydrates, increased serum concentrations of TG and cholesterol in relation to control chow, and these outcomes were not mediated by weight gain. Feeding studies also support hypothesis that carbohydrate-enriched diets increase serum TG concentrations and decrease serum HDL cholesterol and may, therefore, increase the risk of CVD [Fried and Rao, 2003; Krauss et al. 2000]. The higher serum levels of TG and cholesterol observed in this study are due, in part, to higher glycemic load of HC diet, as result of high quantity of sucrose, total carbohydrate and lack of fiber [Chess and Stanley, 2008; Diniz et al. 2008; Sharma et al. 2008; Griel et al. 2006; Fried and Rao, 2003].

Results from recent clinical trials have shown that replacement of dietary fat with carbohydrates resulted in only a modest decrease in HDL and a negligible increase in TG, compared with baseline. Investigators attributed this result to quality of carbohydrates emphasized in the diet: fruits, vegetables and whole grains [Howard et al. 2006; Appel et al. 2005]. Intake of food with higher glycemic index requires higher insulin levels for postprandial metabolism [Griel et al. 2006]. In addition to covalent activation of glycolytic and lipogenic enzymes, high insulin levels may increase TG synthesis by inducing expression of lipogenic genes in liver and adipose tissue by the activation of sterol responsive element bind protein-1 (SREBP-1) [Kersten, 2001]. Also, glucose by itself activates carbohydrate responsive element bind protein (ChREBP), another important transcription factor involved in the induction of lipogenic genes [Yamashita et al. 2001]. In addition to reducing glycemic index, dietary fibers are known to decrease intestine absorption of cholesterol and TG and endogenous production of cholesterol [Lichtenstein et al. 2006].

Functional cardiac LPL activity was reduced in animals fed with HC diet, in relation to control diet. This reduction, apart from decreasing the energy supply to cardiac tissue, may also reduce the clearance of serum TG, since the expression of LPL only in the heart is sufficient to keep the plasma TG level within the normal range [Levak-Frank et al. 1999]. In addition, the increased concentrations of serum TG, as observed in HC rats, have been associated with smaller LDL, which becomes more susceptible to oxidation. Reactive oxygen species may react with protein thiol moieties of this lipoprotein and produce sulfur oxidation states and, therefore, decrease cellular uptake of lipids from the blood [Diniz et al. 2008].

The higher serum levels of FFA and glycerol, suggestive of increased lipolysis in adipose tissue of the HC rats, increase the availability of FA for hepatic synthesis and secretion of TG in these animals [Fried and Rao, 2003]. Therefore, the increased serum level of TG observed in HC animals may be assigned to the additive effects of this diet stimulating liver lipogenesis, increasing intestine absorption of FA and lipolysis in adipose tissue and decreasing serum clearance of TG.

The heart is considered a metabolic omnivore, i.e. it functions best when it oxidizes multiple energy substrates simultaneously [Lopaschuk and Kelly, 2008; Schwenk et al. 2008; Depre et al. 1999]. Regarding cardiac utilization of FA, the refined carbohydrate-enriched diet decreased uptake of FA, both in the hearts only perfused with triolein as well as those perfused concurrently with triolein and glucose. Introduction of glucose in perfusion buffer reduced the cardiac utilization of FA, confirming previous work [Dyck et al. 2004], so that the decrease in the cardiac incorporation of FA caused by HC diet was blunted in the hearts perfused with triolein and glucose concurrently.

Expression of the PPAR_α gene was reduced in the HC hearts, in relation to control hearts. The activity of this nuclear receptor is an important determinant of lipid homeostasis and ATP production in cardiomyocytes because it regulates the transcription of several proteins linked to the transport and metabolism of FA [Carroll and Severson, 2001; Nohammer et al. 2000; Totland et al. 2000]. The decreased activity of PPARα in cardiac tissue of HC animals was evidenced by the reduced expression of its target oxidative enzymes, CPT-1 and ACO. The deleterious effect of the HC diet on FA oxidation has also been observed in previous work [Diniz et al. 2008], leading to major accumulation of TG in the heart. Diniz and colleagues suggested in this study that the depressed fat degradation resulting from the intake of carbohydrate-enriched diet contributes to increased cardiac lipoperoxidation, which is associated with heart failure. However, we did not observe accumulation of TG in cardiac tissue of HC animals in the present study, although it should be emphasized that the earlier study observed more long-term effects and used quantities of carbohydrates that exceeded the distribution percentage of macronutrients recommended for rodents.

In summary, this study reveals that the decrease in the activity of LPL and in the expressions of PPARα, CPT-1 and ACO mRNA, triggered by the HC diet, impaired cardiac uptake and oxidation of FA and, thus, the tissue energy. This is particularly important in the ischemic phase, when the recovery of cardiac function is entirely dependent on the level of energy stores.

Regarding the utilization of glucose, the results obtained revealed that HC diet reduces the cardiac expression of the GLUT4 transporter, therefore decreasing the cardiac uptake and incorporation of the glucose, in relation to control diet. Reduction of the cardiac supply of glucose in the HC animals resulted in a significant decrease of the G-6-P and F-6-P glycolytic intermediates. Previous studies from this laboratory have shown that mice fed a HC diet, similar to that used in these experiments, exhibited elevated circulating TG and hyperglycemia with impairment in glucose tolerance. Plasma concentration of tumor necrosis factor-α (TNF- α) and monocyte chemoattractant protein-1 (MCP-1) and liver concentration of TNF- α were also elevated [Ferreira et al. 2010]. Many lines of evidence have shown that chronic activation of pro-inflammatory pathway within insulin target cells can lead to insulin resistance [Schenk et al. 2008]. Inflammatory signals work in the muscle cell, via the nuclear factor (NF)-κB [Shoelson et al. 2006; Cai et al. 2005] or via the c-Jun N-terminal kinase 1 (JNK1) [Vallerie et al. 2008] signaling cascades, to block an insulin receptor substrate and shut down the insulin signaling pathway. Other studies have shown that the development of systemic insulin resistance is caused, at least in part, by the routing of incoming glucose through the hexosamine biosynthesis pathway [Marshall et al. 1991]. It is postulated that the advanced glycosylation endproduct proteins (AGEs) that form by nonenzymatic glycosylation during hyperglycemic conditions might stimulate TNF-α release [Vlassara et al. 1988].

Taking into account the reasoning exposed above, it is possible to infer that the animals used in this work might present glucose intolerance.

The production of lactate was similar between groups after basal perfusion and reperfusion, data that does not support the results found in the study of Diniz and colleagues [Diniz et al. 2008], which have suggested a myocardial metabolic shifting with enhanced glycolytic pathway in the hearts of rats fed with a HC diet. Thus, our results indicate an impaired cardiac utilization of glucose as a consequence of high intake of refined carbohydrates.

Ischemia increased cardiac production of lactate and decreased concentration of G-6-P and F-6-P in both groups, evidencing the activation of the glycolytic pathway as an alternative in order to produce energy for the myocardium during this phase [Lopaschuk et al. 2010; Schwenk et al. 2008; An et al. 2005]. However, rats that were fed HC diet revealed lower glycolytic activation during ischemia, according to minor production of lactate, in relation to control hearts. In addition to lower glucose uptake, the reduced storage of glycogen in the HC hearts even in the pre-ischemia contributed to impaired ischemic glycolytic flow by the minor substrate supply compared with the control group. The contribution of glycogen to the glycolytic pathway in the ischemic condition is appreciable, with the glycogenolysis providing, in the first 15 minutes of ischemia, about 60 µmol of glucose per gram of weight compared with 35 µmol of glucose per gram of weight from the extracellular uptake [An et al. 2005].

The lower energy efficiency in the HC hearts, in relation to C hearts, confirms the metabolic impairment caused by carbohydrate-enriched diet. The ATP content of the HC hearts was lower than the C group even in pre-ischemia, weakening further energy efficiency during the ischemic phase. This fact is relevant since the cardiac content of ATP is essential to minimizing the postischemia and reperfusion intracellular ionic overload and subsequent cellular and functional impairment. Starting the ischemia with low level of ATP reduces the amount of time it takes for the energy stock to reach a critical level, below which the cellular damage becomes irreversible [Samaja et al. 1998; Jennings and Steenbergen, 1985]. In addition to changes in the cardiac metabolism of FA and glucose observed in this study, Diniz and colleagues showed a direct association between glycemic index and myocardial lipoperoxidation [Diniz et al. 2008], a fact that may also depress energy metabolism by destroying the mitochondrial membrane. This group has emphasized a positive correlation between oxidative stress and loss of myocardial function.

We have proven that the low energy efficiency in HC hearts affected the functional integrity in these organs. The animals that underwent the HC diet presented lower ventricular systolic strength and impairment of the velocity of cardiac contraction and relaxation in both baseline and ischemic conditions, compared with the C group.

It has been established that diet constitutes an important approach to the CVD prevention and treatment strategies. In this study, we clearly demonstrated that a diet rich in refined carbohydrates damages cardiac performance by interfering in several metabolic steps, decreasing the cardiac utilization of glucose and FA and, thus, the ATP pool. This observation is of pathophysiological significance when taking into account the critical role of energy stores in the functional recovery of myocardium after the reperfusion stage.

Footnotes

Funding

This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de Minas Gerais, FAPEMIG (grant numbers CDS-APQ-4738-4.08/07, CDS-785/06 to LMB). A fellowship to L.C.J. Porto from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) is also gratefully acknowledged.

Conflict of interest statement

The authors have no conflicts of interest to declare.

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