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-'''The effect of a low glycemic index diet on blood glucose and lipid metabolism and on cardiac structure in an experimental model of diabetic rats'''
-== Abstract ==
-=== Background: ===
-Hyperglycemia contributes to the development of diabetic cardiomyopathy.  In this study, we investigated the effect of consumption of a low glycemic index (LGI) diet on collagen deposition on heart in rats with experimentally induced diabetes.
-=== Methods: ===
-Male rats were randomly divided into 3 groups: control rats on normal glycemic index diet (C-NGI), streptozotocin (STZ)-induced diabetic rats (55 mg/kg body weight, i.v.) on LGI diet (D-LGI) and STZ-induced diabetic rats on normal glycemic index diet (D-NGI). Biochemical parameters related to blood glucose and lipid metabolism were evaluated in the diabetic groups of rats at the beginning and at the end of the second month of the study, when the animals of both three groups were euthanatized; myocardial tissues were collected, formalin fixed and sectioned. Morphology and collagen deposition were assessed, with histochemical staining.
-=== Results: ===
-At the end of the experimental period, D-LGI group presented significantly ameliorated serum glucose, total cholesterol and triglyceride levels as compared with the D-NGI group. There was a trend for a greater collagen deposition to the heart vessels in D-NGI as compared with D-LGI and C-NGI groups. Indices of cardiac fibrosis like size and length of collagen fiber tended to be greater in the D-NGI than in the D-LGI and C-NGI groups. The patchy collagen deposition was significantly lower in the D-NGI compared with the other two groups, while the density of collagen fibers in cardiac tissues was significantly lower in both STZ-induced diabetic groups in comparison with the control group.
-=== Conclusions: ===
-Consumption of a LGI in a diabetes mellitus Type 1 rat model improves the disturbed serum glucose concentration, ameliorates serum lipid parameters and protects from the development of vascular and interstitial cardiac fibrosis. Diet may display a significant role in the prevention of diabetic cardiomyopathy.
-=== Keywords: ===
- Diabetes mellitus, rats, myocardial interstitial fibrosis
-== Introduction ==
-Hyperglycemia, the primary clinical manifestation of diabetes, is associated with the development of diabetic complications. Several studies have suggested that hyperglycemia accelerates the development of chronic complications via several mechanisms, including the increased aldose reductase related polyol pathway flux, the increased formation of advanced glycation end-products (AGEs), the activation of protein kinase C isoforms, the increased hexosamine pathway flux and overproduction of reactive forms of oxygen [1]. There is strong evidence that AGEs formation is the critical pathogenic link between hyperglycemia and long term micro- and macro-vascular complications of diabetes.
-Persistent hyperglycemia plays an important role to the development of diabetic cardiomyopathy (DCM) [2]. Hyperglycemia induces oxidative stress and activates a number of secondary messenger pathways, leading to cardiac fibrosis and cell death [3]. The link between hyperglycemia and the development of DCM involves the accumulation of AGEs [3]. Within the cells, these and their precursors modify macromolecules, producing irreversible cross-links between extracellular matrix (ECM) proteins, compromising tissue compliance and causing myocardial stiffness. Besides their well known direct toxicity, AGEs also exert their detrimental effect by interacting and up-regulating their receptors, including the receptor for advanced glycation end products (RAGE) and galectin-3 [4]. Through these receptors, AGEs activate several critical molecular pathways, which trigger the production of pro-fibrogenic growth factors, connective tissue growth factor (CTGF) and transformation growth factor β1 (TGFβ1) as well as the inflammatory response. Moreover, the interaction with AGE receptors causes intracellular changes, most notably the activation of redox transcription factors such as the nuclear factor-κB (NFκB) and the activating protein-1 (AP-1), a further element that increases ECM production. Thus, strategies preventing the detrimental effects of AGEs offer promising prospects of improving heart function in diabetic patients [5].
-Therefore, a mode of diabetes treatment independent of blood glucose levels, the inhibition of AGE formation could be useful in the prevention or reduction of certain diabetic complications in both main forms of the disease, Type 1 (T1D) and Type 2 diabetes mellitus (T2D). To accomplish this goal, appropriate experimental models that manifest the same pattern of disease to the clinical situation in humans, should be considered as essential tools for understanding the pathogenesis of diabetes-related complications.
-Previous studies have shown that increased interstitial collagen in ischemic cardiomyopathy is a major determinant of ventricular dysfunction [6]. Early echocardiographic data suggest that fibrosis markedly change the acoustic properties of the heart [7] but failed to provide a definite correlation of collagen deposition to the infraction risk [8]. Currently, collagen content is recognized as a major component of proper cardiac function [9]. However, the potential effect of consumption of diets with different glycemic index on collagen deposition and the development of DCM in experimental diabetes are not known.
-The purpose of this study was to examine the potential effects of two different diets, a normal diet and a low glycemic index diet (LGI) on collagen deposition at the heart in Streptozotocin (STZ)-induced diabetes mellitus Type 1 in rats.
-== Materials and methods ==
-=== Animal treatment ===
-Twenty two adult male Wistar rats weighing 238–342 g (Table 1) were randomly allocated into 3 groups: (1) the control group on normal diet (Group C-NGI) [n = 6]; (2) the diabetic group receiving normal diet (Group D-NGI) [n = 8], and (3) the diabetic group fed with a LGI diet (Hi-Maize, National Starch and Chemical Co, USA) (Group D-LGI) [n = 8]. The animals were housed under standard lab- oratory conditions (23±2oC) and humidity (60%) with 12h light and dark cycle. The experimental protocol was reviewed and approved by the Ethics Committee of the Medical School of the University of Athens and by the Veterinary Service of Athens Regional Unit in compliance with the internationally accepted guidelines on animal research and according to EU legislation.
-Animals in groups D-NGI and D-LGI were injected intravenously with streptozotocin (STZ) with a single dose of (Sigma, St. Louis, USA), 55 mg/kg body weight, dissolved in citrate buffer (10 mM, pH 4.5), as described previously [10] while the animals belonging to control group received vehicle injection. Up to 5 days following STZ administration, the induction of diabetes in all STZ treated animals was confirmed by glucose measurement in blood collected from the tail vein of the rats using an automatic glucometer (glucometer Wellion® Linus, AgaMatrix Inc., Salem, USA). All animals presenting blood glucose levels after a 12-h fasting period higher than 200 mg/dl were considered diabetic.
-The animals were weighed at the beginning of the study and 2 months later, when the rats were euthanatized using an overdose of anesthesia.
-=== Laboratory assays ===
-Apart from blood glucose measurements using the automatic glucometer, fasting blood was drawn from the tail vein of the rats belonging to the D-NGI and D-LGI groups at baseline and at 60 days and the serum was separated by centrifugation for further biochemical analysis. Serum concentration of total cholesterol was estimated by the CHOD-POD enzymatic colorimetric method/Trinder reaction and serum triglycerides were measured with the GPO-POD method. Serum glucose was determined using the Glycerol Phosphate Oxidase-Peroxidase GPO-POD method. All samples were analyzed at the Laboratory of Experimental Surgery and Research of the Medical School of Athens (Athens, Greece), using an automatic analyzer (Technicon RA-XT, Technicon Instruments, Tarrytown, NY).
-=== Determination of fibrosis ===
-After the euthanasia of the animals of the three experimental groups, their hearts were dissected and fixed in 10% formalin at room temperature. The tissues were then embedded in paraffin, sectioned at 5 μm, mounted on glass microscope slides and stained for 1 h in the Picrosirius solution (0.1% solution of Sirius Red F3BA in saturated aqueous picric acid). The stained sections were then washed for 2 min in 0.01 M HCL, dehydrated, cleared and mounted in synthetic resin. The staining was visualized by optical and polarized microscope (Zeiss, Germany) assessed by two independent observers and images were taken under natural and polarized light from at least five areas per slide. Eosin and hematoxylin stained slides (HE) were also prepared to study the morphology of the tissue. Computer-assisted morphometry was performed with a morphology measuring computer programs named Pixcavator [11].
-Given that the depositions in the vesicular lumen must be collagenic, the fibrous element was stained by picrosirius stain and assessed by two different morphometric systems in order to gain most information and eliminate any bias or bug introduced by the computerized measuring algorithms. The methodology of measuring algorithms for these programs has been reviewed and validated in the literature [Intelligent Perception site; Cell profiler site]. Under polarized light only collagen fibers are visible (orange fibers, Figure1) and no reticular fibers are seen (green), as expected, confirming our hypothesis that the narrowing of the lumen must be due to collagen deposition. Data were collected in excel format, processed and compared between groups.
-Using the Pixcavator program the following parameters were assessed: area, length of the perimeter, patchy and density of collagen deposition as well as the length of the collagen fibers in the stained area. All these measurements give an approximation of fibrosis in the cardiac tissue.
-=== Statistical analysis ===
-The normality of the distributions was assessed with Kolmogorov-Smirnov test and graphical methods. Comparisons between two groups were performed with the Student’s t-test for the normal distributions or the non-parametric Mann-Whitney’s U test for the non normal distributions. Differences among multiple groups for parametric data were tested by one-way analysis of variance (ANOVA); when a P-value <0.05 was found in ANOVA, the post hoc least significance difference test (LSD) was used to look for differences between the study groups. Differences among multiple groups for non-parametric data were tested by the Kruskal Wallis test using Mann-Whitney’s U test for post hoc multiple testing. Comparisons between two measurements of the same group were performed using paired samples t-test for normal distributions or Wilcoxon’s signed rank test for non normal distributions. All tests were two-sided. Differences were considered as statistically significant if the null hypothesis could be rejected with >95% confidence (p<0.05). The values of serum glucose levels measured with the automatic glucometer for the confirmation of the induction of diabetes in animals were not included in the statistical analysis in the comparison of measurements of the same group.
-== Results ==
-=== Body weight and serum parameters ===
-No differences were recorded in baseline body weight levels between the three groups. In addition, no differences were observed between the two STZ-treated groups in serum glucose, total cholesterol and triglyceride levels at the beginning of the experiment (0 days).
-D-NGI and C-NGI groups presented significantly increased body weight levels at the 60th day of the experimental period as compared with the initial body weight levels (initial body weight levels vs. final body weight levels, P < 0.001 and P = 0.001 for D-NGI and control group respectively), while the body weight levels of the D-LGI groups remained invariable throughout the study (P = 0.123). At the end of the study, D-LGI group had significantly reduced body weight as compared with the control non-diabetic group (Table 1).
-The intravenous administration of STZ to the rats affected their blood glucose levels (Table 2), leading to increased serum glucose levels (>200 mg/dl using the automatic glucometer in measurements performed up to five days following STZ administration) in all animals of D-NGI and D-LGI groups (average mean ± standard deviation: 426.50 ± 150.52; 376.87 ± 102.64 mg/dl for D-NGI and D-LGI group respectively).
-At the end of the experiment, the animals of D-NGI group presented increased serum glucose levels, total cholesterol and triglyceride levels as compared with the D-LGI group (P = 0.023, P = 0.05 and P = 0.032; serum glucose, total cholesterol and triglyceride levels, respectively).
-In D-NGI group, serum glucose levels were higher in a statistically significant way at the 60th day of the study in comparison to the initial levels (P = 0.008). Regarding serum total cholesterol levels of this group at the end of the experiment, they did not differ to their baseline values (P = 0.591). Serum triglyceride levels were significantly lower at the end of the study as compared to baseline (P= 0.02)(Table 2).
-Serum glucose levels of D-LGI group at the 60th day were significantly elevated in comparison to baseline levels (Baseline vs. 60-days serum glucose levels, P = 0.024). Serum total cholesterol concentration in D-LGI group at the 60th day was not significantly different as compared with its baseline levels (P = 0.205). Finally, serum triglyceride levels in D-LGI group were significantly lower in the 60th day as compared to the beginning of the study (P = 0.001).
-=== Heart histopathology ===
-Using the Haematoxylin Eosin stain the external and internal maximal diameter of the heart vessels was measured. While the external maximal diameter was not different among the three groups, there was a trend for the internal diameter to be smaller in the D-NGI group than in the D-LGI and the C-NGI groups, suggesting a stenosis of the lumen, possibly because of depositions. The ratio of external to internal maximal diameter of the heart vessels was calculated and tended to be higher, although non-significant, in the D-LGI as compared with the D-NGI group (P=0.08) (Figure 2), a finding confirming the visual assessment..
-Moreover, our data from the histological staining with Picrosirius Red suggest that there is a marked increase in all parameters concerning the collagen fibers (namely accumulation, deposition, length, distribution and density) in D-NGI vs. the D-LGI group.The D-LGI group resembles more to the C-NGI group, in respect to the collagen fibers deposited on the cardiac tissue of the rats (Figure 3), although the presence of fibers in the former group is more notable. The mean area occupied by the collagen fibers is larger but does not reach levels of statistical significance, in the D-NGI group as compared with the D-LGI (P=0.07) and the C-NGI (P=0.08) groups, which do not differ (P=0.62) (Figure 3A). The same stands for the mean collagen perimeter which tends to be larger, not reaching levels of statistical significance, in the D-NGI group as compared with the D-LGI (P=0.09) and the C-NGI (P=0.07) groups. D-LGI and C-NGI groups do not differ regarding mean collagen perimeter (P=0.61) (Figure 3B). The mean collagen length in the D-NGI group is higher but not reaching levels of statistical significance, in comparison with the D-LGI (P=0.09) and the C-NGI (P=0.08) groups. There was no difference between these two groups (D-LGI vs control, P=0.83) (Figure 3C).
-The patchy collagen deposition on the other hand, seems to be lower in a statistically significant way in D-NGI than in D-LGI (P=0.04) and the C-NGI (P=0.01) groups and again the two latter exhibit the same staining pattern (P=0.73) (Figure 3D), which leads to the conclusion that the fibers are more focal and grouped together in the D-NGI specimens. However the last parameter assessed, the collagen density, reveals that the grouping of fibers does not necessary means a packed arrangement of the fibers, since the value is lower in D-NGI and the D-LGI (P=0.73), with no significant difference between them, but a marked difference with the C-NGI (D-NGI vs C-NGI and D-LGI vs C-NGI; P=0.04 and P=0.02, accordingly) (Figure 3E). This parameter seems more diabetes specific rather than diet specific, compared to all the previous measured.
-== Discussion ==
-Cardiac failure due to diastolic ventricular dysfunction is a characteristic of diabetic cardiomyopathy and can occur during the early stages of diabetes. In particular, reactive oxygen species (ROS) induce the formation of reactive electrophilic carbonyl species by reacting with lipids and sugars which in turn react with proteins forming irreversible adducts (AGEs, ALEs and EAGLEs) and cross-links [12]. The vascular wall matrix then becomes less distensible, as the formation of these byproducts induces increase in collagen capacity to resist normal turnover. By this mechanism, myocardial fibrosis caused by metabolic abnormalities is considered to be the initial change associated with diabetic cardiomyopathy [13, 14].
-Thus, the possibility was raised that collagen deposition within the cardiac interstitium may be a primary factor contributing to the development of myocardial dysfunction. Studies in human and animal models have demonstrated that changes in myocardial collagen network in ischemic and non-ischemic cardiomyopathy play a major role in the development of ventricular dysfunction [15]. Collagen accumulation is also related to the clinical severity of heart failure, the degree of hemodynamic impairment, hyponatremia, and the need for heart transplantation [16]. In contrast, hypertensive patients treated with angiotensin-converting enzyme inhibitors or angiotensin receptor blockers have shown a reduction in myocardial fibrosis and ventricular hypertrophy, regardless of the decrease in blood pressure, with improvement in systolic and diastolic ventricular function, cardiac arrhythmias, and clinical symptoms, as well as reduced ventricular mass [6].
-It has been long postulated that obesity caused in chemically induced diabetic mice is a negative predisposition factor for cardiac infraction [17]. Recent research reveals that dietary modification such as increasing intake of fiber can improve insulin sensitivity, glucose tolerance and other metabolic disturbances associated with diabetes [18]. This finding is in agreement with studies in humans that show the increased myocardial fibrosis and CML expression, cardiomyocyte hypertrophy, and altered microvasculature structure previously described in diabetic heart disease were a consequence, rather than an initiating cause, of cardiac dysfunction [19].
-Moreover in experimental models of STZ-diabetic rats, oxidative stress and AGE hyper production are maintained through a 6-week period of hyperglycemia. In addition profibrogenic gene expression and increase in ECM deposition, eventually leading to cardiac dysfunction, have been observed. Hyperglycemia has also been found to induce expression of CTGF, which precedes accumulation of ECM [4].
-Since STZ-induced diabetic rats represent a well documented model of experimental type Ι diabetes, we used this animal model of diabetes mellitus type I in order to investigate collagen deposition in the cardiac tissue and gain insight on the possible mechanism of diabetic cardiomyopathy.
-Streptozotocin is reported to induce serum glucose levels alterations in a short period after its administration in Wistar rats [20]. In our study the elevation in serum glucose levels was observed in all treated animal in a 5-days period, following STZ administration.
-The glycemic index of the diet in our study, greatly influenced serum glucose levels in the STZ-treated rats. Serum glucose levels in Group D-LGI were lower at the end of the experimental period in comparison to the other diabetic rat group fed with a normal diet.
-The modifications of the glycemic index of the diet are considered as an effective tool for the dietary management and prevention of diabetes [21]. In accordance to our findings which indicate beneficial effects of LGI diet on serum glucose and body weight levels, the daily incorporation of LGI carbohydrates in meal planning is reported to have a significant impact on both glycemic control and weight management [22].
-The postprandial blood glucose concentrations in patients with diabetes mellitus type I treated with a low–GI and high-fiber diet for a 24-week period resulted in improved serum glucose levels as compared to patients following a high-GI and low-fiber diet [23].
-In another study, the replacement of a wheat starch diet (high glycemic index) with a mung-bean starch diet (LGI) for 5 weeks resulted in decreased non-fasting plasma glucose in normal but not in diabetic rats [24].
-In our study, serum total cholesterol levels at the 60th day of the study for both D-NGI and D-LGI groups did not differ as compared with their baseline levels, while serum triglyceride levels were reduced in both diabetic groups in comparison to their initial values. Some studies report that streptozotocin administration in rats induce a decrease in serum total cholesterol levels some hours after its intraperitoneal administration, while alleviating serum hepatic enzymes levels [25]. Other studies noticed that three administrations of low dose STZ (40 mg/Kg) in CD1 albino male mice resulted in significantly decreased serum cholesterol levels in diabetic as compared with the non-diabetic animals, 28 days following the STZ administration [26].
-On the contrast, other studies have shown significant increase in serum total cholesterol, triglyceride, LDL cholesterol, VLDL cholesterol and significant decrease in serum HDL cholesterol in STZ or alloxan induced diabetic rats [20].
-Low glycemic index diets are reported to reduce plasma free fatty acid levels, triglyceride levels and adipocyte volume in both normal and diabetic rats [24]. In addition, selecting low glycemic index foods has also demonstrated benefits for healthy persons in terms of post-prandial lipid metabolism [27]. Similar to the above conclusions, the administration of a low glycemic index in diabetic rats in our study resulted in an improvement in serum total cholesterol and triglyceride levels in the LGI treated group, as compared with the diabetic rats receiving a diet of normal glycemic index.
-It has been suggested that long-standing diabetes is accompanied by deposition of collagen in the myocardial interstitium. Tang et al. observed that high glucose levels promote the production of collagen types I and III in cardiac fibroblasts [28]. Myocardial fibrosis, which arises from enhanced collagen production in cardiac fibroblasts, is involved in the pathogenesis of diabetic cardiomyopathy [29]. In our study, parameters involving the area occupied, the perimeter covered and the length of the fibers tended to be increased in the diabetic group that received normal diet, in respect to the control group and the diabetic group receiving low glycemic index diet. Our outcomes indicate that collagen deposition that maybe caused due to increased collagen production, is enhanced in metabolically impaired subjects that have not adjusted their diet accordingly. Moreover low glycemic index diet can remedy in part for the diabetic status permitting a rather normal collagen deposition, thus compensating for the impairment.
-In order to strengthen our results and reach levels of statistical significance in all measurements, further investigations may be required, probably by increasing the number of animals to be studied or by expanding the experimental period.
-Interstitial and perivascular fibrosis is a histological hallmark of DCM, and pathological hypertrophy of cardiomyocytes often accompanies it [30]. According to our results in diabetic animals receiving a normal glycemic index diet, not only the quantitative portion of collagen deposition has been increased but there is also a qualitative difference in the distribution of collagen fibers. The patchiness of collagen deposition, depicting the area distribution of collagen, is diminished in the D-NGI group, suggesting that the collagen deposition occurs focally and in a more localized fashion. However, this finding does not imply that the arrangement of collagen is more compact in the areas of grouping, since the density parameter is also decreased.
-Taken together, we could conclude that collagen covers a larger mean area, with larger perimeter covered, but stays focal without increasing in density, most probably because of the longer fibers produced. This is a rather direct evidence of redistribution and remodeling of collagen in diabetic subjects and diet severely influences the pattern.
-Decreased glycogen accumulation in the diabetic tissues may be an indication of impaired energy reserves coupled with reduced functional capacity. Recent studies have reported that decreased glycogen levels in the diabetic heart can be considered as a marker for developing heart dysfunction consequent to induction of diabetes. In this context, other studies suggested a direct association between hyperglycemia and deleterious changes in diabetic myocardium, including myocytes hypertrophy, vascular fibrosis and increased collagen deposition [31]. In our study, the glucose prominent increase of rats, belonging to the D-NGI group as compared with the D-LGI group, is well correlated to the fiber count and total area covered that is in accordance to the already known published data in both humans and mice [32].
-Abnormalities in the lipid metabolism have been described in cardiomyopathy in which the rate of free fatty acid uptake by myocardium is inversely proportional to the severity of the myocardial dysfunction [33].Elevated lipid levels are suggested as one of the major contributing factors in the pathogenesis of cardiomyopathy in diabetic subjects. Ii is quite interesting that in our study the increased myocardial collagen deposition is not accompanied by disturbed serum cholesterol and triglyceride levels. The increase in serum glucose levels may be mainly involved in the metabolic pathways leading to the histological alterations of cardiac tissue.
-In concluding, the glycemic index of diet seems to affect the collagen deposition on the myocardial tissue, in STZ-induced diabetic rats. Expanding these findings, we could infer that LGI diets may benefit both diabetic and non-diabetic persons, since collagen accumulation is an important factor in cardiopathy, regardless the diabetic status of patient.
-== Acknowledgements ==
-The authors would like to thank E. Ntousi and P. Tsakiropoulos for their valuable contribution and technical assistance.
-== Conflict of Interests ==
-The authors declare that there is no conflict of interests regarding the publication of this paper. This research did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector.