Background

The etiology of the metabolic syndrome (MS) includes both genetic and environmental factors. The two most commonly studied animal models of the MS are the high-sucrose diet given to spontaneously hypertensive rats (SHRs) and high-fructose diet given to Sprague Dawley rats (SDRs). This study compares between these two models.

Methods

The two rat strains were examined; within each group, the rats were assigned to either the high-sugar diet (SDRs with fructose-enriched diet and SHRs with sucrose-enriched diet) or standard rat chow (control group). The rats were followed for 7 weeks. The main MS components (obesity, hypertension, impaired glucose tolerance, hyperinsulinemia, hypertriglyceridemia, and hypercholesterolemia) were measured.

Results

At baseline systolic blood pressure (SBP), fasting blood levels of triglycerides and insulin, as well as glucose intolerance, were significantly higher among the SHRs compared to SDRs. Following fructose enrichment, SDRs became hyperinsulinemic, hypertriglyceridemic, hypercholesterolemic, hypertensive, and insulin resistant, whereas SHRs responded to sucrose supplementation by a significant elevation in blood pressure and mild worsening of insulin resistance. Endpoint results revealed superiority of sucrose—SHR model in terms of hypertension and superiority of fructose—SDR model in terms of hyperinsulinemia, hypertriglyceridemia, and hypercholesterolemia. Both models showed similar postintervention degree of glucose tolerance.

Conclusions

The fructose-fed SDR model represents a predominantly environmentally acquired MS, whereas the SHR model is less affected by dietary intervention and better displays the predominantly genetic spontaneous appearance of the syndrome. This fundamental difference should be taken into consideration when choosing an animal model to study the MS.

Methods

Animals, diets, and study design. An interventional comparative study was designed. The experiments were conducted according to the Guidelines for Animal Care and Treatment of the hospital's Animal Ethics Committee.

Twenty male SDRs and 16 male SHRs (Harlan, Israel) weighting 200 20 g were maintained in a temperature-controlled room (22 °C) and kept on a 14/10 h light–dark cycle. Food and water were available ad libitum.

Rats were divided to four groups as follows: (i) 10 SDRs were fed high-fructose (SDR-F) diet and (ii) 10 SDRs were fed standard chow diet (SDR-C) and served as the control group for the SDR-F; (iii) eight SHRs were fed high-sucrose (SHR-S) diet and (iv) eight SHRs were fed standard chow diet (SHR-C) and served as the control group for the SHR-S.

The standard chow diet (Koffolk, Israel) was composed of 50% starch, 21% protein, 4% fat, 4.5% cellulose, and standard vitamins and mineral mix. The high-fructose diet (Harlan Teklad, Madison, WI) was composed of 60% fructose, 21% protein, 5% fat, 8% cellulose, and standard vitamins and mineral mix. The high-sucrose diet was given through 12% sucrose dissolved in the drinking water. The rats were followed up for 7 weeks.

The blood concentrations of triglycerides, insulin, and total cholesterol, as well as systolic blood pressures (SBPs) and glucose tolerance, were monitored at baseline, midpoint (25 days), and at the end of the study.

SBP was measured in conscious rats using the tail cuff technique (Narco Biosystems, Houston, TX). Rats were prewarmed at 37 °C for 30 min before measurements were taken. The mean of five consecutive readings was recorded as SBP. Blood samples were taken from the retro-orbital sinus under light anesthesia with isofluran inhalation for a few seconds. Samples for insulin, triglycerides, and total cholesterol were taken after 5 h of fasting. The plasma was separated and aliquots were stored frozen until tested.

Plasma insulin levels were assayed using RIA kit (DiaSorin, Saluggia, Italy). Triglycerides and total cholesterol levels were assayed with an automated analyzer of an enzymatic colorimetric reaction (Olympyus AU 270, Hamburg, Germany).

Glucose tolerance was tested using oral glucose tolerance test (OGTT). After 12 h fasting, blood glucose was measured using a standard glucometer (Bayer, Leverkusen, Germany). Immediately after this baseline measurement, a hydrous solution of 3.5 g glucose/kg body weight was administered using gastric gavages. Blood glucose levels were measured 5, 10, 15, 30, 60, and 120 min after the glucose load.

Data analysis. Results are presented as mean s.d. Statistical differences between series of data were assessed by two-way paired and unpaired Student's t-tests for group comparison. P values of <0.05 were considered significant.

Results

SDR-fructose model

Feeding SDRs with fructose-enriched diet resulted in a significant increase in SBP, as well as in plasma concentrations of insulin, triglycerides, and total cholesterol (Table 1). In addition, the fructose-fed SDRs displayed impaired glucose tolerance as was expressed by a significant prolonged elevated level of blood glucose following a standard load (Figure 1a, b).

Figure 1.

Effect of dietary intervention on glucose tolerance. (a) Blood glucose levels of the various study groups as a function of time after glucose load and (b) calculated area under the OGTT curve.

Full figure and legend (17K)

Table 1 - Physiological and metabolic components of metabolic syndrome (average s.d.) before and after diet intervention.

Full table (53K)

Control SDR group did not show significant changes in blood pressure, insulin, triglycerides, total cholesterol, and glucose tolerance during the study period (Table 1).

Similar body weight gain was observed among SDR-F and SDR-C groups along the study period (SDR-F from 200.6 5.8 to 313.3 9.1 g, SDR-C from 207.8 2.2 to 306 6.4 g, not significant).

SHR-sucrose model

Enrichment of SHR diet with sucrose did not significantly influence body weight gain, plasma insulin levels, and plasma triglycerides (Table 1). Sucrose-enriched diet increased SBP in SHRs by 22 mm Hg (P = 0.002), whereas standard chow diet increased SBP by only 8.5 mm Hg. The BP increase in the control SHRs is typical for that strain, implying that the increased SBP in SHR-S was only partially contributed by the sugar addition (Table 1). Nevertheless, SBP values of SHR-S at the end of the study were found to be significantly higher than those of SHR-C (P = 0.026), indicating that the dietary maneuver had a significant net effect.

Impaired glucose tolerance was found in sucrose-fed SHRs. OGTT results, expressed as blood glucose concentration as a function of time after glucose load, were higher in SHR-S than in SHR-C (Figure 1a,b).

Total cholesterol levels were found to be slightly, but significantly elevated in both SHR-S and SHR-C groups at the endpoint compared to baseline (Table 1).

Body weight was not significantly affected by sucrose enrichment (Table 1).

Comparison between models

When looking at the metabolic changes obtained by the investigated models, it is obvious that the fructose-SDR model reacted to the dietary intervention. A significant deleterious effect was found on five of six parameters that were measured (SBP, insulin, triglycerides, total cholesterol, and glucose AUC during OGTT). Apparently, the sucrose-SHR model resulted in a limited metabolic deterioration effect since only two of six parameters showed a significant increase compared to control group (SBP and glucose AUC during OGTT).

Regarding the degree of changes, sugar-related SBP elevation was similar in the two models (15 mm Hg), but as for the other metabolic components (triglycerides, insulin, and glucose AUC), fructose-SDR model displayed more intensive response to the dietary manipulation compared with sucrose-SHRs. Thus, insulin elevation was larger in the SDR-F group compared with the SHR-S group (100 22% vs. -11 3%, respectively, P < 0.001), as was the increase in triglycerides (349 23% vs. 30 16%, respectively, P < 0.001). Area under the curve obtained following glucose load was 19.6% higher in SDR-F compared with SDR-C and only 6.3% higher in SHR-S compared with SHR-C group (Figure 2).

Figure 2.

Percentage of change in the various metabolic parameters in SDR-F and SHR-S models.

Full figure and legend (9K)

However, it should be noted that at baseline, most of the MS-related components were significantly higher in the spontaneous hypertensive rats than in the SDRs, regardless of the dietary intervention, and that spontaneous hypertensive rats were also spontaneously hyperinsulinemic as well as insulin resistant (Table 1, Figure 1a,b). Nonetheless, analysis of endpoint results between the two sugar-treated groups reveals superiority of SHR-C model in terms of hypertension and superiority of SDR-F model in terms of hyperinsulinemia, hypertriglyceridemia, and hypercholesterolemia. Both models showed similar postintervention degree of glucose tolerance.

Discussion

Being a global epidemic that is accompanied by hazardous consequences, MS has been recognized by an expert panel of the National Cholesterol Education Program as a target for risk reduction therapy.²⁰ Understanding the pathophysiology of the MS and investigating ways to prevent and treat it is an important challenge to the medical science in the near future.

An indispensable method to expand our clinical knowledge is to use animal models that mimic the investigated disease. Because the MS comprises several elements and is thought to be a result of genetic and epigenetic factors, transgenic animal models may not fully address this need. Preferred animal models for the MS are those that appropriately combine genetic tendency with nutritional intervention to induce the characteristic manifestation of the syndrome, namely, hypertension, impaired glucose metabolism, and dyslipidemia. In view of that, it is important to characterize and compare the commonly used animal models of the MS to determine which the optimal model is, and to point to the weak and strong elements in each model. Such comparison will enable rational choice between the available models to study a particular aspect of the MS.

Our data indicate that both investigated models manifest the major characteristics of the MS. Fructose-enriched diet given to SDRs clearly induce the MS in those animals with coappearance of hypertension, hypertriglyceridemia, and decreased insulin sensitivity as reflected by the combination of elevated serum insulin and impaired OGTT. SHR strain spontaneously represents moderate MS, whereas the sucrose enrichment had small additive deleterious effect: a further elevation in blood pressure and mild worsening of insulin resistance.

Comparison between the models indicates that giving sucrose to SHRs is less effective in inducing the MS compared with giving high-fructose diet to apparently normal SDRs. Two fundamental differences between these models should be analyzed while trying to interpret these results. First, is the use of normotensive and nonglucose tolerant (although susceptible) animals (SDRs) vs. animals showing spontaneous hypertension and impaired glucose tolerance prior to diet intervention (SHRs), and the second is the difference in type of carbohydrate (fructose vs. sucrose). Recently, few researchers administered sucrose solution to normal Wistar rats for several weeks to establish MS.^21,22,23,24 This new model may help to elucidate the question whether the use of preaffected animals in SHR-S model is the key factor that caused the less intensive MS compared with SDR-F, or alternatively, is sucrose a less potent trigger than fructose in terms of inducing the MS? Unfortunately, results obtained using this model are not unified: in response to sucrose, Amamoto et al.²² found hyperinsulinemia, whereas Lombardo et al.²³ reported that insulin level remain unchanged. Moreover, none of these studies measured blood pressure, thus this central component of the MS is not comparable. In addition, major methodological differences regarding concentration of sucrose (15–63%) and duration of intervention (2–30 weeks) complicate the evaluation of this model. Few studies looked at the effects of chronic (>10 weeks) fructose diet given to SHRs with conflicting results. Yoshida et al. showed an increase in SBP, whereas Girard et al. showed no change in SBP despite impairment of glucose metabolism.^25,26

Kanarek and Orthen-Gambill compared the effects of identical concentrations of glucose, fructose, and sucrose given to SDRs on lipids and glucose metabolism.²⁷ This study, conducted before the term MS was introduced, lasted for 7 weeks, similar to our follow-up, and found higher triglycerides levels in rats fed with fructose than in rats fed with sucrose. Both groups gained similar body weight during the experiment. In addition, similar fasting insulin levels and similar reaction toward glucose load in OGTT were observed in fructose and sucrose-fed animals. Our results are in accordance with this study and also with another study that compared the effects of sucrose supplement to SHRs and to its native normotensive control strain Wistar–Kyoto (WKY) rats.²⁸ This study revealed that SHRs have higher triglycerides level than WKY, and that both strains reacted to sucrose addition by similar mild elevation of triglycerides. Neither SHRs nor WKY significantly elevated total cholesterol in response to sucrose enrichment. At baseline, SHRs, but not WKY, were insulin resistant and hyperinsulinemic, as reflected by HOMA-IR index and serum insulin level, and this was further worsened by sucrose consumption.

Collectively, these two studies, together with our data, strongly suggest that the differences between SDR-F and SHR-S models stem from the combination of different action of sugars and genetic differences between rat strains. Our study extends and completes the former studies by measuring blood pressure, an important component of MS, and by the direct comparison of the two most commonly used models. We cannot conclusively point to a superiority of one model over the other but rather typify the models to a predominantly environmental (SDR-F) and predominantly genetic (SHR-C) model and thus allow logical selection of the appropriate model to a particular study.

The issue of choosing between predominantly environmental and predominantly genetic model of the MS is important. The mechanism of insulin resistance in the various models is closely linked to the applicability of an animal model to the clinical setting for which the model is used.²⁹ Some reports suggest that dietary factors play a central role in the development and consequences of MS. A large body of evidence points out a linkage between the increased fructose consumption in the western world diet and the parallel elevation of the MS prevalence. On the other hand, some experts point to the genetic tendency as the most important factor that underlies the MS and its outcome. Studies of familial aggregates, candidate genes, and genome wide linkage scans point to a genetic predisposition as an essential element in the development of MS and its consequences.³⁰ Indeed, major characteristics of the MS are expressed clearly in genetic models of the MS.^31,32

In summary, in this study we compared the two standard animal models that mimic the human MS with its hemodynamic and metabolic components. Both induced MS but the two models differ in the dominant feature of the syndrome. The fructose-fed SDR model presents a predominantly environmentally acquired MS, whereas the SHR model is less affected by dietary intervention and better displays the predominantly genetic spontaneous appearance of the syndrome. Future research in that field should take into account these differences between the models and accordingly select the proper animal model that addresses particular aspects of the human MS.

Disclosure:

The authors declared no conflict of interest.

References

1. Reaven GM. Banting lecture 1988. Role of insulin resistance in human disease. Diabetes 1988; 37:1595–1607. | Article | PubMed | ISI | ChemPort |
2. Meigs JB. Invited commentary: insulin resistance syndrome? Syndrome X? Multiple metabolic syndrome? A syndrome at all? Factor analysis reveals patterns in the fabric of correlated metabolic risk factors. Am J Epidemiol 2000; 152:908–911; discussion 912. | Article | PubMed | ISI | ChemPort |
3. Meigs JB, Rutter MK, Sullivan LM, Fox CS, D'Agostino RB Sr, Wilson PW. Impact of insulin resistance on risk of type 2 diabetes and cardiovascular disease in people with metabolic syndrome. Diabetes Care 2007; 30:1219–1225. | Article | PubMed | ChemPort |
4. Ford ES, Giles WH, Mokdad AH. Increasing prevalence of the metabolic syndrome among U.S. adults. Diabetes Care 2004; 27:2444–2449. | Article | PubMed | ISI |
5. Isomaa B. A major health hazard: the metabolic syndrome. Life Sci 2003; 73:2395–2411. | Article | PubMed | ChemPort |
6. Reaven GM. Syndrome X: 6 years later. J Intern Med Suppl 1994; 736:13–22. | PubMed | ChemPort |
7. Johnson RJ, Segal MS, Sautin Y, Nakagawa T, Feig DI, Kang DH, Gersch MS, Benner S, Sánchez-Lozada LG. Potential role of sugar (fructose) in the epidemic of hypertension, obesity and the metabolic syndrome, diabetes, kidney disease, and cardiovascular disease. Am J Clin Nutr 2007; 86:899–906. | PubMed | ChemPort |
8. Bray GA, Nielsen SJ, Popkin BM. Consumption of high-fructose corn syrup in beverages may play a role in the epidemic of obesity. Am J Clin Nutr 2004; 79:537–543. | PubMed | ISI | ChemPort |
9. Akinyanju PA, Qureshi RU, Salter AJ, Yudkin J. Effect of an "atherogenic" diet containing starch or sucrose on the blood lipids of young men. Nature 1968; 218:975–977. | Article | PubMed | ChemPort |
10. Bantle JP, Raatz SK, Thomas W, Georgopoulos A. Effects of dietary fructose on plasma lipids in healthy subjects. Am J Clin Nutr 2000; 72:1128–1134. | PubMed | ISI | ChemPort |
11. Crapo PA, Reaven G, Olefsky J. Plasma glucose and insulin responses to orally administered simple and complex carbohydrates. Diabetes 1976; 25:741–747. | Article | PubMed | ChemPort |
12. Faeh D, Minehira K, Schwarz JM, Periasamy R, Park S, Tappy L. Effect of fructose overfeeding and fish oil administration on hepatic de novo lipogenesis and insulin sensitivity in healthy men. Diabetes 2005; 54:1907–1913. | Article | PubMed | ChemPort |
13. Blouet C, Mariotti F, Mathe V, Tome D, Huneau JF. Nitric oxide bioavailability and not production is first altered during the onset of insulin resistance in sucrose-fed rats. Exp Biol Med (Maywood) 2007; 232:1458–1464. | Article | PubMed | ChemPort |
14. Sharabi Y, Oron-Herman M, Kamari Y, Avni I, Peleg E, Shabtay Z, Grossman E, Shamiss A. Effect of PPAR- agonist on adiponectin levels in the metabolic syndrome: lessons from the high fructose fed rat model. Am J Hypertens 2007; 20:206–210. | Article | PubMed | ChemPort |
15. Gallou-Kabani C, Junien C. Nutritional epigenomics of metabolic syndrome: new perspective against the epidemic. Diabetes 2005; 54:1899–1906. | Article | PubMed | ChemPort |
16. Kovacs P, van den Brandt J, Kloting I. Genetic dissection of the syndrome X in the rat. Biochem Biophys Res Commun 2000; 269:660–665. | Article | PubMed | ChemPort |
17. Elkayam A, Mirelman D, Peleg E, Wilchek M, Miron T, Rabinkov A, Oron-Herman M, Rosenthal T. The effects of allicin on weight in fructose-induced hyperinsulinemic, hyperlipidemic, hypertensive rats. Am J Hypertens 2003; 16:1053–1056. | Article | PubMed | ChemPort |
18. Erlich Y, Rosenthal T. Effect of angiotensin-converting enzyme inhibitors on fructose induced hypertension and hyperinsulinaemia in rats. Clin Exp Pharmacol Physiol Suppl 1995; 22:S347–S349. | Article | PubMed | ChemPort |
19. Pagliassotti MJ, Shahrokhi KA, Moscarello M. Involvement of liver and skeletal muscle in sucrose-induced insulin resistance: dose-response studies. Am J Physiol 1994; 266(5 Pt 2):R1637–R1644.
20. Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, And Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III). JAMA 2001; 285:2486–2497. | Article | PubMed | ISI |
21. Aguilera AA, Diaz GH, Barcelata ML, Guerrero OA, Ros RM. Effects of fish oil on hypertension, plasma lipids, and tumor necrosis factor- in rats with sucrose-induced metabolic syndrome. J Nutr Biochem 2004; 15:350–357. | Article | PubMed | ChemPort |
22. Amamoto T, Kumai T, Nakaya S, Matsumoto N, Tsuzuki Y, Kobayashi S. The elucidation of the mechanism of weight gain and glucose tolerance abnormalities induced by chlorpromazine. J Pharmacol Sci 2006; 102:213–219. | Article | PubMed | ChemPort |
23. Lombardo YB, Hein G, Chicco A. Metabolic syndrome: effects of n-3 PUFAs on a model of dyslipidemia, insulin resistance and adiposity. Lipids 2007; 42:427–437. | Article | PubMed | ChemPort |
24. Torres IP, El Hafidi M, Zamora-Gonzalez J, Infante O, Chavira R, Banos G. Modulation of aortic vascular reactivity by sex hormones in a male rat model of metabolic syndrome. Life Sci 2007; 80:2170–2180. | Article | PubMed | ChemPort |
25. Girard A, Madani S, Boukortt F, Cherkaoui-Malki M, Belleville J, Prost J. Fructose-enriched diet modifies antioxidant status and lipid metabolism in spontaneously hypertensive rats. Nutrition 2006; 22:758–766. | Article | PubMed | ChemPort |
26. Yoshida K, Kawamura T, Xu HL, Ji L, Mori N, Kohzuki M. Effects of exercise training on glomerular structure in fructose-fed spontaneously hypertensive rats. Hypertens Res 2003; 26:907–914. | Article | PubMed | ChemPort |
27. Kanarek RB, Orthen-Gambill N. Differential effects of sucrose, fructose and glucose on carbohydrate-induced obesity in rats. J Nutr 1982; 112:1546–1554. | PubMed | ChemPort |
28. Umeda M, Kanda T, Murakami M. Effects of angiotensin II receptor antagonists on insulin resistance syndrome and leptin in sucrose-fed spontaneously hypertensive rats. Hypertens Res 2003; 26:485–492. | Article | PubMed | ChemPort |
29. Shimamoto K, Ura N. Mechanisms of insulin resistance in hypertensive rats. Clin Exp Hypertens 2006; 28:543–552. | Article | PubMed | ChemPort |
30. Teran-Garcia M, Bouchard C. Genetics of the metabolic syndrome. Appl Physiol Nutr Metab 2007; 32:89–114. | Article | PubMed | ChemPort |
31. Yamaguchi Y, Yoshikawa N, Kagota S, Nakamura K, Haginaka J, Kunitomo M. Elevated circulating levels of markers of oxidative-nitrative stress and inflammation in a genetic rat model of metabolic syndrome. Nitric Oxide 2006; 15:380–386. | Article | PubMed | ChemPort |
32. Kloting N, Bluher M, Kloting I. The polygenetically inherited metabolic syndrome of WOKW rats is associated with insulin resistance and altered gene expression in adipose tissue. Diabetes Metab Res Rev 2006; 22:146–154. | Article | PubMed | ChemPort |