Obese Zucker rats

Obese Zucker rats are the best known and most widely used animal model of genetic obesity. The fa mutation was discovered in 1961 by Lois Zucker in a cross between Merck M-strain and Sherman rats⁽Reference Zucker and Zucker²⁾. The animals that are homozygous for the fa allele, the fa/fa Zucker rats, better known as obese Zucker rats, become noticeably obese between the third and the fifth week of life. These animals present a mutation in the leptin receptor, which is the molecular base of their characteristic phenotype⁽Reference Chua, Chung and Wupeng^3–Reference Phillips, Liu and Hammond⁵⁾. Leptin is produced by adipose tissue and plays an important role in the central regulation of energy balance⁽Reference Zhang, Proenca and Maffei⁶⁾. This hormone is released into the circulatory system by the adipose tissue in proportion to the amount of lipids stored and acts in the brain on the leptin receptors, determining a decrease in food intake and an increase in energy expenditure⁽Reference Ahima and Flier^7–Reference Palou, Serra and Bonet⁹⁾. A direct or indirect consequence of the lack of a leptin receptors-mediated counter-regulation is that obese Zucker rats display markedly elevated circulating leptin levels compared with their lean counterparts⁽Reference Hardie, Rayner and Holmes^10, Reference Picó, Sánchez and Oliver¹¹⁾. Old classical orexigenic peptides such as neuropeptide Y, galanin, orexins and melanin-concentrating hormone are upregulated in obese Zucker rats⁽Reference Beck^12–Reference Stricker-Krongrad, Dimitrov and Beck¹⁵⁾. Concretely, this strain is characterised by an increased expression of ghrelin both at the peripheral and central levels⁽Reference Beck, Richy and Stricker-Krongrad^16, Reference Beck, Richy and Stricker-Krongrad¹⁷⁾. This fact could be participating in the development of extra weight in the obese Zucker rats.

The obese Zucker rats develop severe obesity associated with hyperphagia, defective non-shivering thermogenesis and preferential deposition of energy in adipose tissue⁽Reference Chua, Chung and Wupeng³⁾. By 14 weeks of life, body composition of the obese Zucker rats is approximately 40 % weight lipid⁽Reference Zucker and Zucker^18–Reference Zucker and Antoniades²⁰⁾. The affected rats develop hyperplasia and adipocyte hypertrophy⁽Reference Johnson, Zucker and Cruce²¹⁾.

In addition to their characteristic obesity, obese Zucker rats present a range of endocrinological abnormalities. In reality, these animals are a widely extended model of insulin resistance, presenting very similar features to those characterising human metabolic syndrome. In fact, as well as resistance to the metabolic actions of insulin, these animals present dyslipidaemia, mild glucose intolerance and hyperinsulinaemia⁽Reference Zucker and Zucker^18–Reference Muller and Cleary²⁵⁾. Hyperinsulinaemia is detectable at 3 weeks and persists throughout the animals' lives, the islets of Langerhans' hypertrophy moderately and increase in number. In addition, the animals present renal damage⁽Reference Kasiske, O'Donnell and Keane²⁶⁾.

At 17 d, obese Zucker rats can already be seen to eat more compared with lean animals from the same litter⁽Reference Stern and Johnson²⁷⁾. Hyperphagia is particularly apparent during the growth period of the obese animals, i.e. during the first 16 weeks of life⁽Reference Vasselli, Cleary and Jen²⁸⁾. Some pharmacological treatments, naloxone⁽Reference Thornhill, Taylor and Marshall²⁹⁾, d-amphetamine and fenfluramine⁽Reference Grinker, Drewnowski and Enns³⁰⁾, acarbose⁽Reference Vasselli, Haraczkiewicz and Maggio³¹⁾ and cholecystokinin⁽Reference Maggio, Haraczkiewicz and Vasselli³²⁾ among others and dietary manipulations have succeeded in reducing hyperphagia in these animals to a varying degree, but have not managed to normalise the obese body composition. Lifelong food intake restriction results in a reduction in these animals' body weight, but the bodies of obese Zucker rats always continue to maintain a proportion of lipids of approximately 50 %. This percentage is much greater than the percentage of lipids found in the bodies of lean littermates (20 %)⁽Reference Cleary, Vasselli and Greenwood³³⁾. We also know that, when energy intake is reduced, these animals respond with a decrease in the number of fat cells rather than a decrease in the volume of these cells⁽Reference Hausman, Fine and Tagra³⁴⁾.

Different studies suggest that the activity of adipose tissue lipoprotein lipase activity, which is significantly correlated with enhanced TAG uptake by adipose tissue, is one of the candidates for the primary lesion produced by the presence of the fa gene in Zucker rats. The increase in this enzyme's activity may correlate with enhanced TAG uptake by adipose tissue⁽Reference Maggio and Greenwood³⁵⁾. Lipase lipoprotein activity, which controls lipid filling of adipocytes, is elevated in 12-d-old animals, in other words well before the animals can be visually identified as obese⁽Reference Gruen, Hietanen and Greenwood³⁶⁾. This change precedes other determining factors of obesity, such as enhanced liver lipogenesis and hyperinsulinaemia⁽Reference Turkenkopf, Olsen and Moray^37–Reference Greenwood³⁹⁾.

The amount of blood per unit of body weight in obese Zucker rats is lower than normal. The plasma of these animals is milky in appearance, as its fatty acid and cholesterol contents are ten and four times greater than normal, respectively. In reality, these rats present a hepatic overproduction of lipoproteins. The increase of lipids and lipoproteins in plasma is also one of the first anomalies to be observed in the rats⁽Reference Zucker^40–Reference Witztum and Schonfeld⁴⁵⁾. They show an increase in VLDL and in HDL but although they present a decrease in the expression of hepatic receptors for LDL, they show no increase in LDL-cholesterol and cannot be used as a model of atherogenesis⁽Reference Liao, Angelin and Rudling⁴⁶⁾. Like other rodents, they have larger amounts of HDL than LDL, but an increase in LDL-cholesterol can be induced in these animals by means of dietary supplements of saturated fats and cholesterol⁽Reference Vaskonen, Mervaala and Seppänen-Laakso⁴⁷⁾. Thus the increase in TAG concentration in plasma exhibited by obese Zucker rats is due to the accumulation of VLDL, and the increase in cholesterol is due to the increase in cholesterol in the VLDL and HDL fractions. The increase in HDL-cholesterol is particularly manifest in the male rats⁽Reference Lin⁴⁸⁾. In fact, in 1985, Lin described clear differences between obese males and females. This researcher showed that the increase in the serum cholesterol of obese females was caused principally by its high content of non-esterified cholesterol associated with VLDL. By contrast, in males, serum cholesterol was chiefly transported as esters of cholesterol with HDL.

These rat glucose levels are in reality normal or only slightly higher than normal. Therefore, these animals are not the best model to study the effective treatments to control alterations of glucose homeostasis. Nevertheless, some researchers have succeeded in identifying several vascular changes characteristic of diabetes in these rats⁽Reference Lash, Sherman and Hamlin⁴⁹⁾. The lipid profile of lean Zucker rats is similar to that of Sprague–Dawley⁽Reference Zucker^40, Reference Barry and Bray⁴¹⁾ and Wistar⁽Reference Schonfeld and Pfleger⁴²⁾ rats. These animals are sensitive to insulin, are normotensive and have a normal glucose tolerance.

The link between obesity and hypertension has been recognised for some time. Several studies have reported conflicting results about whether obese Zucker rats are hypertensive compared with their lean controls⁽Reference Ernsberger and Nelson^50–Reference Kurtz, Morris and Pershadsingh⁶²⁾. Systolic arterial blood pressure in obese rats is lower than that in control lean rats of between 8 and 12 weeks of life. At 24 weeks, the phenomenon goes into reverse, and at 28 weeks, systolic arterial blood pressure in obese rats is significantly higher than in their lean counterparts. With these observations in mind, Kurtz et al. ⁽⁵³⁾ indicated that obese Zucker rats could be considered a model of obesity and hypertension. These animals could constitute an experimental model in which hypertension was specifically associated with the genotype for obesity. The increase in arterial blood pressure in the obese animals is not due to an increase in renal Na retention⁽Reference Kurtz, Morris and Pershadsingh⁶²⁾. The impaired vascular responses to acetylcholine that has been observed in some studies in the oldest obese Zucker rats indicate that endothelial dysfunction could justify, at least in part, the increased arterial blood pressure in these animals⁽Reference Subramanian and MacLeod⁶³⁾. There is evidence for a local angiotensin II-generating system in adipose tissue⁽Reference Harte, McTernan and Chetty^64–Reference Griendling, Sorescu and Ushio-Fukai⁶⁶⁾, implying that the vasoactive component angiotensin II may be produced by adipose tissue. Angiotensin II is a powerful stimulus for the generation of reactive oxygen species in the blood vessels⁽Reference Dzau^67, Reference Unger and Gohlke⁶⁸⁾. This increased oxidative stress may interact with NO function, leading to endothelial dysfunction⁽Reference De Gasparo⁶⁹⁾. Therefore, we can also assume that the increased proportion of adipose tissue in the obese Zucker rats, and consequently the increased production of angiotensin II and reactive oxygen species, could facilitate the development of hypertension and endothelial dysfunction in these animals.

Obesity is also associated with a state of chronic inflammation characterised by abnormal production of proinflammatory mediators⁽Reference Ouchi, Kihara and Funahashi⁷⁰⁾, including TNF-α⁽Reference Hotamisligil, Shargill and Spiegelman^71, Reference Hotamisligil, Arner and Caro⁷²⁾and inducible NO synthase⁽Reference Perreault and Marette⁷³⁾. This inflammatory state is associated with a deficit of energy in the form of ATP⁽Reference Wlodek and Gonzales^74, Reference Boudina, Sena and O'Neill⁷⁵⁾and simultaneous overproduction of fat and leptin, which is accompanied by leptin resistance in the brain⁽Reference Wlodek and Gonzales^74, Reference Munzberg and Myers⁷⁶⁾. Recent studies have shown that fat tissue is not a simple energy storage organ, but exerts important endocrine and immune functions. These are achieved predominantly through the release of several factors termed ‘adipocytokines’, which include several novel and highly active molecules released abundantly by adipocytes like above-mentioned leptin, as well as some more classical cytokines released possibly by inflammatory cell infiltrating fat like, TNF-α, IL-6, monocyte chemotactic protein-1 and IL-1⁽Reference Tilg and Moschen⁷⁷⁾. In this context, TNF-α, a proinflammatory cytokine, is overexpressed in obesity and likely mediates insulin resistance in the major animal models of obesity⁽Reference Hotamisligil, Shargill and Spiegelman⁷¹⁾, including obese Zucker rats⁽Reference Picchi, Gao and Belmadani⁷⁸⁾. Both research groups postulated that overexpression of TNF-α induces the activation of NADPH oxidase and production of superoxide anion leading to endothelial dysfunction in obese Zucker rats.

Obese spontaneously hypertensive rats

The SHR, a well-known experimental model to study hypertension, have been also proposed as a model of insulin resistance. These rats show hypertriacylglycerolaemia, abdominal obesity and hypertension⁽Reference Reaven, Chang and Hoffman^79, Reference Kvetnanský, Rusnák and Gasperíková⁸³⁾. In the background of SHR, different strains of corpulent SHR, such as obese SHR named, Koletsky rats, SHR/N-corpulent rats and SHR/NDmc-corpulent rats, seem to be even more adequate to study the metabolic syndrome than the SHR. The leptin receptor gene is also knocked out in these rats.

Stroke-prone–SHR fatty (fa/fa) rats

Stroke-prone SHR (SHRSP) are a rat model that develops severe hypertension. SHRSP rats develop hypertension-related disorders, such as nephropathy, cardiac hypertrophy and atherosclerosis, similar to human essential hypertension and 100 % die to stroke⁽Reference Yamori, Ohtaka and Horie¹⁰⁰⁾. As SHR rats, SHRSP is also a model of insulin resistance syndrome⁽Reference Reaven, Chang and Hoffman^79, Reference Collins, Rodenbaugh and DiCarlo¹⁰¹⁾. In spite of SHRSP being a good model of hypertension and insulin resistance, SHRSP weigh less than their normotensive control, Wistar–Kyoto rats, and have reduced plasma total cholesterol and NEFA levels. Very recently, Hiraoka-Yamamoto et al. ⁽Reference Hiraoka-Yamamoto, Nara and Yasui¹⁰²⁾ have produced a new animal model of the metabolic syndrome, by introducing a segment of the mutant leptin receptor gene from the Zucker line heterozygous for the fa gene mutation, into the genetic background of the SHRSP. Therefore, a new congenic strain, SHRSP fatty (fa/fa) rats, was derived by replacing the fa locus of chromosome from Zucker (fa/fa) rats. The SHRSP fatty rats are characterised by the spontaneous development of hypertension, obesity, hyperleptinaemia and several metabolic disorders such as hyperlipidaemia and hyperinsulinaemia.

Sterol-regulatory element-binding protein–spontaneously hypertensive rats

The relationship between the metabolic syndrome and non-alcoholic fatty liver disease has recently begun to attract considerable attention⁽Reference Marchesini, Brizi and Bianchi^103–Reference den Boer, Voshol and Kuipers¹⁰⁵⁾. In subjects with clinical features of the metabolic syndrome, the prevalence of non-alcoholic fatty liver disease can be very high even in the absence of diabetes, obesity or abnormal liver enzymes. Moreover, 50 % of subjects with pure fatty liver and up to 90 % of subjects with non-alcoholic steatohepatitis have the metabolic syndrome according to Adult treatment panel III criteria⁽Reference Marchesini, Bianchi and Merli¹⁰⁴⁾. Although insulin resistance can be determinant of fatty liver, it has also been suggested that hepatic steatosis may play a role in the pathogenesis of the metabolic syndrome and promote insulin resistance in liver and skeletal muscle⁽Reference Diehl^106–Reference Samuel, Liu and Qu¹⁰⁸⁾. Some investigators have further proposed that non-alcoholic fatty liver disease may be considered an additional feature of the metabolic syndrome⁽¹²⁰⁾. Therefore, the availability of animal models with hepatic steatosis, as well as insulin resistance, dyslipidaemia and hypertension, could be valuable for studying the pathogenesis and treatment of the metabolic syndrome and its relationship to non-alcoholic fatty liver disease. Very recently, Qi et al. ⁽Reference Horton, Goldstein and Brown¹⁰⁹⁾ have created a non-obese rat model with hypertension, fatty liver and characteristics of the metabolic syndrome by transgenic overexpression of a sterol-regulatory element-binding protein in the SHR rats. Sterol-regulatory element-binding proteins are transcription factors involved in the regulation of fatty acid and lipid metabolism and can activate the expression of multiple genes involved in the hepatic synthesis of cholesterol, TAG, fatty acids and phospholipids⁽Reference Horton, Goldstein and Brown^110, Reference Horton, Shimomura and Ikemoto¹¹¹⁾. This indicates hepatic steatosis and multiple biochemical features of the metabolic syndrome, including hyperinsulinaemia, hyperglycaemia and hypertriacylglycerolaemia in the absence of obesity. The sterol-regulatory element binding protein–SHR model could therefore provide valuable opportunities for investigating pathogenetic mechanisms that may relate fatty liver disease to the metabolic syndrome.

Wistar Ottawa Karlsburg W rats

In 1995, a new inbred rat strain was developed, termed Wistar Ottawa Karlsburg W (WOKW) rats. These animals derived from a Wistar rat outbred strain of the BioBreeding Laboratories (Ottawa, Ont., Canada). The WOKW strain provides a good animal model expressing the metabolic syndrome. It is especially useful because their metabolic syndrome is under polygenic control, as in human subjects, and not due to a single-gene mutation⁽Reference Filippetti, Kloting and Massi¹¹²⁾. The dark agouti rats are usually used as control animals of WOKW⁽Reference van den Brandt, Kovács and Klöting¹¹³⁾. WOKW compared with dark agouti rats show hyperphagia, and are heavier and fatter. Segregating populations derived from this strain and inbred dark agouti rats have been successfully used to identify quantitative trait loci for major components of the metabolic syndrome, such as insulin resistance on WOKW chromosome 3 and hypertriacylglycerolaemia on WOKW chromosomes 4 and 6⁽Reference van den Brandt, Kovács and Klöting^114, Reference Klöting, Kovács and van den Brandt¹¹⁵⁾. The WOKW develops a nearly complete metabolic syndrome with obesity, moderate hypertension, dyslipidaemia, hyperinsulinaemia and impaired glucose tolerance⁽Reference van den Brandt, Kovács and Klöting^114, Reference Kovács, van den Brandt and Klöting^116, Reference Klöting, Vogt and Serikawa¹¹⁷⁾. A cross-sectional comparative study indicated that the WOKW rat begins to manifest the signs of the metabolic syndrome between 8 and 10 weeks of age⁽Reference van den Brandt, Kovács and Klöting¹¹³⁾. Very recently, the metabolic syndrome in WOKW rats has been also associated with coronary dysfunction⁽Reference Grisk, Frauendorf and Schlüter¹¹⁸⁾. The dark agouti strain does not show any of these characteristics and has been considered as the control strain for the WOKW rats⁽Reference Filippetti, Kloting and Massi^112, Reference van den Brandt, Kovács and Klöting¹¹³⁾.

Low-capacity runner rats

Very recently, Wisløff et al. ⁽Reference Wisløff, Najjar and Ellingsen¹¹⁹⁾ have generated an animal model of the metabolic syndrome. To obtain this model, rats were selectively bred based on their ability to perform on a treadmill endurance running task. Accordingly, rats that have a high intrinsic aerobic capacity and are capable of running comparatively long distances are classified as high-capacity runner rats and are bred together. On the other hand, rats with a low intrinsic aerobic capacity that are only capable of running relatively short distances are classified as low-capacity runner (LCR) rats and are bred with each other. Eleven generations of selective breeding resulted in elevated blood pressure in LCR rats when compared with high-capacity runner rats. The LCR rats also show endothelial dysfunction, insulin resistance and hyperinsulinaemia, visceral adiposity, hypertriacylglycerolaemia and elevated plasma NEFA. Therefore, one advantage of this new experimental model is that elevated blood pressure in the LCR rats occurs together with the other hallmarks of the metabolic syndrome⁽Reference Wisløff, Najjar and Ellingsen¹¹⁹⁾.