Metabolic and oxidative stress markers in Wistar rats after 2 months on a high-fat diet

The consumption of diets high in energy (protein, fat and carbohydrates) has become a habit in modern societies despite the reduction in daily energetic needs from 3000 to 2200 kcal [1]. These preferences for high-calorie or high-fat diets (HFDs) lead to an obesogenic environment for humans, increasing the risk of developing diseases associated with metabolic disorders such as obesity and metabolic syndrome (MS). MS has become a public health problem throughout the world and is defined by a combination of several metabolic abnormalities: impaired glucose tolerance or insulin resistance, impaired blood pressure, dyslipidemia (increase in triglycerides and high-density lipoprotein cholesterol), cardiovascular alterations and central obesity [2, 3]. These metabolic disorders have been identified as risk factors for the development of severe complications. In obese patients, ectopic storage of fat occurs in particular organs and tissues that are not normally involved in fat storage, such as cardiovascular tissues including blood vessels [4]. Moreover, fat-engorged adipocytes and fat-laden myocytes are resistant to the signalling effects of insulin, leading to the storage of lipids and triglycerides in several tissues, including the pancreas and liver [5]. The presence of saturated fat in the liver in turn causes insulin resistance in this organ [6, 7], leading to NAFLDs (non-alcoholic fatty liver diseases), particularly steatosis [8]. Other metabolic alterations may also be added to the list of known and identified factors leading to MS, with oxidative stress (OS) as one candidate factor [9]. Close links exist between MS and OS [10‐12] due to an imbalance between pro-oxidant and anti-oxidant species in favour of oxidised entities [13]. Indeed, patients with metabolic syndrome exhibit damage due to OS [14]. Insulin resistance and inflammation increase reactive oxygen species (ROS) production and OS [15] through superoxide anion production and the decreased levels of antioxidative enzymes such as superoxide dismutase and glutathione peroxidase [16]. This leads to end products of lipid peroxidation such as malondialdehyde (MDA) and carbonylated proteins [17] in the plasma and to the accumulation of ROS in blood vessels [18]. Moreover, the accumulation of insulin in MS stimulates lipogenesis and further hepatic glycogen synthesis [19, 20].

Faced with the widespread morbidity induced by MS, scientists are trying to understand the impact of environmental factors on humans using in vivo models. Many animal models of MS have been developed, including spontaneous transgenic models and diet-induced models. The most common spontaneous or transgenic MS models are i) ob/ob mice (autosomal recessive disease) [21, 22]; ii) obese “yellow mice” (autosomal dominant disease) [23]; iii) NZO (New Zealand Obese) obese mice (polygenic disease) [24], and iv) obese Zucker rats [25]. Dietary models combining high-calorie diets with reductions in physical activity are easier and cheaper and reflect the natural history of metabolic syndrome. For example, in the spiny mouse (Acomys cahirinus), a rich sucrose diet induces glucose intolerance and hyperinsulinemia without hyperglycemia or obesity, while a high-lipid diet induces obesity and hyperglycemia [26]. The sand rat (Psammomys obesus) develops obesity and hyperinsulinism associated with glucose intolerance after three months on a high-calorie diet. Moreover, after six months, diet can induce insulin-dependence [27]. HFD is well-known to induce some metabolic disorders, and the consequences of HFD are completely dependent on the composition and duration of the diet [28, 29]. Therefore, these data suggest that it is possible to develop a model of MS by changing the composition of the diet. However, it is important to take the age of the rats in these models into account. The age of Wistar rats appears to be related to the development of metabolic syndrome [3]. Few studies have characterised the metabolic alterations and their consequences during a short-term diet. Therefore, the dual effects of age and the duration of the diet could create a bias in these models.

The aim of this study was to develop a rapid and easy model of MS and to characterise the metabolic and tissue alterations in that model by subtracting age-related effects. MS was induced by feeding young and healthy Wistar rats a HFD containing high levels of lipids. The animals were then followed for two months to understand the impact of HFD over a short period on the appearance of MS and its consequences for metabolic parameters and tissue alterations.

In this study, we developed a rapidly and easily induced model of MS by feeding healthy Wistar rats with a HFD. After only 2 months, this special diet containing seven fold more fat (21%) and almost the same level of carbohydrates (50%) as the common diet (3% and 60%, respectively), provoked an obesogenic environment which in turn caused several metabolic disorders: obesity, insulin resistance, hypertriglyceridemia and widespread alterations in the tissues studied here, including hyperplasia of pancreatic islets and hepatic steatosis. These data correspond with the accepted definition of MS. Moreover, these characteristics were associated with OS in these tissues and in blood vessels, but not in plasma as assessed by lipid peroxidation and protein carbonylation.

The metabolic follow-up showed that short-term HFD induced a significant weight gain in rats after one month, which was confirmed after 2 months. This weight gain correlates well to the literature, where many studies report weight gains in HFD rats treated for different periods [34‐39]. In parallel with this weight gain, HFD rats exhibit increases in abdominal fat mass (data not shown), indicating the development of obesity in these rats. This obesity is accompanied by a significant increase in C-peptide levels in fasting and re-feeding conditions without changes in glycemia but associated with an insulin resistance confirmed by a significant increase of HOMA2. The same profiles were reported in Wistar rats fed for 8 weeks with high-fat, low-carbohydrate diets [39]. Other studies reported both hyperglycemia and hyperinsulinemia after only 7 weeks of a HFD containing higher fat than in our study (30-35%) [35]. This suggests that after 8 weeks of HFD with only 21% fat, we were able to induce hyperinsulinism without any change in glycemia. These results also suggest that to induce hyperglycemia, we should increase the level of fat in our diet to 35%. Furthermore, a diet rich in fat (40%) but poor in carbohydrates (14%) was not sufficient to increase glycemia after 2 months [39]. In fact, short-term MS can be induced by a diet containing at least 20% fat combined with approximately 50% carbohydrates.

In our study, hyperinsulinemia was associated with hypertriglyceridemia, which was characterised by high plasma triglyceride levels in HFD rats. The same profile was also observed by Sasidharan et al.[40] with a diet containing a comparable fat level. Conversely, the HFD induced a dramatic increase in hepatic glycogen storage reflecting the activation of glycogenesis, characteristic of MS. In contrast, de Castro et al.[41] required more time (13 weeks) to observe an increase in glycogen deposition in the liver using a diet with a high level of fat (40%) and a lower level of carbohydrates (21%). Surprisingly, Gauthier and colleagues [42] reported a decrease in hepatic glycogen storage in rats fed with a diet with the same level of carbohydrates (42%) and a higher level of fat (36%) without requiring the same long time period.

Short term HFD also induced important alterations in pancreatic and hepatic tissues and in blood vessels. In the pancreas, islet hyperplasia was observed in HFD rats in response to hyperinsulinism, with observations including enlarged cells, holes and an increase in the islet surface area. The same trend was also reported in mice fed with a diet with a higher level of fat (60%) for 8, 10 and 12 weeks [43‐45] and in rats [46] fed a higher level of fat (60%) for 10 weeks. In the liver, the hepatocytes of HFD rats exhibit small and widespread vacuoles in the cytoplasm which are smaller than the nuclei of hepatic cells. This suggests the presence of microvesicular lipid deposition characterising hepatic steatosis [32]. This accumulation of lipids only occurs at hepatic levels. Vacuoles were also observed in the livers of rats fed with HFD with higher levels of fat (58% and 45%) for nearly the same period of time [34, 47].

HFD rats also exhibited an increase in the quantities of superoxide anions in pancreatic and hepatic tissue and the mesenteric artery as revealed by dihydroethidium (DHE) staining compared to rats fed with the ND. This indicates the development of OS combined with MS, as previously reported [10‐12]. However, OS was only present in tissues, as we reported no effects of short-term HFD plasma OS (protein carbonylation and lipid peroxidation). A longer period of treatment with HFD (more than 2 months) led to the detection of plasma OS due to increases in the levels of oxidised proteins and lipids (data not shown). Some studies reported the appearance of lipid peroxidation in the plasma after 8 weeks [48] or 10 weeks [49] of HFD containing more fat (32 and 36%) than in our study. In contrast, few or no studies have reported the analysis of carbonylated plasma proteins in HFD model rats. Only Cole and colleagues [50] showed no effect of high-fat (55%), low-carbohydrate (16%) diets on mitochondrial proteins after 3 weeks.

The model developed here is essentially linked to the hypercaloric diet with high fat (21%) and high carbohydrate contents (50%), without age-effects. Ghezzi et al.[3] demonstrated that aging causes the spontaneous development of obesity and insulin resistance in adult and mature rats fed with ND. Other studies have also reported aging-related endothelial dysfunction associated with oxidative stress [33]. Moreover, most MS models result in complications that are often irreversible. In contrast, our model leads to reversible tissue alterations (data not shown). In fact, we have shown in our laboratory that the stop of the HFD feeding or the introduction of antioxidant in the diet could reduce tissue oxidative stress, restore normal insulin and decrease lipid accumulation in the liver what makes this model an ideal tool for the development of particular diet or the validation of biological effectiveness of antioxidant compounds.

This study demonstrates that our rapid and simple model of MS induction by feeding young Wistar rats with a HFD is a true model of MS as reported in humans. The model was associated with metabolic disorders, reversible damage, tissue alterations and OS in the pancreas, liver and blood vessels, without age-linked effects. This model presents many advantages, making it a good tool for studies of MS prevention, including studies assessing new therapeutic strategies such as antioxidative molecules.