Animals

An interventional comparative study was designed. The experiments were conducted according to the Guidelines for Animal Care. The Animal Care Ethics Committee approved the study protocol and issued an ethical clearance (No. 346 of 28.02.2023) from the Bulgarian Food Safety Agency.

The rats were housed, maintained and euthanized in accordance with the relevant international rules and recommendations as outlined in the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (ETS 123).

Sixteen male Wistar rats at three months of age (200–300 g) were used. Rats were housed in Plexiglas cages (4 per cage) in a 12/12 light/dark cycle under standard laboratory conditions (ambient temperature 20 ± 2 °C and humidity 72 ± 4%). All animals were purchased from the National Breeding Center, Sofia, Bulgaria and allowed a minimum of 7 days to acclimate to the new conditions before the start of the study. Food and fresh drinking water were available ad libitum throughout the experimental period of 10 weeks.

The rats in the control group were fed a standard diet appropriate to their species and age, and had access to clean drinking water in unlimited quantities. The diet of rats in the HFD group was prepared by mechanically mixing standard rodent chow with 17% lard. The rats of the third group (HF-HC) received a diet formulated by the Department of Pharmaceutical Technology. The fourth group of rats was fed a so-called cafeteria diet, which included a variety of packaged foods such as wafers, corn balls, fried peanuts, potato chips, and others purchased from the supermarket. The nutrient composition of CFD and HFD is presented in Table 1.

The HF-HCD (Table 2) was prepared by moulding from a melt. Preparation of the base started with the melting of lard at 40 °C. Then cocoa butter was added, which should be melted at 35–36 °C because of the presence of a significant amount of oleic acid that causes unstable crystalline modifications. Suppose that the temperature regime is not observed after cooling. In that case, the melting temperature of the cocoa butter drops to 23–24 °С, and it isn’t easy to apply and store the resulting food at room temperature.

As sucrose, fructose, starch, protein, and standard rat chow are insoluble in the base (lard and cocoa butter), they are suspended in the melted base.

Pouring in the moulds is carried out in the so-called “cream” state when the processes of recrystallization in the base have already started. This method guarantees the absence of sedimentation of the dispersed materials in the base and their uniform distribution throughout the entire volume of the mould.

The displacement value (f) of solid substances in the base is initially determined to ensure the exact amounts of ingredients by using the formula:

$\text{f}=\frac{100(N.E-G_n)}{G_n.X}$

where,

N – number of pieces

E – the average mass of one piece without dispersed substances, g;

G_n – total mass of pieces with dispersed substances, g;

X – the content of dispersed substances in weight %.

The formula determines the calculation of the required amount of base using the displacement value:

M = F – (f₁.S₁+ f₂.S₂+……f_n.S_n) ,

where,

M - the amount of base for preparing the exact number of flat cylinders;

F - the capacity of the mould (g);

f - displacement coefficient of the substances that will be included in the base;

S - the amount of substance included in the base

The weekly measurement of blood glucose levels was performed using a blood drop from the tail vein and mini glucometer Accu-Chek^® (Roche diagnostics).

Biochemical markers such as glucose, urea, creatinine, uric acid (UA), total protein, albumin, transaminases L-aspartate aminotransferase (ASAT) and L-alanine aminotransferase (ALAT), total bilirubin, direct bilirubin, amylase, total cholesterol, triglycerides, creatine kinase (CK), and creatine kinase-myocardial band (CK-MB) were assessed using commercial kits for biochemical analyser “Mindray BS-120” as described by the manufacturer.

The blood pressure and the heart rate were measured at the beginning and at the end of the experiment using CODA non-invasive blood pressure system from Kent Scientific. Changes in the tail volume are detected using volume-pressure recording technology. Those changes correspond to systolic and diastolic blood pressure. The method relies to two tail-cuffs to measure blood pressure: occlusion cuffs (O-cuff) and volume-pressure recording cuffs (VPR-cuff). At the beginning, the animals are restrained in specific cages to limit their ability to move. The placement of the animals in the restraining cages should be very gentle to minimize the stress of the animals. The animals are left for 5 to 10 minutes to acclimatize and calm down. During this time, the restraining cage, together with the animals are put on a heating plate in order to increase the temperature in the tail for more accurate measurement of the blood pressure. The heating plate is set at 35 degrees, and the testing animals are covered with a specific blanket to maintain this temperature. The measurement consists of 15 cycles, with 5 seconds between the cycles. Mean values of the blood pressure and heart rate are than calculated and used to compare between the groups (Daugherty et al. 2009; DiPetrillo 2009; Wang et al. 2017).

Lipid peroxidation was determined by measuring the formation rate of thiobarbituric acid (TBARS) (expressed as malondialdehyde (MDA) equivalents) as described by Polizio and Pena (2005). The amount of MDA was calculated and expressed in nmol/g wet tissue.

GSH was estimated by measuring non-protein sulfhydryls after protein precipitation with trichloracetic acid (TCA) using the method described by Bump et al. (1983). Absorbance was determined, and results were expressed in nmol/g wet tissue.

The excised livers were rinsed in ice-cold physiological saline and minced with scissors. 10% homogenates were prepared in 0.05 M phosphate buffer (pH=7.4), centrifuged at 7,000 × g, and the supernatant was used for antioxidant enzymes assay. The protein content of liver homogenate was measured by the method of Lowry (1951).

Catalase activity (CAT) (Aebi 1974). Briefly, 10 µL of homogenate was added to 1990 µL of H₂O₂ solution. CAT activity was determined by measuring the decrease in absorbance at 240 nm. The enzyme activity was expressed as nmol/mg/min.

Superoxide dismutase activity (SOD) was measured according to the method of Misra and Fridovich (1972), following spectrophotometrically the autoxidation of epinephrine at pH=10.4 at 30 °C, using the molar extinction coefficient of 4.02 mM^-1cm^-1.

Glutathione peroxidase activity (GPx) was measured by NADPH oxidation, using a coupled reaction system consisting of glutathione, glutathione reductase and cumene hydroperoxide (Tappel 1978). Results are expressed as nmol/mg/min.

For light microscopic evaluation, liver tissues and pancreas were fixed in 10% buffered formalin, and, then, thin sections (4 μm) were subsequently stained with hematoxylin/eosin to determine general histological features (Bancroft et al. 2008). Sections were studied under a light microscope Leica DM 500.

Weekly monitoring of blood glucose levels showed that at the end of the experimental period, blood glucose was elevated to the highest degree in animals fed HF-HCD and CFD by 32% and 53% respectively, compared to the control animals. In the past, peaks of postprandial hyperglycemia were attributed only to simple carbohydrates such as mono- or disaccharides like fructose or sucrose, but in recent years it has been found that some complex carbohydrates can exhibit the same hyperglycemic peaks when rapidly digested (Bessesen et al. 2001). The rapid catabolism of the highly refined corn starch used to make the corn balls, as well as the wafers administered in CFD, may explain the high fasting glucose levels seen in this group of animals.

Serum biochemical parameters are presented in Table 3. Urea was lowered by 66%, 44%, and 70% in the HF-HC, HFD, and CFD-fed animals, respectively, relative to controls, but within the reference range. The uric acid level was higher in HF-HCD and in CFD by 44% and 102%, respectively, relative to control rats, probably due to high fructose intake which normally is metabolized to uric acid. Increased serum uric acid levels are commonly seen in patients with metabolic syndrome and are widely accepted as risk factors for hypertension, gout, non-alcoholic fatty liver disease, chronic kidney disease (CKD), and cardiovascular diseases. The high values of total protein and albumin are remarkable. Total protein was increased by 19, 21, and 24% relative to controls in HF-HCD, HFD, and CFD, respectively, and albumin was elevated by 61, 70, and 59%, respectively (Cho et al. 2012) found that the higher serum albumin levels are positively associated with METS, probably through increased abdominal obesity, high fasting blood glucose, and triglycerides. Transaminases were also elevated relative to both controls and reference values. ALAT activity was increased by more than 200% in all diet groups, and ASAT activity was increased by more than 300% in all groups of experimental animals. Studies have demonstrated the association of METS with elevated ALAT and ASAT levels (Hanley et al. 2005; Liangpunsakul et al. 2005; Andre et al. 2007; Nikniaz et al. 2018).

Total bilirubin was elevated by 71% relative to control only in the group fed with the diet prepared by our Faculty. Amylase activity was many times lower, relative to control, by about 90% in all groups. Low serum amylase activity has been reported in certain common conditions such as obesity, diabetes (regardless of type), and metabolic syndrome, all of which appear to have a common etiology of insufficient insulin action due to insulin resistance and/or diminished insulin secretion (Nakajima et al. 2016). All these abnormalities in biochemical parameters indicate an extremely severe impairment of renal, hepatic, and pancreatic function, as well as damage to the musculoskeletal system and connective tissue, probably associated with myositis, rhabdomyolysis, and hence renal involvement. These results reveal a very severe nutritional and metabolic disorder, probably with endocrine dysfunction and hypothyroidism, which is also reflected in the weight change of the animals (Fig. 1).

Figure 1. 

Weekly blood glucose (mmol/L) changes. Abbreviations: SCD (control group fed with a standard chow), HFD (lard-based high-fat diet), HF-HCD (high fat - high carbohydrates diet), and CFD (fat, salt, and sugar-rich supermarket foods (cafeteria diet). ^ap< 0.05 vs control SCD; ^bp< 0.05 vs HFD diet; ^cp< 0.05 vs HF-HCD; ^dp< 0.05 vs CFD.

It is worth noticed that cholesterol and triglycerides were in the normal reference range in all animal groups, which is most likely related to body weight reduction (Fig. 1).

The activity of CK and CK-MB fractions was extremely elevated in all groups of animals, which is probably related to the method of euthanasia and the abrupt release of these fractions from the muscle tissue of the animals before decapitation (O’Brien et al. 1997).

The change in body weight of the experimental animals is reflected in Fig. 2. At the beginning of the experiment, all the animals included in the four study groups had similar body weights. Body weight gain in the HFD group was progressively higher than in the other groups and showed weight gain, about 42%, respectively, from baseline weight. The animals fed the normal rodent chow without any other supplements gained by 29% (p< 0.05) compared to the beginning of the experiment. The HF-HCD and CFD groups showed a reduction of the weight with 18% and 16%, respectively compared with the first day of the experiment. We assumed that the weight loss in these groups is related to the intake of a small amount of food due to the unpleasant taste and lack of consistency of the food they are used to. Differences in the consistency of the food produced differences in food consumption when compared to standard chow (SC) and HFD, which in fact was a normal SC covered with lard.

Figure 2. 

Weekly body weight changes (in grams). Abbreviations: SC (control group fed with a standard chow), HF-HC (high fat - high carbohydrates diet), HFD (lard-based high-fat diet), and CFD (fat, salt, and sugar-rich supermarket foods (cafeteria diet). ^ap< 0.05 vs control SCD; ^bp< 0.05 vs HF-HC diet; ^cp< 0.05 vs HFD; ^dp< 0.05 vs CFD.

The measurement of arterial blood pressure at the beginning and at the end of the experiment showed that systolic blood pressure (SBP) was increased to the highest degree in animals fed HF-CD and HFD, by 36 and by about 20%, respectively (Table 4). Hypertension is one of the main constituents of METS. Hypertension is strongly associated with metabolic syndrome through the pathophysiology, which involves obesity (Stanciu et al. 2023). Dietary fat contributes to the elevation of blood pressure and increases the risk of stroke and coronary artery disease. The increase of plasma fatty acids may contribute to the development of hypertension via a process involving the elevation of Ca²⁺ current density and an alteration of channel kinetics in the vascular smooth muscle membrane (Wilde et al. 2000).

In the HF-HCD group, the amount of MDA (Fig. 3) was the highest by 188% compared to the controls and to a smaller extent to other groups, while the level of GSH (Fig. 4) was the lowest (75%), compared to the controls, indicating that this group was the most affected by oxidative stress. This pro-oxidant state was confirmed by the data obtained by measuring the activity of antioxidant enzymes (Fig. 5).

Figure 3. 

Malondialdehyde quantity in the liver homogenate from rats fed with different diets; ^ap< 0.05 vs control SCD; ^bp< 0.05 vs HF-HC diet; ^cp< 0.05 vs HFD; ^dp< 0.05 vs CFD.

Figure 4. 

Level of reduced glutathione (GSH) in the liver homogenate from rats fed with different diets; ^ap< 0.05 vs control SCD; ^bp< 0.05 vs HF-HC diet; ^cp< 0.05 vs HFD; ^dp< 0.05 vs CFD.

Figure 5. 

Changes (%) in the activity of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the liver homogenate from rats fed with different diets, compared with control rats, fed with the normal rat chow. ^ap< 0.05 vs control SCD; ^bp< 0.05 vs HF-HC diet; ^cp< 0.05 vs HFD; ^dp< 0.05 vs CFD.

The activities of the antioxidant enzymes CAT, SOD, and GPx were statistically significantly reduced in the group fed HF-HCD by 26%, 22%, and 28%, respectively, compared to control animals (Fig. 5). In the HFD group, we also observed an albeit smaller decrease in the activity of these enzymes, by 10, 15, and 12%, respectively. Many pieces of evidence suggest that increased oxidative stress may be considered the key to the generation of insulin resistance, diabetes, and cardiovascular disease. Data support the concept that free radical overgeneration may play an important role in METS and related manifestations (Ceriello et al. 2004; Mahjoub et al. 2012).

Oxidative stress could be an early event in the pathology of these chronic diseases rather than merely a consequence (Roberts et al. 2009). No change in the activity of antioxidant enzymes was observed in CFD-fed animals. This is probably related to the availability of antioxidant supplements to these foods available in supermarkets.

Morphological changes in the livers and pancreas are presented in Fig. 6. In the control animal group, histopathological changes in both investigated organs were not seen. The liver and Langerhans islets of the pancreas were without fatty deposits, and the structure of organs was normal. In the HF-HCD group fatty and glycogen degeneration was seen. Massive deposition of adipose tissue between the pancreatic lobules was visible. Nonalcoholic steatohepatitis (NASH) is considered the hepatic manifestation of METS. In the HFD group, weak morphological changes in the liver were shown. Slightly increased fatty tissue between the pancreatic lobules was also visible (Patton et al. 2010). The fatty pancreas also showed a strong correlation with metabolic syndrome. Animal studies reported that lipid deposition in pancreas islet cells due to a high fat/high glucose diet could damage pancreatic beta cells and make them hyperglycemic (Yin et al. 2004). Metabolic conditions give rise to pancreatitis (Kota et al. 2013). CFD group was presented with slight degenerative changes in hepatocytes, predominantly in the periphery of the lobules, and the pancreas was with preserved structure.

Figure 6. 

Histological profile.