The aim of this study was to describe the retina oxidative parameters and the carnosine influence on diet-induced obesity model. The experimental model proposed in this study promoted metabolic changes, represented by increased levels of fasting blood glucose and of plasmatic triglycerides. Carnosine supplementation influenced on plasma triglyceride. A greater weight gain and a different body composition represented by the adiposity index were also observed. In the retinal tissue, the hypercaloric diet did not influence the redox state. Nevertheless, carnosine exerted a reducer effect, with higher concentrations of TAC, GSH, and GSH:GSSG ratio. Oxidative damages associated with the hypocaloric diet, characterized by a higher concentration of carbonylated proteins and lower concentrations of the sulfhydryl groups, were observed. However, treatment with carnosine only induced a non-significant increase in the sulfhydryl type levels. Figure 4 summarizes these findings.
Simple carbohydrate and saturated fatty acid are associated with the development of the metabolic syndrome and multiple comorbidities (40–42). In relation to vision, the Beaver Dam Eye Study associated high intake of saturated fat and cholesterol with an increased risk for early age-related maculopathy (43). Another study corroborated these findings, observing that ingestion of high glycemic index foods also contributed to the development of AMD 2.71 times more than the ingestion of low glycemic index foods (fruits, cereals, vegetables, whole wheat bread) (44). The present study used a hypercaloric diet enriched with simple carbohydrates with 25% sucrose solution. It is an innovative model that mimics the development of obesity with metabolic complications, according to a protocol previously adopted by our group (36). This diet can induce hyperadiposity, insulin resistance with hyperglycemia, dyslipidemia, and even arterial hypertension, as Wistar rats hardly develop hypertension via diet models (36). In this model, echocardiographic and renal function alterations were observed (36). For clarification purposes, it is important to point out that the “control” animals (SD) are usually fed ad libitum, which frequently leads to over-eating and excessive gain of body weight (45–46). This fact was confirmed at the end of the study when weight gain was observed in the animals of the four groups in relation to the start of the experiment. The amount of energy intake by the animals fed ad libitum many times significantly exceeds their energy expenditure, resulting in a substantial gain of body weight or positive energy balance, frequently associated with early diseases (47). Consequently, the group that ingests food ad-libitum may be subject to harmful factors induced by the intake above the body requirements (48). For this reason, our study comprised four groups: SD group; HcD group; SD + Car group; and HcD + Car group. As expected, the hypercaloric diet induced a significant increase of glucose in relation to SD group, corroborating literature findings (49). However, the addition of carnosine (HcD + Car) was not able to revert this condition. The hypercaloric diet also induced a significant increase in serum triglyceride (TG) levels. Similar results have been shown in studies of diets high in refined sugar (3,50). Carnosine supplementation led to a decrease in triglycerides in relation to HcD, although with no statistical significance.
The high sugar-fat diet group received carnosine supplementation with the objective to test its antioxidant effects on the retina. The intake of hypercaloric diets associated with a western lifestyle has been associated with an excessive generation of ROS (3,51–52). It is known that a sucrose-based diet interferes in the performance of the antioxidant system, overloading the organism's defense system, leading to oxidative stress (53–55). It has also been demonstrated that the high fructose and high-fat diets affect the function and structure of the rat retina (56). In our study, the hypercaloric diet did not interfere in the production of hydrogen peroxide in the retina, as shown in Fig. 1, with no significant statistical difference among groups.
The levels of total antioxidant activity (TAC), expressed in mM Trolox equivalent, were higher in the retina of the groups that received carnosine supplementation when compared to the SD groups (Fig. 2a). It is important to point out that a low TAC value directly indicates the deficit of its specific composing substances (57), considered an important biomarker of tissue antioxidant system (58). Patients with AMD had lower plasma TAC levels when compared to the control group (57, 59–64). It is a likely indication that the oxidoreduction disturbance may be involved in the pathogenesis of AMD and that the increase in TAC levels may be associated with a protective factor of the retina. In this regard, carnosine may have played a protective role in the retina of the rats fed the hypercaloric diet.
The hypercaloric diet group (HcD) presented a decrease in reduced GSH level in relation to SD group, however, with no statistical relevance. It has been observed that a high sucrose diet (545 g/Kg of sucrose) administered for more than three months, induced a significant decrease of GSH in the rat brain (55). Probably, the smaller amount of sucrose (80 g/kg) offered to HcD animals in the present study may account for the discreet reduction of GSH, corroborating the findings of another study (65). Of great importance is the finding that glutathione deficiency contributes to oxidative stress and, therefore, may play a key role in the pathogenesis of many diseases (66). Conversely, HcD + Car group had a significant increase in the GSH levels in relation to SD + Car group (Fig. 2b). It is important to remember that glutathione has several major physiological functions, such as: protection of cells against destructive effects of reactive oxygen intermediates and free radicals, detoxication of external substances such as drugs and environmental pollutants, maintenance of red cell membrane stability, and enhancement of immunological function through its effects on lymphocytes (67). It was shown that oxidation induces apoptosis of RPE cells may be protected by GSH (68). Another study reported a significantly lower plasma GSH in older individuals affected by AMD, diabetes, and controls (elderly with no diabetes or AMD) than in younger individuals (69). Considering the increase of GSH in the HcD + Car group, it is possible to suggest that carnosine played a protective role in the retina.
A decrease of the GSH:GSSG ratio in the HcD group was observed when compared with HcD + Car group, although with no significant differences. Aging, chronic diseases, hypercaloric diet, and even AMD may potentially reduce the GSH:GSSG ratio (69,70–72). It is a known fact that the activity of GSH as an antioxidant can be expressed in two ways: as a function of GSH concentration and as a function of the redox state of the GSH:GSSG ratio (69). In the present study, the hypercaloric diet did not influence the retinal redox state. Nevertheless, the treatment with carnosine revealed higher GSH concentrations, observed in the GSH:GSSG ratio and, consequently, in a higher antioxidant power.
The present research also establishes a mapping of protein carbonylation of the Wistar rat retina fed the hypercaloric diets, as high values of protein carbonyl groups have been found in patients with AMD (59,73). The reactive carbonyl species are important cytotoxic mediators produced due to the oxidative damage of biomolecules (lipids and sugars), leading to alterations in the cell-signaling mechanisms to the nucleus, positively regulating the redox-sensitive transcription factors, and inducing the irreversible structural modification in important molecules [proteins, peptides (cysteine, lysine, histidine), lipids, DNA] (74). Protein carbonyls are the most widely studied markers of protein oxidation and are frequently used as markers of oxidative stress, being indicators of the amount of protein that has been oxidized by highly reactive free radicals (75–76). Due to this ROS overproduction, increased protein carbonylation levels have been described along with these diet-induced disorders (77–78). Our results show that long-term intake of an HcD diet was associated with formation of carbonyl functional groups in relation to groups SD groups (Fig. 3a). It has been demonstrated that a high-caloric diet promotes similar results in the plasma and liver (73). Studies indicate that carnosine acts by a direct antioxidant mechanism and by sequestering reactive carbonyls (RCS), the byproducts of lipid and glucose oxidation, thus inhibiting AGE and ALE which are the reaction products of RCS with proteins (79). Although the in vitro capacity of carnosine to scavenge acrolein and form 3-methylpyridinium carnosine adduct has been demonstrated (8,27), in the present study, the dietary intervention of rats supplemented with carnosine did not show specificity by down-regulating carbonylation in the retina. On the other hand, an increase in carbonyl protein levels was observed in the HcD + Carn group. This does not mean that carnosine induced higher levels of carbonyl in HcD, as there was no increase in the level of SD + Car in relation to SD.
Besides the oxidative damage to proteins that a hypercaloric diet causes, it was observed that this diet also leads to a significant loss of the sulfhydryl group (Fig. 3b). Supplementation of carnosine to hypercaloric diet group restored values of sulfhydryl levels in the retina similar to SD (Fig. 3b). This is possible due to the increased GSH concentrations (80). It is important to remember that sulfhydryl groups, also biomarkers of oxidative stress, are considered the most powerful and most frequent antioxidants in the plasma (81), and their expression is reduced in AMD patients (82).
The diet prepared for this study, which mimics modern eating habits, induced oxidative changes in the retina. Carnosine seems likely to induce a potential antioxidant effect, elevating TAC and GSH concentrations in retinal tissue. Although some studies associate carnosine with the prevention and even the cure of cataract, the action of this antioxidant was not tested in oxidative and inflammatory markers of age-related macular degeneration. Further studies are required to determine the antioxidative effect of carnosine on AMD.