Dexamethasone, a potent synthetic glucocorticoid, is known to lead to changes in the metabolisms of carbohydrates, lipids, and proteins and to act in other tissues, in a time and dose-dependent manner . Additionally, chronic glucocorticoid excess disrupts the internal milieu, resulting in central obesity, muscle atrophy, and fatty liver . Given the severity of this disease, we sought to standardize a rat model of Cushing’s syndrome induced by chronic glucocorticoid over levels and then to assess the potential effects of the administration of the branched-chain amino acid leucine on the pathophysiological process since there are few studies regarding the systemic effects of this amino acid.
In the present study, we observed the development of characteristic metabolic patterns of CS, such as visceral obesity, adrenal atrophy, and biochemical alterations. These effects accompanied lower body mass and food intake. In addition, the administration of leucine potentiated some of these effects and promoted liver damage associated with reduced antioxidant defenses.
A neurohumoral system-mediated primarily by the hypothalamus is in charge of hunger and satiety control by increasing the number of circulating hormones, such as leptin and insulin, which are responsible for initiating the signs of satiety in the central nervous system [34, 35]. Furthermore, the inhibition of ghrelin activity by glucocorticoids is directly related to the reduction in body mass . Another important aspect is the reduction in body mass and muscle atrophy, both known characteristics of chronic use of dexamethasone, due to the increase in muscle proteolysis and the inhibition of protein synthesis, which is an additional effect resulting from the increase in lipolysis [37, 38].
In this context, the administration of dexamethasone, in all doses tested, promoted a reduction in food intake but not water from the second week of treatment. In addition, there was a reduction in body weight evolution in these animals, which is directly related to lower food intake.
It has been shownm, in a literature review , that obesity and weight gain were present in 95% of patients with CS; however, despite this apparent discrepancy, the data obtained in the present work demonstrate a decrease in body mass in animal models of CS, and for these models weight gain is not common. These results agree with previous data, in which the CS model showed significant body mass loss in the first weeks, while in the control group, there was an increase in body mass. In subsequent weeks of this study, the group with CS showed a gradual increase in body weight . The lipogenic effects on the visceral adipose tissue and the catabolic effects on the skeletal muscle may explain those results .
The effects of the supplementation of branched-chain amino acids (BCAA), such as L-leucine effects on metabolic syndromes and body composition, are diverse. Several studies have shown that their use can result in a decrease in total body weight in obese rats, with decreased adiposity, in addition to improving insulin sensitivity and reducing plasma cholesterol concentrations [16, 20].
When assessing the influence of leucine supplementation in the three tested doses, we found an increase in the anorexigenic effect without changes in water intake despite the weight loss remaining similar to what we observed on the treatment with dexamethasone alone.
Some previous research study an association of leucine supplementation and carbohydrates, proteins, or other amino acids. However, when supplemented in the absence of other BCAAs (isoleucine and valine) or with a low protein and carbohydrate diet, animals show a decreased food consumption, body mass, and growth .
These effects may be associated with reduced concentrations of ghrelin and increased concentrations of leptin, which are responsible for the perception of hunger and satiety, respectively, thus contributing to the reduction in body mass [41, 42], as it has been previously described for dexamethasone . Likewise, The literature has shown that leucine promotes protein anabolism and lipolysis (43), contributing to these observed results.
Adrenal atrophy is a classic effect in CS that results from chronic treatment with glucocorticoids . Glucocorticoid treatment inhibits the hypothalamus-pituitary-adrenal (HPA) axis, thus, the hypothalamus stops releasing the corticotrophin-releasing hormone, responsible for synthesizing the adrenocorticotropic hormone (ACTH), so it does not act by promoting cell tropism on the adrenals, resulting in the atrophy of this gland . Furthermore, the chronic use of glucocorticoids favors greater visceral adiposity . Likewise, glucocorticoid acts on pre-adipocytes inducing differentiation and increase in adipose mass, in addition to increasing the activity of lipoprotein lipase (LPL), which induces fatty acids storage on tissues .
As expected for elevated glucocorticoid levels, we observed that the administration of dexamethasone 1.0 mg.kg− 1 promoted a reduction in the adrenal mass, indicating inhibition of the HPA axis. Furthermore, there was a greater deposition of visceral fat, a result confirmed by a higher adiposity index. Moreover, the administration of leucine (0.5, 1.0, and 1.5%) potentiated these effects, indicating a non-beneficial action of this amino acid in the context of CS.
The glucocorticoid excess, either endogenous (Cushing’s Syndrome) or exogenous (long-term glucocorticoid treatment), results in visceral adipose tissue deposition, muscle atrophy, fatty liver, hypertension, hyperglycemia, dyslipidemia, and insulin resistance [2, 46]. In this study, we observed that administration alone of dexamethasone 1.0 mg.kg− 1 promoted hyperalbuminemia and that, when associated with leucine supplementation, the rats developed mixed dyslipidemia, illustrated by the elevation of TAG, cholesterol, and VLDL levels. Moreover, leucine supplementation maintained hyperalbuminemia.
In CS, the secretion of the hormones insulin and glucagon is compromised. These hormones are responsible for controlling, in addition to glycemic levels, the intracellular concentration of cholesterol, which if altered could lead to an increase in cholesterol levels and trigger a reduction in the activity of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a limiting enzyme in cholesterol biosynthesis .
Furthermore, hyperalbuminemia can also be explained by the inhibition of the HPA axis, leading to a reduction in aldosterone production, which is responsible for regulating sodium homeostasis . The increase in albumin levels may result from the decrease in circulating volume, leading to a higher concentration of plasma proteins.
Fat deposition and liver damage are characteristics of excess glucocorticoids in the body, as previous research has shown [2, 46]. In this study, the administration of leucine, together with dexamethasone, led to an increase in liver TAG deposition (Fig. 4A). In addition, we observed an increase in the levels of the AST enzyme, but not ALT, in this association (Fig. 4B-C). This result is a highly sensitive indicator of hepatocellular damage and, to a certain extent, indicates liver damage .
Some studies have shown that glucocorticoids promote the decrease of glucose transport stimulated by insulin [49, 50]. When in excess, they lead to a decreased uptake and oxidation of this substrate in skeletal muscle , which are likely responsible for promoting glucose intolerance and insulin resistance. When administered for a prolonged period or even in high doses, glucocorticoids promote glucose intolerance, as the high synthesis and secretion by pancreatic β-cells of insulin cannot compensate for the metabolic demand . In this work, dexamethasone 1.0 mg/kg promoted a reduction in glucose tolerance (Figs. 5A and C), which may be associated with a morphological adaptation of the pancreas, as glucocorticoid favors hypertrophy of pancreatic β cells and consequently an increase in insulin secretion [24, 53]. Furthermore, these effects accompany a pattern of insulin resistance in the animals in the group that received the highest dose of DEXA (Figs. 5B and D).
Those data agree with the expected results for CS models, as it is well established that this disease promotes metabolic disorders associated with lower glucose uptake due to insulin resistance [30, 5].
Based on the parameters described so far, we observed that the administration of dexamethasone 1.0 mg.kg− 1 proved to be the most effective in promoting the development of CS in rats and that supplementation with leucine promoted a worsening effect in the observed parameters, including reduced body and muscle mass and increased visceral adiposity, hyperalbuminemia, and dyslipidemia.
The antioxidant defense system works by reducing the damage caused by free radicals and these reactive species, and when there is lipid peroxidation resulting from increased fat, it causes an imbalance in the oxidative and antioxidant system . With the increase in fat, there may be progressive and cumulative cell damage because of pressure due to the large body mass, where cell injury, which releases pro-inflammatory cytokines, generating reactive oxygen species .
MDA is a biomarker and one of the secondary products of lipid peroxidation, derived from the β-rupture of endo-cyclization of polyunsaturated fatty acids, such as linoleic, arachidonic, and docosahexaenoic acid . The triggering of inflammatory processes in CS is often associated with myeloperoxidase (MPO), an important inflammatory indicator, considering that the release of this enzyme is due to macrophages activated during inflammation and that the reactive species produced by them can activate factors of transcription, including the NF-kB signaling pathway (transcription factor involved in the synthesis of pro-inflammatory cytokines) increasing the inflammatory process .
In this work, the concentrations of MDA in the liver increased in the DEXA group but did not change in the group that received leucine, indicating a reduction in lipid peroxidation mediated by the action of this amino acid. However, these values should be observed with caution, as the group that received leucine had significantly higher values of MPO. Therefore, these data cannot indicate whether leucine in isolation could control the entire cellular antioxidant system.
Oxidative stress can trigger cell adaptation or injury. When adapted, the cells tolerate oxidative stress by regulating the synthesis of antioxidant defenses until the balance is restored . This picture of oxidative stress can cause damage to the most diverse types of biomolecules − including DNA, proteins, and lipids − since the target of oxidative stress varies depending on the cell, type of exposure, or even the intensity of the stress .
Several enzymes are part of the defense mechanism, including SOD, CAT, and glutathione peroxidase (GPx), in addition to others that are not directly related to the process, such as glucose-6-phosphate dehydrogenase (G6DP), and non-enzymatic antioxidants such as vitamin E, vitamin C and flavonoids .
SOD and CAT act mainly in hydrophilic regions, SOD with specificity for O2 − dismutation, generating hydrogen peroxide, while CAT decomposes H2O2. Since SOD and CAT are two enzymes that cooperate, the activities of these two enzymes should change synchronously and simultaneously. GSH is important for acting in thiolic homeostasis, maintaining the cell redox balance, and defending against electrophilic agents. This antioxidant benefit occurs through the reactive thiol group (SH) of its cysteine .
We observed that the activity of these antioxidant enzymes did not change by dexamethasone or leucine administration, indicating no counterbalancing antioxidant response induced by oxidative stress indicated by the high levels of MDA and MPO.
However, we observed that, although SOD mRNA was unaffected by glucocorticoid and leucine, the mRNA transcript for CAT decreased with leucine supplementation and, controversially, NADPH oxidase 4 (NOX4), oxidative enzyme-producing superoxide anion, was also reduced. In addition, the fatty acid transporter protein type 2 (FATP2), important in the transport of fatty acids and metabolism, did not show statistically significant differences between the groups, although this marker is commonly associated with fatty liver .