Branched-chain amino acids (BCAA—leucine, isoleucine and valine) supplementation, especially leucine, has been described as a potential therapeutic tool capable to attenuate skeletal muscle atrophy induced by several catabolic conditions, such as cancer, sepsis, muscular diseases [1
] and glucocorticoid treatment [2
]. Since leucine is considered as the second more potent insulin secretagogue amongst all the amino acids (AA) [3
], it has been studied for its capacity to modulate whole body glucose homeostasis [4
Indeed, leucine supplementation might exert positive systemic effects in conditions characterized by increases in glucose homeostasis disturbance, such as high fat diet (HFD) induced insulin resistance, but the effects are controversial. For example, Zhang and coworkers [5
] found increases in the glucose metabolism of leucine supplemented mice, whereas Lynch and coworkers [6
] did not observe improvements nor decreases in the glucose homeostasis. However, this may have been due to the lack of standardization of doses and forms of administration. On the other hand, in healthy rats and humans, oral leucine feeding has shown to rapidly inhibit skeletal muscle protein degradation [7
] and also promote robust increases in skeletal muscle protein synthesis [9
], demonstrating overall a potential capacity to handle disturbances in glucose metabolism and spare skeletal muscle mass, especially under atrophic conditions [8
]. However, its chronic effects remain elusive, specifically during insulin resistant states from different physiopathological backgrounds, such as glucocorticoid treatment (and even HFD treatment).
Dexamethasone (DEXA) is a synthetic glucocorticoid form of the endogenous hormone cortisone, which exhibits potent immunosupressant and anti-inflammatory properties [10
]. The successful therapeutic benefits of this drug in a wide range of inflammatory diseases is, however, limited as it presents several side effects [11
], such as insulin resistance and skeletal muscle atrophy [12
]. Therefore, in order to benefit from the desired effects of long term DEXA treatment, the deleterious responses must be reduced. In this context, leucine supplementation may represent an interesting intervention with a clinical perspective, due to the reasons justified from above.
Since there is still a lack of evidence regarding the effects of leucine supplementation on DEXA-induced insulin resistance and skeletal muscle atrophy, we decided to investigate: (1) whether a supplementation, with a low dose of leucine (not capable of increasing insulin levels or muscle protein synthesis) or a high dose of leucine (capable of maximally increasing both insulin concentration and muscle protein synthesis) given through gavage or drinking water is able to improve glucose metabolism, as well as spare the muscle mass and, consequently, voluntary muscular strength in healthy (control pair-fed and energy restricted) rats; (2) investigate to see if leucine supplementation via gavage or via drinking water exerts some positive effect on glucose metabolism and muscle sparing/strength effects under DEXA treatment; in other words, besides the dosage effect, would frequent nutritional stimuli (leucine provided through drinking water) be different from that provided by a pulsatile pattern (leucine provided through gavage) with both groups consuming the same daily dosage?
The major findings of the present study are that under DEXA treatment, leucine supplementation through gavage in both low and high doses was not capable of changing metabolic parameters (i.e., triacylglycerol, fasting insulin levels and fasting glucose levels), but was capable of decreasing maximal voluntary strength function. On the other hand, when administered to leucine supplementated rats via drinking water and under DEXA treatment, even at low dosages, it was capable of inducing a massive diabetic state (and also decreasing the EDL mass), when compared with leucine supplemented rats via gavage, even at low doses. This result clearly demonstrates that not only the daily dosage, but also the administration form and leucine kinetics of this supplement are important players to be considered under DEXA treatment induced insulin resistance.
As previously described, leucine supplementation has been shown to spare skeletal muscle mass during several atrophic states, including insulin resistance [5
]. Recently, it was demonstrated that supplementation with 0.6 g/kg of body mass of BCAA (46% leucine, 28% valine and 23% isovaline) was capable to attenuate the soleus muscle atrophy induced by DEXA (0.6 mg/kg given intraperitoneally—I.P., during 5 days) in Sprague Dawley rats [2
]. However, in the same study, the authors did not report any information regarding insulin resistance. On the other hand, in our study, we chose the dosage of 5 mg DEXA/kg of body mass, since 1 mg/kg of body mass was unable to induce measurable skeletal muscle atrophy in our Wistar rats (data not shown). Moreover, when compared to previous studies of our group also using Wistar rats, DEXA given I.P. compared to DEXA given via drinking water [20
], DEXA I.P. was slightly superior in increasing fasting glucose levels and inferior in inducing skeletal muscle atrophy [20
]. From the above information, we conclude that: (1) glucose metabolism should be evaluated together with the possible sparing effect of leucine supplementation under glucocorticoid treatment; and (2) the administration pathway exerts a determinant effect on the magnitude of decrements in glucose homeostasis and skeletal muscle atrophy, not necessarily linked to each other.
As stated before, our major expectation was that leucine supplementation in low versus
high dosages, due to its different physiological effects, would be capable of inducing profound changes in glucose homeostasis parameters, as well as skeletal muscle atrophy in both treated and non-treated DEXA groups. In fact, our expectation was that low dosages (due to the fact of non stimulating insulin secretion) [9
] would be beneficial to DEXA treated rats, because glucose metabolism is already profoundly affected by glucocorticoid treatment. On the other hand, in energy restricted healthy rats, we were confident that high dosages would be better, because data from the literature suggests that higher leucine dosages would be more effective at decreasing muscle proteolysis [8
]. Surprisingly, leucine supplementation offered via gavage was innocuous to glucose homeostasis and skeletal muscle mass under DEXA treatment in both low and high doses. This result demonstrated the capacity of the whole body to deal with low and even very high amounts of leucine administered as a bolus
, which is somewhat surprising, because leucine, especially in high amounts, is capable of modifying insulin secretion (and consequently glucose uptake by peripheral tissues) and induces skeletal muscle protein synthesis, while decreasing muscle proteolysis [1
]. Following the same line of reasoning and contrariwise to our initial hypothesis, in the healthy group (non DEXA treated group), both low and high doses showed similar effects on skeletal muscle atrophy. However, serum glucose, fasting insulin concentrations and also HOMA-IR index significantly decreased, suggesting an improvement in glucose metabolism in the low dose leucine supplemented group, which could be of clinical significance during weight loss diets, as suggested by Layman [21
]. This result could explain why BCAA supplementation, in addition to dexamethasone treatment, was so effective in the treatment of skeletal muscle atrophy in the study conducted by Yamamoto and coworkers [2
]. Interestingly, in a study of mice consuming high fat diet, the consumption of a chow containing 20% protein (with 1.5% leucine in w/v) increased oxygen consumption (and increasing resting energy expenditure) [5
], and in C2C12 myocytes, leucine (0.5 mM) increased mitonchondrial mass by 30% and stimulated genes related to mitochondrial biogenesis [22
]. Additionally, only leucine supplementation was able to protect animals from the deleterious effects of a high fat diet, such as insulin resistance and increased LDL cholesterol [5
]. Although not directly measured in our study, we also observed a decrease in the blood TAG concentration in the low dose control group.
We then undertook a second study comparing the effects of DEXA plus leucine treatment with low and high doses of this amino acid used via bolus, as previously described, against the same daily concentration offered in the drinking water. Since the dosage effect was not different when comparing rats presenting insulin resistance mediated by DEXA treatment as shown in Figure 1
A, would frequent nutritional stimuli be different from that provided by a pulsatile pattern to aggravate insulin resistance caused by DEXA treatment?
To test this hypothesis, we supplemented four groups of DEXA-treated rats, which consumed the same daily dose: the first two groups consumed the low dose of leucine in a pulsatile form (via gavage)versus
a non-pulsatile form (via drinking water), and the second two groups followed the same schedule but consumed the high dosage. Our results were notable: rats supplemented through short periods of time (offered in drinking water), in a non-pulsatile form presented a markedly higher fasting glycemia compared with rats supplemented with the same daily dosage, in a pulsatile form (Figure 4
A). These results suggest that tissues need time to terminate the leucine signal. Moreover, these results show that the continuous presence of this AA in the whole body, in an DEXA-induced insulin resistant state, would be capable of transforming to a clear diabetes state even with such a small leucine dose (i.e.
, not capable of affecting glucose homeostasis when supplied via bolus). Importantly, this outcome also occurred in the high dosage group, which proves that the threshold of leucine supplementation capable of inducing diabetes, in a previous DEXA-induced insulin resistance, is extremely low when supplied via drinking water; this would be a completely novel result. On the contrary, the results clearly demonstrated that leucine supplementation with low dose via drinking water did not modify muscle mass of DEXA-treated animals when compared with gavage and the same pattern was observed with a high dose of leucine. This would mean that skeletal muscle at our end point, would not suffer the effects of this disturbance on glucose homeostasis. In fact, as shown below, GLUT-4 gene expression was unaltered in the muscles analyzed in this study. Interestingly, leucine supplementation (1.5% in drinking water for eight months) carried out in the polygenic mouse model NONcNZO10/LtJ (RCS10), which is predisposed to beta cell failure and type 2 diabetes, is able to improve the glycemic control that was associated with an increased insulin response to food challenge in control mice [23
]. In our study, in the presence of DEXA plus high doses of leucine in the drinking water, we observed a significant decrease in the insulin level measured in fasted animals. Such a decrease may be associated with a failure of the beta cells to respond to high leucine concentration for insulin secretion in this group of animals. However, such an effect was not observed in the control animals supplemented with high leucine dose. Although insulin levels decreased in both situations, these results would indicate diametrically opposite situations. Under healthy conditions, a low dose of supplemented leucine would be capable of increasing glucose homeostasis and reducing insulin plasmatic levels. On the other hand, when given chronically at low and high doses in the presence of DEXA-induced insulin resistance, leucine supplementation promotes a clear diabetic state, and the diminishment of insulin levels observed with high doses would indicate a beta cell failure function. However, this hypothesis needs further research in order to be confirmed.
In muscle cells, glucose transport is mainly controlled by the stimulation of insulin, leading to the translocation of GLUT-4 from late and early endosome vesicles to the plasmatic membrane, as well as through control of gene expression. Indeed, multiple and complex mechanisms control the GLUT-4 transporter function [24
]. In addition, it is acknowledged that DEXA treatment affects several steps of the insulin signaling cascade, leading to impaired glucose transport inside muscle cells [13
]. However, there is very little information about the involvement of leucine supplementation in DEX-treated animals on GLUT-4 gene expression in different muscle tissues.
In study 1, we detected in EDL muscles a marked impairment of GLUT-4 gene expression in DEXA treated groups (Figure 2
). EDL muscles are primarily composed of fast twitch muscle fibers [25
]. This result may be linked with the modification of the genomic expression that can lead to impaired glucose transport [10
]. This would mean that during conditions of supplementation with high doses of leucine and in short term periods, GLUT-4 expression is strongly controlled by hormonal inputs. In order to test such a concept, we compared in study 2 the effect of leucine supplementation in DEXA treated animals (Figure 5
). The results obtained are compatible with the idea that with high doses of leucine in short-term periods, GLUT-4 expression is mainly controlled by hormonal inputs, not genetic ones. For example, Hu and coworkers [12
] recently showed that under insulin resistant conditions (e.g., stressed rats showing increased glucocorticoid levels), the cortisol receptor binds to and inactivates the insulin receptor, demonstrating the strong impact of glucocorticoids during periods of insulin resistance on cell signaling. In another study by Doi and coworkers [26
] examining the effects of isoleucine, the investigators found in C2
cells that the isoleucine effect on glucose uptake was mediated by phosphatidylinositol 3-kinase (PI3K). These results suggest that isoleucine stimulates the insulin-independent glucose uptake in skeletal muscle cells, which may contribute to the plasma glucose-lowering effect of isoleucine in normal rats. Collectively, our results suggest that healthy adult rats are capable of metabolizing very high amounts of leucine, and that the threshold of leucine supplementation needed to transform a protein synthesis signal into an insulin-resistant one is very high during normal states, but abnormal under glucocorticoid induced insulin resistance states, especially when supplied via drinking water.
The role of leucine supplementation in this scenario is uncertain because, when compared with Control-NS group, only Control-LL group showed a decreased GLUT-4 expression, and this decrease is evidenced only in the EDL muscle (Figure 2
B). This result points out that GLUT-4 gene expression in EDL muscles may be altered not only by DEX treatment, but also by leucine supplementation, although this parameter is not predictive of changes in the whole body glucose metabolism and additional measurements, such as total GLUT-4, membrane-bound and glucose uptake in isolated muscles should provide more conclusive results.
Finally, when we evaluated the muscle function of these animals, we observed that animals treated with DEXA and receiving high dose of leucine presented a significant reduction in mean ambulation when compared with the control group. Surprisingly, control animals supplemented with high doses of leucine also presented lower mean grip strength when compared with the low dose group, suggesting that a high leucine dose, applied via bolus, is not innocuous in this experimental model after seven days of treatment.