The S35 mice gained more weight than the S20 mice. These results confirm that S mice have an increased need for energy from dietary protein compared with the C mice. In contrast, the C35 mice had less weight gain than the C20 group. This study supports our hypothesis and shows for the first time, to our knowledge, that increased dietary protein alone improves weight gain in the Berkeley S mouse model. The 20% protein diet inadvertently contained lower fat and sucrose than the other diets. However, there was a consistent increase in the rate of weight gain for S mice with increasing dietary protein (15, 20, 25, and 35% of energy), whereas the opposite result was observed for the C mice. Hence, we conclude that the protein:energy ratio is the major determinant of the dietary effects observed in these studies.
The significantly slower weight gain for C35 mice supports the notion that removal of a level of nitrogen that presents a toxic metabolic load to the system is occurring (33
). There is evidence to suggest that at very high levels of protein consumption in control mice, the availability of some nonessential amino acids may be limited by the extent to which they are consumed in the detoxification of excess essential amino acids (33
), thus limiting growth. Conversely, the high protein diet may be increasing the availability of amino acids that are limiting in sickle cell disease and required for growth and repair. Adults with HbSS are reported to have a reduced plasma essential: nonessential amino acids ratio (34
), although urinary losses of amino acids are significantly reduced, compared with matched HbAA controls. Plasma arginine, leucine, valine, and histidine were among the most severely reduced essential amino acids. Both arginine and histidine are essential for growth (35
). Low circulating levels of other amino acids have been documented, such as cysteine (36
), the rate-limiting precursor for glutathione synthesis. Low cysteine levels in sickle cell disease may be related to low plasma pyridoxine levels (37
), because pyridoxal phosphate is a required cofactor for the conversion of homocysteine to cystathionine, a precursor for cysteine synthesis.
These results also demonstrate that sickle mice fed a high protein diet at weaning had lower circulating and liver mRNA levels of the sensitive markers of inflammation, CRP, and IL-6. Activated monocytes, endothelial cells, and fibroblasts produce IL-6, which functions in both innate and adaptive immunity. IL-6 then stimulates synthesis of acute phase proteins such as CRP via hepatocytes. Both inflammatory markers are key indicators of a predominantly proinflammatory pathway, activated by other proinflammatory cytokines such as tumor necrosis factor-α
). Although the cytokine profile of steady-state HbSS-mediated inflammation is still unfolding (39
), this study is the first report to our knowledge of a role for antiinflammatory effects of a high protein diet in a transgenic sickle cell mouse model.
Although determining the mechanism for the reduced inflammatory proteins was not the main purpose of this study, we tested the hypothesis that the sickle cell mice had increased L-arginine availability from the high protein diet for nitric oxide production and to prevent microvascular injury. Arginine deficiency in sickle cell disease (40
) is gaining interest, particularly because arginine is a substrate for the synthesis of nitric oxide, which is a potent vasodilator. Low nitric oxide availability is considered a central feature of endothelial dysfunction and ultimate vasoocclusive crises in sickle cell disease. A shift in arginine metabolism toward enhanced urea production via the arginase pathway vs. nitric oxide production via the nitric oxide synthase pathway has been demonstrated (9
). These observations concur with our previous findings of increased urea production rate and urea nitrogen retention in adults with homozygous sickle cell disease (5
) and our present results suggest that a high protein diet may correct the shift observed in arginine metabolism, as demonstrated by reduced liver arginase activity in sickle cell anemia. Kaul et al. (27
) recently demonstrated that arginine supplementation in sickle cell mouse models markedly increased nitric oxide generation. These results are consistent with arginine supplementation enhancing nitric oxide production in a clinical trial (42
). However, the 35% energy from protein supplied in our studies did not raise arginine levels in sickle mice, just as the Comprehensive Sickle Cell Center study of 0.05–0.1 g/kg did not increase arginine levels in sickle cell patients (43
), suggesting that the requirement for increased arginine is extremely high.
In summary, a high protein diet improved weight gain and reduced inflammatory proteins CRP and IL-6 in the Berkeley mouse model of sickle cell disease. We believe that the high protein diet is increasing the availability of amino acids, which have become limited in sickle cell disease, because of an increased demand on protein synthesis for red cell replacement. This increased metabolic demand is not satisfied by a diet that is adequate for healthy individuals with normal hemoglobin.