Diet and gender influence survival of transgenic Berkley sickle cell mice

The prevalence of sickle cell disease (SCD) is highest in regions in which there is global malnutrition with high infant and early childhood mortality. In sub-Saharan Africa, 5-year olds are at the highest risk of death. In tribal communities of India, 20% of children with SCD die by the age of 2 years and 30% die before reaching adulthood. Additionally, individuals with SCD may suffer from disease-related malnutrition as a result of increased demands for absorbed nutrients, caused by multiple pathological factors including higher resting basal metabolic rate. Deficiencies of many nutrients, including vitamins, zinc, magnesium, and fatty acids are well-documented in SCD. Nutritional deficiencies may begin during gestation because of poor parental diet, which adds to the existing challenge of examining the effect of diet on survival of individuals with SCD. Transgenic mouse models of SCD including homozygous Berkley (HbSS-BERK) expressing exclusively human sickle hemoglobin replicate the pathophysiology of human SCD and provide an opportunity to examine the effect of dietary interventions across generations. We examined whether a diet enriched in protein, amino acids, fatty acids, and specific critical micronutrients (Table 1) fed to parents and/or offspring would improve the survival of offsprings with SCD. We used protocols approved by the Institutional Animal Care and Use Committee of the University of Minnesota. HbSS-BERK mice offspring (henceforth called pups) expressing >99% human sickle hemoglobin were used. Because homozygous BERK female mice often do not breed/survive pregnancy, we used heterozygous BERK female (HbAS-BERK) and male HbSS-BERK as breeding pairs (Figure 1). Breeders and pups were randomly assigned to either a sickle diet (SD) or regular diet (RD) (Table 1) to differentiate groups by pup diet, parental diet, and gender (Figure 1). After phenotyping, only the homozygous pups were included in the study. Breeder pairs were divided into two dietary groups, SD and RD, as shown in Figure 1. Compared to the RD, the SD has a higher content of protein and fat calories and contains higher amounts of specific minerals, vitamins, amino acids, and omega-3-fatty acids (Table 1). Pups were weaned 3 weeks after birth and fed the SD for 10 days after weaning and then randomly assigned to either the SD or RD until 150 days of age. A multivariate analysis was performed on the relationship between rates of survival and the following factors of interest: pup gender, pup diet, and parental diet. The outcome variable of interest, survival in days, was examined using KaplanMeier plots of estimated survival and Cox regression to obtain hazard ratios (HR) with 95% confidence intervals (95% CI). For Cox regression, the significance of individual factors in multivariate analysis and the significance of overall survival differences were based on Wald and logrank tests, respectively. Data were analyzed using the ‘survival’ package in ‘R 3.4. 0’ (R Foundation for Statistical Computing; Vienna, Austria). P values <0.05 were considered statistically significant. We first studied the effect of diet on survival of HbSSBERK pups. Of 601 pups, 482 were given the SD and 119 were given the RD. At 150 days, 77% of pups on the SD and 51% of pups on the RD were alive (Figure 2A). According to the multivariate analysis, pups fed the SD had significantly prolonged survival compared to pups on the RD (HR: 0.3988; 95% CI: 0.2860-0.5561; P=6.03x10). Dietary supplementation with individual nutrients has been shown to attenuate pathogenic mechanisms and reduce morbidity associated with SCD. A high protein diet attenuated ischemic end-organ damage, increased muscle mass, bone mineral density, and grip strength, and decreased vascular leakage in HbSS-BERK or Townes sickle mice. Omega-3-fatty acid supplementation decreased NF-κB activation, adhesion molecule expression, and inflammatory markers and reduced the frequency of vaso-occlusive crises and transfusion requirements in patients with SCD. Zinc supplementation improved growth and decreased the incidence of infection. Folic acid is critical for DNA synthesis and does not have body stores. A compensatory increase in erythropoiesis in SCD results in folate deficiency due to over-utilization. In HbSS-BERK mice, arginine supplementation markedly enhanced nitric oxide bioavailability, and decreased both hemolysis and oxidative stress. Arginine supplementation alleviated pain induced by vaso-occlusive crises and decreased narcotic use by >50% in a randomized placebo-controlled clinical trial. In a comparison with placebo administration, L-glutamine supplementation (0.6 g/Kg/day) reduced the average cumulative recurrence of vaso-occlusive crises but significantly increased musculoskeletal, abdominal and chest pain in patients with SCD in a phase III clinical trial. The SD includes these nutrients but in significantly smaller amounts, which may synergize to meet the nutritional requirements in SCD without causing off-target effects. Therefore, supplementation of diet with a combination of multiple nutrients may be more beneficial than the use of individual nutrients at high doses. We next studied the effects of gender on survival of HbSS-BERK pups. We compared the survival of 338 female pups and 263 male pups independent of their