Scienze biologiche

Dietary proteins are the source of the amino acids required by the body for tissue growth and maintenance. The Population Reference Intake (PRI) for proteins, as defined by the European Food Safety Authority (EFSA) for healthy adults, including the elderly, is 0.83 g/kg body weight/day. This amount is defined on the net balance of body protein (or “nitrogen balance”, given by the difference between dietary nitrogen intake and losses) equivalent to 0.66 g/kg/day plus a safety factor for interpersonal variability and differences in proteins quality of mixed diets. The PRI, however, is the minimum daily amount of protein needed to maintain the nitrogen balance and avoid a progressive loss of lean body mass in healthy people with moderate physical activity. Therefore nitrogen balance may not be adequate to define protein requirement in adults and especially in ageing characterized by loss of muscle mass and function (sarcopenia). Furthermore until recently the prevalent idea was that a protein intake above PRI had no further benefits and on the contrary could impair health. These believes are now under discussion, diets with higher protein intake have been shown beneficial in the prevention and treatment of conditions such as sarcopenia, COPD and type 2 diabetes mellitus. There is a need of more precise methods to define protein requirement. AIM. The aim of the present thesis is to investigate in human healthy volunteers new biomarkers adequate to define optimal protein intake. Recent studies have determined protein needs by measuring whole-body protein metabolism using stable labeled isotope-amino acids. METHODS. Our research group has applied two different metabolic methods based on the most widely used tracer, i.e. D5-Phe stable isotope, in two experimental bed rest campaigns (FP7 PLANHAB and INTERREG PANGaA) in healthy volunteers. BR is a suitable model to investigate physiologic adaptation to inactivity. MAIN RESUTLTS. FP7 PLANHAB. We applied the stable isotope infusion technique, to assess the effect of physical inactivity and/or hypoxic condition on whole body protein turnover as previously described in Biolo et al 2008. Chronic hypoxia has been associated with an overall reduction in protein synthesis and in total plasma and skeletal muscle protein content. During the PLANHAB study we investigated, through a crossover randomization, the net effects of 10 days normobaric hypoxia (4000 mt.), associated with either ambulatory conditions or BR, in 11 young (age 24±4 yr), healthy and normal weight male subjects maintained on eucaloric diets. Main results. Hypoxia in ambulatory conditions significantly decreased whole body protein turnover by reducing both protein synthesis (-8±2%) and protein degradation (-8±3%). Hypoxia during bed rest did not caused significant changes in protein metabolism. INTERREG PANGaA. The skeletal muscle loss in aging is caused mainly by the “anabolic resistance” i.e. the inadequate increase in the rate of protein synthesis in response to nutritional-metabolic stimuli, including exercise, protein and amino acid intake as well as insulin and insulin-like growth factor stimulation. As a consequence, the net protein balance becomes negative leading to sarcopenia. The effects of ageing on the anabolic resistance induced by inactivity are poorly investigated. During the PANGeA study we had the opportunity to perform the second documented experimental BR in in healthy elderly volunteers and the first comparing aged with young subjects. To evaluate the anabolic resistance associated with ageing and inactivity, we enrolled 7 young (23±1yr) and 8 elderly (59±1yr) normal weight individuals, in a 14-d experimental BR protocol. We replaced our previous infusion method with a new, simpler, safer and quicker technique, by which tracers are given orally instead of parenterally, the all procedure is completed in two hours, instead of 6, and only two blood draws versus 7 are sufficient. Main results. At baseline parameters of anabolic sensitivity were comparable between young and elderly individuals. The anabolic resistance significantly increased after BR in both groups (bed-rest effect p<0.01), with a statistically significant bed-rest×group interaction (p=0.01). Anabolic resistance increased significantly in elderly (18.5%±7.3%) more than in young (5.2%±9.4%) subjects. DISCUSSION. In the PLANHAB study, hypoxia in ambulatory conditions reduced by the same level both protein synthesis and catabolism, as measured by isotope infusions, suggesting an adaptive mechanism: the lower energy production and availability induced by hypoxia associated with ambulatory condition. These modifications could not have been revealed by the use of nitrogen balance method, showing the relevance of more sophisticated analysis. The direct evaluation of the muscle protein metabolism through an infusion of stable-labeled isotope tracer, considered the golden standard methodology, gave us, in the PLANHAB study, reliable results in the early protein metabolism changes during hypoxia and/or BR. This method however has the limit of being complex, onerous and invasive, therefore being unsuitable for clinical evaluation. In the PANGeA study we could confirm the presence of a reduced sensitivity to anabolic stimuli in the elderly population compared to the young men. The elderly subjects are therefore, more at risk to develop changes of protein metabolism induced by inactivity. The simpler, timesaving and less invasive method we have developed for the PANGeA study, on the other hand, could be applied to a wider ranges of experimental conditions and clinical settings.

Aims: The mechanisms involved in sarcopenia, the decline in muscle mass with aging coupled with loss of force and function, has been actively investigated in animal and human models over the last years [reviewed in Di Iorio et al., Sarcopenia: age-related skeletal muscle changes from determinants to physical disability, Int. J. Immunopathol. Pharmacol. 19 (2006) 703-719]. An important age-associated deficit may be the alteration of the mechanisms controlling Ca2+ handling. Moreover, it has already been proposed that defective fibres in old humans could result from a reduced efficiency of aged satellite cells (a distinct muscle cell subtype, responsible for post-natal growth and repair of damaged fibres) in properly differentiating into myotubes with a mature E-C coupling mechanism [see: Lorenzon et al., Aging affects the differentiation potential of human myoblasts, Exp. Gerontol. 39 (2004) 1545-1554]. Proceeding from these results, the main goal of the present Ph.D. thesis was to investigate whether the inefficiency of aged satellite cells to generate functional skeletal muscle fibres could be partly due to defective voltage-dependent Ca2+ currents. Methods: The whole-cell patch clamp and the videoimaging techniques were employed to measure respectively T- and L-type Ca2+ currents and [Ca2+]i transients in myoblasts and/or myotubes derived from murine and human satellite cells, obtained respectively from young murine skeletal muscle and then aged in vitro under culture conditions, and from human skeletal muscle tissue of healthy donors aged 2, 12, 76 and 86 years. Results: First of all, I confirmed that both murine and human senescent satellite cells fuse more slowly and less efficiently, leading to smaller and thinner myotubes, as known from previous work. Moreover, I showed for the first time that both in myotubes derived from in vitro aged murine satellite cells and in human myotubes derived from satellite cells of old donors the functional expression and the biophysical properties of T- and L-type voltage-dependent Ca2+ channels are impaired. In fact, extensively, less Ca2+ can be available via T-type and L-type channels in old myotubes than in the young ones, and this can be put in relation to the age-related decrease in the quality of myoblast fusion. I also confirmed a specific responsibility of the decrease of the L-type channel number and/or activity for the age-related lowering of intracellular Ca2+ release (the so-called E-C uncoupling; see: Delbono et al., Excitation-calcium release uncoupling in aged single human skeletal muscle fibers, J. Membr. Biol. 148 (1995) 211-222]. Conclusions: From these results one can infer a clear parallelism between the results obtained with the in vitro aging of murine satellite cells model and that concerning the physiological process of human skeletal muscle aging in vivo. In the final analysis, aging effects on voltage-dependent L- and T-type currents could be one of the causes of the inability of old satellite cells to efficiently counteract age-related impairment in muscle force. So, a further strong evidence has been given that in humans, as in other mammals, the satellite cells and the regulation of Ca2+ homeostasis have a decisive role in the physiological process of skeletal muscle aging.