Francesco Agostini

Effects of E2F1-cyclin E1-E2 circuit down regulation in hepatocellular carcinoma cells

BACKGROUND No effective therapy is available for hepatocellular carcinoma. To identify novel therapeutic strategies, we explored the effects of the depletion of E2F1, cyclin E1-E2 whose inter-relationships in hepatocellular carcinoma cell proliferation have never been defined. METHODS siRNA-mediated depletion of the targets was studied in the hepatocellular carcinoma cells HepG2, HuH7 and JHH6 characterized by high, medium and low hepatocyte differentiation grade, respectively; a model of normal human hepatocytes was also considered. RESULTS The depletion of each target mRNA reduced the levels of the other two mRNAs, thus demonstrating a close regulatory control, also confirmed by over-expression experiments. At the protein level, an exception to this trend was observed for cyclinE1 whose amount increased upon cyclin E2 (HepG2, HuH7, JHH6) and E2F1 (HepG2) depletion. In HepG2, reduced cyclinE1 proteolysis accounted for this observation. Additionally, cyclin E1-E2-E2F1 targeting decreased the levels of cyclin A2 mRNA and of the hyper-phosphorylated form of pRb thus leading to an S-phase cell decrease; migration was impaired as well. Finally, the model of human hepatocytes used was clearly less affected by target mRNAs depletion than hepatocellular carcinoma cells. CONCLUSION Our data provide novel mutual relationships amongst cyclin E1-E2-E2F1 and indicate their role in sustaining hepatocellular carcinoma cell proliferation/migration, validating the concept of an anti-cyclin E1-E2-E2F1 therapeutic approach for hepatocellular carcinoma.

Effect of physical activity on glutamine metabolism

Purpose of reviewGlutamine is largely synthesized in skeletal muscles and provides fuel to rapidly dividing cells of the immune system and precursors to gluconeogenesis in the liver. Physical exercise is known to affect glutamine synthesis and to modulate glutamine uptake. Overtraining is frequently associated with reduced availability of glutamine and decreased immunocompetence. Inactivity affects glutamine metabolism, but this subject was poorly investigated. Recent findingsStrenuous physical exercise as well as exhaustive training programs lead to glutamine depletion due to lowered synthesis and enhanced uptake by liver and immune cells. Evidence suggests that postexercise glutamine depletion is associated with immunodepression. Counterwise, moderate training leads to improved glutamine availability due to a positive balance between muscle synthesis and peripheral clearance. Physical inactivity, as investigated by experimental bed rest in healthy volunteers, reduced glutamine synthesis and availability. SummaryAfter exercise, a reduced glutamine availability may be considered as a marker of overtraining. An increased glutamine availability may contribute to decreased inflammation and health benefits associated with optimal training. Thus, glutamine supplementation may enhance immunocompetence after strenuous exercise. The potential of glutamine supplementation during physical inactivity needs to be explored.

Effects of inactivity on human muscle glutathione synthesis by a double‐tracer and single‐biopsy approach

Oxidative stress is often associated to inactivity‐mediated skeletal muscle atrophy. Glutathione is one of the major antioxidant systems stimulated, both at muscular and systemic level, by activation of oxidative processes. We measured changes in glutathione availability, oxidative stress induction and the extent of atrophy mediated by 35 days of experimental bed rest in vastus lateralis muscle of healthy human volunteers. To assess muscle glutathione synthesis, we applied a novel single‐biopsy and double‐tracer ([2H2]glycine and [15N]glycine) approach based on evaluation of steady‐state precursor incorporation in product. The correlations between the traditional (multiple‐samples, one‐tracer) and new (one‐sample, double‐tracer infusion) methods were analysed in erythrocytes by Passing–Bablok and Altman–Bland tests. Muscle glutathione absolute synthesis rate increased following bed rest from 5.5 ± 1.1 to 11.0 ± 1.5 mmol (kg wet tissue)−1 day−1 (mean ±s.e.m.; n= 9; P= 0.02) while glutathione concentration failed to change significantly. Bed rest induced vastus lateralis muscle atrophy, as assessed by pennation angle changes measured by ultrasonography (from 18.6 ± 1.0 to 15.3 ± 0.9 deg; P= 0.01) and thickness changes (from 2.3 ± 0.2 to 1.9 ± 0.1 cm; P < 0.001). Moreover, bed rest increased protein oxidative stress, as measured by muscle protein carbonylation changes (from 0.6 ± 0.1 to 1.00 ± 0.1 Oxydized‐to‐total protein ratio; P < 0.04). In conclusion, we developed in erythrocytes a new minimally invasive method to determine peptide synthesis rate in human tissues. Application of the new method to skeletal muscle suggests that disuse atrophy is associated to oxidative stress induction as well as to compensatory activation of the glutathione system.

Inactivity-mediated insulin resistance is associated with upregulated pro-inflammatory fatty acids in human cell membranes.

BACKGROUND & AIMS Low-grade systemic inflammation and pro-inflammatory pattern of cell membrane fatty acid composition characterize patients affected by type 2 diabetes and metabolic syndrome. We hypothesize that inactivity-induced insulin resistance could affect levels of pro-inflammatory fatty acids in cell membranes. METHODS Thirty healthy, male, young volunteers were investigated before and after 35-day experimental bed rest. Diet composition was adapted to previous dietary habits. Fatty acid composition of erythrocyte membranes was analyzed by gas-chromatography using flame ionization detector. RESULTS Following bed rest, the HOMA index of insulin resistance significantly increased by +51+/-11% (P<0.01). Bed rest was associated with increased n-6 polyunsaturated (+4.7+/-2.2%; P<0.01) and decreased monounsaturated (-4.8+/-1.5%; P<0.01) fatty acid content in erythrocyte membranes. Fractional content of arachidonic acid increased by +14+/-12% (P=0.01) following inactivity. Delta5 and Delta9 desaturase indexes, as estimated from product-to-precursor ratios, significantly diminished following bed rest from 9.6+/-0.4 to 8.4+/-0.3 (P<0.001) and from 0.72+/-0.02 to 0.69+/-0.01 (P<0.05), respectively. The n-3 fatty acids, alpha-linolenic and eicosapentaenoic, were decreased (P=0.05) following inactivity by 4.7+/-13.2% and 3.8+/-5.2%, respectively. CONCLUSIONS Inactivity-mediated insulin resistance was associated with altered Delta5 and Delta9 desaturase indexes and with pro-inflammatory fatty acid pattern in erythrocyte membranes. These abnormalities could contribute to the low-grade inflammation associated to inactivity.

Prolonged Inactivity Up-Regulates Cholesteryl Ester Transfer Protein Independently of Body Fat Changes in Humans

CONTEXT Physical inactivity is associated with insulin resistance and decreased high-density lipoprotein (HDL) cholesterol. Cholesteryl ester transfer protein (CETP) is involved in cholesterol metabolism, being responsible for the transfer of cholesteryl esters from HDL to very low- and low-density lipoproteins. OBJECTIVE We hypothesized that physical inactivity could decrease HDL cholesterol through changes in CETP availability. DESIGN AND PARTICIPANTS Twenty-four healthy, male volunteers (aged 23.1 +/- 0.5 yr) were investigated in eucaloric conditions before and at the end of 35 d of experimental bed rest. MEASURES Changes in body composition were monitored by bioimpedance throughout the study. Before and at the end of the experimental period, plasma insulin and glucose and plasma lipid pattern as well as CETP concentrations were determined. RESULTS Our results demonstrated that during bed rest, fat mass did not change significantly, whereas fat-free mass decreased by 3.9 +/- 0.4% (P < 0.01). The homeostatic model assessment index of insulin resistance significantly (P < 0.001) increased by 47 +/- 11% after bed rest. Bed rest decreased HDL cholesterol by 12 +/- 3% (P < 0.05), increased triglycerides by 51 +/- 10% (P < 0.05), whereas it did not change significantly low-density lipoprotein cholesterol. Plasma CETP concentration increased after bed rest by 27 +/- 9% (P < 0.01). Bed rest-induced changes in CETP concentrations inversely correlated with changes in the ratio between HDL and non-HDL cholesterol (n = 24; R = -0.43; P < 0.05). CONCLUSIONS Physical inactivity decreases HDL cholesterol, at least in part, through CETP up-regulation.

Physical inactivity decreases whole body glutamine turnover independently from changes in proteolysis

Glutamine synthesis and utilization are strictly linked to energy metabolism and physical activity. To investigate the interaction between bed rest and moderate energy restriction on whole body glutamine kinetics in healthy volunteers, we performed a four‐period study in which each subject randomly underwent a bed rest or ambulatory 14 day period both in eucaloric or hypocaloric (−20% of energy requirement) conditions. Glutamine kinetics were measured by l‐[5‐15N]glutamine and l‐[1‐13C]leucine primed continuous infusions in the postabsorptive state and during a 3 h infusion of a glutamine‐free amino acid mixture (0.13 g amino acids (kg lean body mass (LBM))−1 h−1). Bed rest decreased glutamine de novo synthesis in the postabsorptive state both in eucaloric (from 4.17 ± 0.14 to 3.56 ± 0.13 μmol (kg LBM)−1 min−1; P < 0.001) and in hypocaloric (from 3.79 ± 0.19 to 3.49 ± 0.14 μmol (kg LBM)−1 min−1; P < 0.001) conditions, independently of changes in whole body proteolysis. Bed rest did not affect glutamine clearance. We failed to detect either significant effects of energy intake or energy × activity interactions on glutamine kinetics. Bed rest significantly decreased postabsorptive plasma glutamine concentrations (P < 0.05). Amino acid infusion increased glutamine de novo synthesis rate and plasma glutamine concentrations in all conditions, without significant effects of bed rest or energy levels. We conclude that inactivity is associated with decreased whole body glutamine availability due to down‐regulated de novo synthesis.