Bone fragility and associated fracture risk are major problems in aging. Oxidative stress and mitochondrial dysfunction play a key role in the development of bone fragility. Mitochondrial dysfunction is closely associated with excessive production of reactive oxygen species (ROS). L-Carnitine (L-C), a fundamental cofactor in lipid metabolism, has an important antioxidant property. Several studies have shown how L-C enhances osteoblastic proliferation and activity. In the current study, we investigated the potential effects of L-C on mitochondrial activity, ROS production, and gene expression involved in osteoblastic differentiation using osteoblast-like cells (hOBs) derived from elderly patients. The effect of 5mM L-C treatment on mitochondrial activity and L-C antioxidant activity was studied by ROS production evaluation and cell-based antioxidant activity assay. The possible effects of L-C on hOBs differentiation were assessed by analyzing gene and protein expression by Real Time PCR and western blotting, respectively. L-C enhanced mitochondrial activity and improved antioxidant defense of hOBs. Furthermore, L-C increased the phosphorylation of Ca2+/calmodulin-dependent protein kinase II. Additionally, L-C induced the phosphorylation of ERK1/2 and AKT and the main kinases involved in osteoblastic differentiation and upregulated the expression of osteogenic related genes, RUNX2, osterix (OSX), bone sialoprotein (BSP), and osteopontin (OPN) as well as OPN protein synthesis, suggesting that L-C exerts a positive modulation of key osteogenic factors. In conclusion, L-C supplementation could represent a possible adjuvant in the treatment of bone fragility, counteracting oxidative phenomena and promoting bone quality maintenance.

BackgroundBetaine (BET), a component of many foods, is an essential osmolyte and a source of methyl groups; it also shows an antioxidant activity. Moreover, BET stimulates muscle differentiation via insulin like growth factor I (IGF-I). The processes of myogenesis and osteogenesis involve common mechanisms with skeletal muscle cells and osteoblasts sharing the same precursor. Therefore, we have hypothesized that BET might be effective on osteoblast cell differentiation.MethodsThe effect of BET was tested in human osteoblasts (hObs) derived from trabecular bone samples obtained from waste material of orthopedic surgery. Cells were treated with 10 mM BET at 5, 15, 60 min and 3, 6 and 24 h. The possible effects of BET on hObs differentiation were evaluated by real time PCR, western blot and immunofluorescence analysis. Calcium imaging was used to monitor intracellular calcium changes.ResultsReal time PCR results showed that BET stimulated significantly the expression of RUNX2, osterix, bone sialoprotein and osteopontin. Western blot and immunofluorescence confirmed BET stimulation of osteopontin protein synthesis. BET stimulated ERK signaling, key pathway involved in osteoblastogenesis and calcium signaling. BET induced a rise of intracellular calcium by means of the calcium ions influx from the extracellular milieu through the L-type calcium channels and CaMKII signaling activation. A significant rise in IGF-I mRNA at 3 and 6 h and a significant increase of IGF-I protein at 6 and 24 h after BET stimulus was detected. Furthermore, BET was able to increase significantly both SOD2 gene expression and protein content.ConclusionsOur study showed that three signaling pathways, i.e. cytosolic calcium influx, ERK activation and IGF-I production, are enhanced by BET in human osteoblasts. These pathways could have synergistic effects on osteogenic gene expression and protein synthesis, thus potentially leading to enhanced bone formation. Taken together, these results suggest that BET could be a promising nutraceutical therapeutic agent in the strategy to counteract the concomitant and interacting impact of sarcopenia and osteoporosis, i.e. the major determinants of senile frailty and related mortality.

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Recent evidences indicate early, diabetes-driven, retinal neurodegeneration as the origin of diabetic retinopathy. To verify the possibility to prevent the disease, we investigated in a mouse model of type 1 diabetes the effect of long-lasting hyperglycemia on retinal and choroidal structures and, in parallel, we tested the effect of topical treatment with the neuroprotective agent citicoline. Forty wild-type C57B6 mice were included in this study. Diabetes was induced by a single intravenous injection of alloxan. Five animals were considered as sham-treated controls, 15 animals as sham-treated diabetic mice, 5 animals as citicoline-treated controls and 15 animals as citicoline-treated diabetic mice. After eight months of diabetes in vivo analysis of the retina was performed using the Spectralis HRA (Heidelberg Retinal Angiography) + OCT. Neuroretinal abnormalities, in particular a significant narrowing of Retinal Nerve Fiber Layer (19.3 ± 2.2 vs 23.3 ± 2.4, ?m ± SD, p=0.01), Ganglion Cells/Inner Plexiform Layer (54.3 ± 5.1 vs 62.6 ± 4.0, p=0.03), Ganglion Cells Complex (73.9 ± 4.8 vs 83.8 ± 3.4, p=0.003) and Retinal thickness (223.8 ± 3.9 vs 236.7 ± 5.8, p=0.0004) were detected in the diabetic mouse that showed also a significant reduction of Choroidal thickness (67.4 ± 3.3 vs 84.7 ± 1.9, p=0.0001). In line with the hypothesis that neuroprotection might help preventing diabetic retinopathy, neuroretinal but not choroidal (choroid lacks a neuronal component) dysfunctions were prevented by citicoline. Altogether these findings demonstrate that diabetes-driven neuroretinal dysfunctions can be monitored in vivo by OCT in the mouse. Retinal neuroprotection as obtained by topical citicoline protects from these abnormalities suggesting this approach as a possible way to prevent diabetic retinopathy.

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