Aleardo Koverech

Cellular stress responses, hormetic phytochemicals and vitagenes in aging and longevity.

Modulation of endogenous cellular defense mechanisms represents an innovative approach to therapeutic intervention in diseases causing chronic tissue damage, such as in neurodegeneration. This paper introduces the emerging role of exogenous molecules in hormetic-based neuroprotection and the mitochondrial redox signaling concept of hormesis and its applications to the field of neuroprotection and longevity. Maintenance of optimal long-term health conditions is accomplished by a complex network of longevity assurance processes that are controlled by vitagenes, a group of genes involved in preserving cellular homeostasis during stressful conditions. Vitagenes encode for heat shock proteins (Hsp) Hsp32, Hsp70, the thioredoxin and the sirtuin protein systems. Dietary antioxidants, such as polyphenols and L-carnitine/acetyl-L-carnitine, have recently been demonstrated to be neuroprotective through the activation of hormetic pathways, including vitagenes. Hormesis provides the central underpinning of neuroprotective responses, providing a framework for explaining the common quantitative features of their dose response relationships, their mechanistic foundations, their relationship to the concept of biological plasticity as well as providing a key insight for improving the accuracy of the therapeutic dose of pharmaceutical agents within the highly heterogeneous human population. This paper describes in mechanistic detail how hormetic dose responses are mediated for endogenous cellular defense pathways including sirtuin, Nrfs and related pathways that integrate adaptive stress responses in the prevention of neurodegenerative diseases. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.

CARNITINE REPLACEMENT IN END-STAGE RENAL DISEASE AND HEMODIALYSIS

Abstract: In patients with chronic renal failure, not yet undergoing hemodialysis (HD), plasma acylcarnitines accumulate in part due to a decreased renal clearance of esterified carnitine moieties. In these patients, a high acylcarnitine/free‐carnitine ratio is usually found in plasma. Patients undergoing maintenance HD, usually present with plasma carnitine insufficiency, due to accumulation of metabolic intermediates combined with impaired carnitine biosynthesis, reduced protein intake and increased removal via HD. Plasma carnitine concentrations rapidly decrease to 40% of baseline level during the dialysis session, with a slow restoration of the carnitine concentration during the interdialytic period, mainly from organs of storage (skeletal muscle). Dietary intake also plays an important role in carnitine homeostasis of HD patients since the prevalence of malnutrition ranges from 18% to 75% of these cases. This could differentially affect various body compartments, with clinical consequences such as impaired muscle function, decreased wound healing, altered ventilatory response, and abnormal immune function. Repeated hemodialytic treatments are associated with decreased carnitine stores in skeletal muscle. The administration of intravenous l‐carnitine (LC) postdialysis replenishes the free carnitine removed from the blood and contributes to replenishment of muscle carnitine content. LC supplementation in selected uremic patients may yield clinical benefits by ameliorating several conditions, such as erythropoietin‐resistant anemia, decreased cardiac performance, intradialytic hypotension, muscle symptoms, as well as impaired exercise and functional capacities. Furthermore, LC may positively influence the nutritional status of HD patients by promoting a positive protein balance, and by reducing insulin resistance and chronic inflammation, possibly through an effect on leptin resistance.

Upregulation of mGlu2 Receptors via NF-κB p65 Acetylation Is Involved in the Proneurogenic and Antidepressant Effects of Acetyl-L-Carnitine

Acetyl-L-carnitine (ALC) is a naturally occurring molecule with an important role in cellular bioenergetics and as donor of acetyl groups to proteins, including NF-κB p65. In humans, exogenously administered ALC has been shown to be effective in mood disturbances, with a good tolerability profile. No current information is available on the antidepressant effect of ALC in animal models of depression and on the putative mechanism involved in such effect. Here we report that ALC is a proneurogenic molecule, whose effect on neuronal differentiation of adult hippocampal neural progenitors is independent of its neuroprotective activity. The in vitro proneurogenic effects of ALC appear to be mediated by activation of the NF-κB pathway, and in particular by p65 acetylation, and subsequent NF-κB-mediated upregulation of metabotropic glutamate receptor 2 (mGlu2) expression. When tested in vivo, chronic ALC treatment could revert depressive-like behavior caused by unpredictable chronic mild stress, a rodent model of depression with high face validity and predictivity, and its behavioral effect correlated with upregulated expression of mGlu2 receptor in hippocampi of stressed mice. Moreover, chronic, but not acute or subchronic, drug treatment significantly increased adult born neurons in hippocampi of stressed and unstressed mice. We now propose that this mechanism could be potentially involved in the antidepressant effect of ALC in humans. These results are potentially relevant from a clinical perspective, as for its high tolerability profile ALC may be ideally employed in patient subpopulations who are sensitive to the side effects associated with classical antidepressants.

Carnitine-Acyltransferase System Inhibition, Cancer Cell Death, and Prevention of Myc-Induced Lymphomagenesis

BACKGROUND The metabolic alterations of cancer cells represent an opportunity for developing selective antineoplastic treatments. We investigated the therapeutic potential of ST1326, an inhibitor of carnitine-palmitoyl transferase 1A (CPT1A), the rate-limiting enzyme for fatty acid (FA) import into mitochondria. METHODS ST1326 was tested on in vitro and in vivo models of Burkitts lymphoma, in which c-myc, which drives cellular demand for FA metabolism, is highly overexpressed. We performed assays to evaluate the effect of ST1326 on proliferation, FA oxidation, and FA mitochondrial channeling in Raji cells. The therapeutic efficacy of ST1326 was tested by treating Eµ-myc mice (control: n = 29; treatment: n = 24 per group), an established model of c-myc-mediated lymphomagenesis. Experiments were performed on spleen-derived c-myc-overexpressing B cells to clarify the role of c-myc in conferring sensitivity to ST1326. Survival was evaluated with Kaplan-Meier analyses. All statistical tests were two-sided. RESULTS ST1326 blocked both long- and short-chain FA oxidation and showed a strong cytotoxic effect on Burkitts lymphoma cells (on Raji cells at 72 hours: half maximal inhibitory concentration = 8.6 μM). ST1326 treatment induced massive cytoplasmic lipid accumulation, impairment of proper mitochondrial FA channeling, and reduced availability of cytosolic acetyl coenzyme A, a fundamental substrate for de novo lipogenesis. Moreover, treatment with ST1326 in Eµ-myc transgenic mice prevented tumor formation (P = .01), by selectively impairing the growth of spleen-derived primary B cells overexpressing c-myc (wild-type cells + ST1326 vs. Eµ-myc cells + ST1326: 99.75% vs. 57.5%, difference = 42.25, 95% confidence interval of difference = 14% to 70%; P = .01). CONCLUSIONS Our data indicate that it is possible to tackle c-myc-driven tumorigenesis by altering lipid metabolism and exploiting the neoplastic cell addiction to FA oxidation.

Acetyl-l-carnitine increases artemin level and prevents neurotrophic factor alterations during neuropathy

Damages to the nervous system are the primarily cause of neuropathy and chronic pain. Current pharmacological treatments for neuropathic pain are not able to prevent or revert morphological and molecular consequences of tissue injury. On the other hand, many neurotrophins, like nerve growth factor (NGF), paired off restorative effects with hyperalgesia. Interestingly, the glial cell line-derived neurotrophic factors GDNF and Artemin (ARTN) seem to support neuron survival and to normalize abnormal pain behaviour. In the present research protein levels of NGF, GDNF and ARTN were evaluated in a rat model of peripheral neuropathy, the chronic constriction injury (CCI). NGF was increased by CCI in the ipsilateral dorsal root ganglia (DRG), in the spinal cord and in the periaqueductal grey matter (PAG). On the contrary, ARTN was decreased bilaterally in DRG, spinal cord and PAG. GDNF levels decreased in ipsilateral DRG, whereas the constriction did not modify its expression in the central nervous system districts. Repeated treatments with the antihyperalgesic and neuroregenerative compound acetyl-l-carnitine (ALCAR; 100 mgkg(-1) i.p. twice daily for 15 days) was able to prevent the increase of NGF levels. In conditions of pain relief ALCAR normalized peripheral and central alterations of GDNF and ARTN levels. Characteristically, sham animals that underwent the same ALCAR treatment, showed increased levels of ARTN both in the DRG and in the spinal cord. These data offer a new point of view on the mechanism of the antihyperalgesic as well as the neuroprotective effect of ALCAR.

Preliminary observations on the use of propionyl-L-carnitine in combination with sildenafil in patients with erectile dysfunction and diabetes.

SUMMARY Purpose: To investigate the efficacy and tolerability of oral propionyl-L-carnitine (PLC) plus sildenafil in men with erectile dysfunction (ED) and diabetes unresponsive to sildenafil monotherapy. Materials and methods: Patients with medically documented ED of organic or mixed aetiology and diabetes (type 1 and 2) were randomised to receive oral PLC (2 g/day) plus sildenafil (50 mg twice weekly) (20 patients, Group 1) or sildenafil alone (20 patients, Group 2), in a double-blind, fixed-dose study. All patients had been previously treated unsuccessfully with a minimum of eight administrations of sildenafil. Efficacy was evaluated using the International Index of Erectile Function (IIEF) questionnaire: total score, subscores for questions 3 (Q3; achieving an erection) and 4 (Q4; maintaining an erection) and global efficacy question (GEQ: ‘Has treatment improved your erections?’). Patients Event Logs were also used. Results: After 24 weeks of treatment, mean scores for IIEF Q3 and Q4 had improved significantly in patients of Group 1 (4.25 ± 0.63 and 3.95 ± 1.0) compared with Group 2 (2.9 ± 0.71 and 2.7 ± 0.96) ( p < 0.01). Moreover, the percentage of patients with improved erections (GEQ 68% vs. 23%) and successful intercourse attempts (76% vs. 34%) was significantly increased in Group 1 compared with Group 2 ( p < 0.01). Fourteen (70%) patients in Group 1 and four (20%) in Group 2 reported an increase in mean IIEF EF domain score of ≥ 4 ( p < 0.01). Treatments were well tolerated and no patient discontinued study medication. Two patients in Group 1 reported mild gastric pain. Conclusions: Salvage therapy with PLC plus sildenafil was more effective than sildenafil in the treatment of ED in patients with diabetes refractory to sildenafil monotherapy.

L-carnitine and short chain ester in tears from patients with dry eye.

Purpose. The tear film is essential for the integrity of the ocular surface. In ocular diseases such as dry eye syndrome (DES), tear film osmolarity is increased relative to normal physiological conditions. DES can be caused by deficiency in lachrymation, hyperevaporation, or surface alterations. Carnitines, shown to have osmoregulatory properties, are thought to regulate tear film osmolarity, thus protecting the corneal surface from damage. We investigated the presence of carnitine in tears, compared tear carnitine concentrations in healthy subjects and in DES patients and speculate on carnitines potential role as a protective agent in the tear film. Methods. Tears were collected from 10 healthy subjects and 10 DES patients. Carnitine levels were assessed by high performance liquid chromatography-mass spectrometry. Results. Carnitine and its derivatives were detected in the tear samples. In DES patients, concentrations were substantially lower than in healthy subjects; the mean concentrations were l-carnitine, 3.27 ± 0.80 and 8.94 ± 0.50 &mgr;Mol/L; l-acetylcarnitine, 1.66 ± 0.50 and 3.05 ± 0.65 &mgr;Mol/L; and l-propionylcarnitine, 0.30 ± 0.11 and 0.57 ± 0.13 &mgr;Mol/L, in DES patients and healthy subjects, respectively. Conclusions. Although increased tear film osmolarity has been previously observed in DES patients, our study showed lower carnitine levels in DES patients than in healthy subjects, rather than the increased levels expected, although a causal relationship between carnitine levels and hyperosmolarity has not been established. The damage to ocular surface cells because of exposure to hypertonic tear film observed in DES may be partially because of an imbalance in the concentration of carnitine molecules in the tear film relative to the ocular surface cells. We propose, therefore, that carnitine solutions may have a role in preventing the adverse effects of observed hyperosmolarity and suggest that further studies are now warranted to investigate the clinical application of carnitine in the treatment of DES.

Effect of propionyl-l-carnitine, l-arginine and nicotinic acid on the efficacy of vardenafil in the treatment of erectile dysfunction in diabetes

ABSTRACT Objective: The association of diabetes-related vascular damage and the role of metabolic factors in erectile dysfunction are well known in the literature. The compounds propionyl-l-carnitine (PLC), l-arginine (l-Arg) and nicotinic acid have numerous metabolic actions which have been reported to improve endothelial function. This study investigated the administration of the combination of these three compounds alone and in association with an inhibitor of 5-phosphodiesterase (5PDE), vardenafil, on endothelial function in diabetic patients with erectile dysfunction. Methods: A total of 40 patients aged between 50 and 60 years with insulin-dependent diabetes (IDDM) for 3–4 years were selected from 509 patients presenting with erectile dysfunction. The patients were randomly subdivided into four groups of ten to be treated for 12 weeks. Group A was administered one sachet each day of test formulation containing PLC, l-Arg and nicotinic acid (Ezerex); group B with one 20 mg capsule of vardenafil (Levitra) twice a week; group C was treated with one sachet each day of the test formulation plus vardenafil 20 mg twice a week. Group D was administered placebo capsules twice weekly. Endothelial function was evaluated by examining flow-mediated dilation (FMD) and erectile function was estimated with the International Index of Erectile Function (IIEF5) questionnaire in all subjects. Results: At the end of treatment group A showed an increment of 2 points in the IIEF5; group B showed an increment of 4 points; group C, the group which was administered all the treatments, showed an increment of 5 points, and group D, treated with placebo, showed no increment in the IIEF5. Conclusion: Although there was a small number of subjects in this study the data suggest that the test formulation may improve the endothelial situation in diabetes. The test formulation together with vardenafil was better than the 5PDE inhibitor alone, but further studies are needed to confirm these findings.

l-Carnitine enhances extracellular matrix synthesis in human primary chondrocytes

Osteoarthritis (OA) is one of the most common degenerative joint disease for which there is no cure. It is treated mainly with non-steroidal anti-inflammatory drugs to control the symptoms and some supplements, such as glucosamine and chondroitin sulphate in order to obtain structure-modifying effects. Aim of this study is to investigate the effects of l-carnitine, a molecule with a role in cellular energy metabolism, on extracellular matrix synthesis in human primary chondrocytes (HPCs). Dose-dependent effect of l-carnitine on cartilage matrix production, cell proliferation and ATP synthesis was examined by incubating HPCs with various amounts of molecule in monolayer (2D) and in hydromatrix scaffold (3D). l-Carnitine affected extracellular matrix synthesis in 3D in a dose-dependent manner; moreover, l-carnitine was very effective to stimulate cell proliferation and to induce ATP synthesis, mainly in 3D culture condition. In conclusion, l-carnitine enhances cartilage matrix glycosaminoglycan component production and cell proliferation, suggesting that this molecule could be useful in the treatment of pathologies where extracellular matrix is degraded, such as OA. To our knowledge, this is the first study where the effects of l-carnitine are evaluated in HPCs.

Propionyl-L-carnitine in Leriche-Fontaine stage II peripheral arterial obstructive disease.

Peripheral arterial obstructive disease (PAOD) of the lower limbs affects 5% of the adult population. Uncontrolled arteriopathy is established due to a microcirculatory deficit, which may be present despite a good Winsor index and which leads to exhaustion of the functional microcirculatory reserve. The target of this study was to examine possible improvements in microvascular and tissue homeostasis by the administration of propionyl-L-carnitine (PLC). A total of 26 patients were enrolled in this study, aged 65 +/- 15 years; two males were diagnosed at stage IIA and 17 males and seven females at stage IIB PAOD. The main criterion of inclusion was the worsening of walking distance during the last month. In this study the duration of therapy was 33 days. PLC was administered in three flasks, each containing 300 mg in 250 cc saline by continuous infusion. The following parameters were measured before and after treatment: pain-free and maximum walking distance (measured on a treadmill at 3.2 km/hr with a gradient of 12%), recovery time from pain after maximum walking distance, ankle-brachial index by means of the Doppler apparatus, and evaluation of the microcirculation using capillaroscopy. The results showed that therapy with PLC was effective at restoring activity of skeletal muscle in ischemic conditions. In particular, capillaroscopy showed improvement in the angioarchitecture in the microcirculation fields, expressed as increased numbers of visible capillaries and diminution in the time of loss of sodium fluorescein marker. The clinical data showed increased walking distance and diminished time to recover from pain, and the clinical improvement correlated with improved microcirculatory function. From these preliminary data has emerged an indication of therapy with PLC for chronic obstructive arteriopathy of the lower limbs at stage II. Further studies with higher numbers of patients and more controlled variables are planned.