Menotti Calvani

Nitric oxide in the central nervous system: neuroprotection versus neurotoxicity

At the end of the 1980s, it was clearly demonstrated that cells produce nitric oxide and that this gaseous molecule is involved in the regulation of the cardiovascular, immune and nervous systems, rather than simply being a toxic pollutant. In the CNS, nitric oxide has an array of functions, such as the regulation of synaptic plasticity, the sleep–wake cycle and hormone secretion. Particularly interesting is the role of nitric oxide as a Janus molecule in the cell death or survival mechanisms in brain cells. In fact, physiological amounts of this gas are neuroprotective, whereas higher concentrations are clearly neurotoxic.

Polyunsaturated fatty acids: Biochemical, nutritional and epigenetic properties

Dietary polyunsaturated fatty acids (PUFA) have effects on diverse physiological processes impacting normal health and chronic diseases, such as the regulation of plasma lipid levels, cardiovascular and immune function, insulin action and neuronal development and visual function. Ingestion of PUFA will lead to their distribution to virtually every cell in the body with effects on membrane composition and function, eicosanoid synthesis, cellular signaling and regulation of gene expression. Cell specific lipid metabolism, as well as the expression of fatty acid-regulated transcription factors, likely play an important role in determining how cells respond to changes in PUFA composition. This review will focus on recent advances on the essentiality of these molecules and on their interplay in cell physiology, leading to new perspective in different therapeutic fields.

Mechanisms of recovery from type 2 diabetes after malabsorptive bariatric surgery.

Currently, there are no data in the literature regarding the pathophysiological mechanisms involved in the rapid resolution of type 2 diabetes after bariatric surgery, which was reported as an additional benefit of the surgical treatment for morbid obesity. With this question in mind, insulin sensitivity, using euglycemic-hyperinsulinemic clamp, and insulin secretion, by the C-peptide deconvolution method after an oral glucose load, together with the circulating levels of intestinal incretins and adipocytokines, have been studied in 10 diabetic morbidly obese subjects before and shortly after biliopancreatic diversion (BPD) to avoid the weight loss interference. Diabetes disappeared 1 week after BPD, while insulin sensitivity (32.96 ± 4.3 to 65.73 ± 3.22 μmol · kg fat-free mass−1 · min−1 at 1 week and to 64.73 ± 3.42 μmol · kg fat-free mass−1 · min−1 at 4 weeks; P < 0.0001) was fully normalized. Fasting insulin secretion rate (148.16 ± 20.07 to 70.0.2 ± 8.14 and 83.24 ± 8.28 pmol/min per m2; P < 0.01) and total insulin output (43.76 ± 4.07 to 25.48 ± 1.69 and 30.50 ± 4.71 nmol/m2; P < 0.05) dramatically decreased, while a significant improvement in β-cell glucose sensitivity was observed. Both fasting and glucose-stimulated gastrointestinal polypeptide (13.40 ± 1.99 to 6.58 ± 1.72 pmol/l at 1 week and 5.83 ± 0.80 pmol/l at 4 weeks) significantly (P < 0.001) decreased, while glucagon-like peptide 1 significantly increased (1.75 ± 0.16 to 3.42 ± 0.41 pmol/l at 1 week and 3.62 ± 0.21 pmol/l at 4 weeks; P < 0.001). BPD determines a prompt reversibility of type 2 diabetes by normalizing peripheral insulin sensitivity and enhancing β-cell sensitivity to glucose, these changes occurring very early after the operation. This operation may affect the enteroinsular axis function by diverting nutrients away from the proximal gastrointestinal tract and by delivering incompletely digested nutrients to the ileum.

Effects of dietary fatty acids on insulin sensitivity and secretion

Globalization and global market have contributed to increased consumption of high‐fat, energy‐dense diets, particularly rich in saturated fatty acids( SFAs). Polyunsaturated fatty acids (PUFAs) regulate fuel partitioning within the cells by inducing their own oxidation through the reduction of lipogenic gene expression and the enhancement of the expression of those genes controlling lipid oxidation and thermogenesis. Moreover, PUFAs prevent insulin resistance by increasing membrane fluidity and GLUT4 transport. In contrast, SFAs are stored in non‐adipocyte cells as triglycerides (TG) leading to cellular damage as a sequence of their lipotoxicity. Triglyceride accumulation in skeletal muscle cells (IMTG) derives from increased FA uptake coupled with deficient FA oxidation. High levels of circulating FAs enhance the expression of FA translocase the FA transport proteins within the myocites. The biochemical mechanisms responsible for lower fatty acid oxidation involve reduced carnitine palmitoyl transferase (CPT) activity, as a likely consequence of increased intracellular concentrations of malonyl‐CoA; reduced glycogen synthase activity; and impairment of insulin signalling and glucose transport. The depletion of IMTG depots is strictly associated with an improvement of insulin sensitivity, via a reduced acetyl‐CoA carboxylase (ACC) mRNA expression and an increased GLUT4 expression and pyruvate dehydrogenase (PDH) activity. In pancreatic islets, TG accumulation causes impairment of insulin secretion. In rat models, β‐cell dysfunction is related to increased triacylglycerol content in islets, increased production of nitric oxide, ceramide synthesis and β‐cell apoptosis. The decreased insulin gene promoter activity and binding of the pancreas‐duodenum homeobox‐1 (PDX‐1) transcription factor to the insulin gene seem to mediate TG effect in islets. In humans, acute and prolonged effects of FAs on glucose‐stimulated insulin secretion have been widely investigated as well as the effect of high‐fat diets on insulin sensitivity and secretion and on the development of type 2 diabetes.

Acetylcarnitine induces heme oxygenase in rat astrocytes and protects against oxidative stress: involvement of the transcription factor Nrf2.

Efficient functioning of maintenance and repair processes seem to be crucial for both survival and physical quality of life. This is accomplished by a complex network of the so‐called longevity assurance processes, under control of several genes termed vitagenes. These include members of the heat shock protein system, and there is now evidence that the heat shock response contributes to establishing a cytoprotective state in a wide variety of human conditions, including inflammation, neurodegenerative disorders, and aging. Among the various heat shock proteins, heme oxygenase‐1 has received considerable attention; it has been recently demonstrated that heme oxygenase‐1 induction, by generating the vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, could represent a protective system potentially active against brain oxidative injury. Acetyl‐L‐carnitine is proposed as a therapeutic agent for several neurodegenerative disorders. Accordingly, we report here that treatment of astrocytes with acetyl‐L‐carnitine induces heme oxygenase‐1 in a dose‐ and time‐dependent manner and that this effect was associated with up‐regulation of heat shock protein 60 as well as high expression of the redox‐sensitive transcription factor Nrf2 in the nuclear fraction of treated cells. In addition, we show that addition of acetyl‐L‐carnitine to astrocytes, prior to proinflammatory lipopolysaccharide‐ and interferon‐γ‐induced nitrosative stress, prevents changes in mitochondrial respiratory chain complex activity, protein nitrosation and antioxidant status induced by inflammatory cytokine insult. Given the broad cytoprotective properties of the heat shock response, molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. Particularly, manipulation of endogenous cellular defense mechanisms via acetyl‐L‐carnitine may represent an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration. We hypothesize that maintenance or recovery of the activity of vitagenes may delay the aging process and decrease the risk of age‐related diseases.

Acetyl-L-carnitine-induced up-regulation of heat shock proteins protects cortical neurons against amyloid-beta peptide 1–42-mediated oxidative stress and neurotoxicity: Implications for Alzheimer's disease

Alzheimers disease (AD) is a progressive neurodegenerative disorder characterized by loss of memory and cognition and by senile plaques and neurofibrillary tangles in brain. Amyloid‐beta peptide, particularly the 42‐amino‐acid peptide (Aβ1–42), is a principal component of senile plaques and is thought to be central to the pathogenesis of the disease. The AD brain is under significant oxidative stress, and Aβ1–42 peptide is known to cause oxidative stress in vitro and in vivo. Acetyl‐L‐carnitine (ALCAR) is an endogenous mitochondrial membrane compound that helps to maintain mitochondrial bioenergetics and lowers the increased oxidative stress associated with aging. Glutathione (GSH) is an important endogenous antioxidant, and its levels have been shown to decrease with aging. Administration of ALCAR increases cellular levels of GSH in rat astrocytes. In the current study, we investigated whether ALCAR plays a protective role in cortical neuronal cells against Aβ1–42‐mediated oxidative stress and neurotoxicity. Decreased cell survival in neuronal cultures treated with Aβ1–42 correlated with an increase in protein oxidation (protein carbonyl, 3‐nitrotyrosine) and lipid peroxidation (4‐hydroxy‐2‐nonenal) formation. Pretreatment of primary cortical neuronal cultures with ALCAR significantly attenuated Aβ1–42‐induced cytotoxicity, protein oxidation, lipid peroxidation, and apoptosis in a dose‐dependent manner. Addition of ALCAR to neurons also led to an elevated cellular GSH and heat shock proteins (HSPs) levels compared with untreated control cells. Our results suggest that ALCAR exerts protective effects against Aβ1–42 toxicity and oxidative stress in part by up‐regulating the levels of GSH and HSPs. This evidence supports the pharmacological potential of acetyl carnitine in the management of Aβ1–42‐induced oxidative stress and neurotoxicity. Therefore, ALCAR may be useful as a possible therapeutic strategy for patients with AD.

Insulin Clearance in Obesity

Insulin uptake and degradation is a complex and not yet completely understood process involving not only insulin sensitive tissues. The most important degradative system is insulin degrading enzyme which is a highly conserved metalloendopeptidase requiring Zn++ for its proteolytic action, although protein disulfide isomerase and cathepsin D are also involved in insulin metabolism. The liver and the kidney are the principal sites for insulin clearance. In obese subjects with hyperinsulinemia and high levels of free fatty acids, insulin hepatic clearance is impaired, while the glomerular filtration rate, renal plasma flow and albumin excretion are increased, suggesting a state of renal vasodilatation leading to an abnormally transmitted arterial pressure to the glomerular capillaries through a dilated afferent arteriole. Insulin can be cleared also by muscle, adipocytes, gastrointestinal cells, fibroblasts, monocytes and lymphocytes which contain insulin receptors and internalization and regulation mechanism for insulin metabolism.

Cancer and anticancer therapy-induced modifications on metabolism mediated by carnitine system.

An efficient regulation of fuel metabolism in response to internal and environmental stimuli is a vital task that requires an intact carnitine system. The carnitine system, comprehensive of carnitine, its derivatives, and proteins involved in its transformation and transport, is indispensable for glucose and lipid metabolism in cells. Two major functions have been identified for the carnitine system: (1) to facilitate entry of long‐chain fatty acids into mitochondria for their utilization in energy‐generating processes; (2) to facilitate removal from mitochondria of short‐chain and medium‐chain fatty acids that accumulate as a result of normal and abnormal metabolism. In cancer patients, abnormalities of tumor tissue as well as nontumor tissue metabolism have been observed. Such abnormalities are supposed to contribute to deterioration of clinical status of patients, or might induce cancerogenesis by themselves. The carnitine system appears abnormally expressed both in tumor tissue, in such a way as to greatly reduce fatty acid beta‐oxidation, and in nontumor tissue. In this view, the study of the carnitine system represents a tool to understand the molecular basis underlying the metabolism in normal and cancer cells. Some important anticancer drugs contribute to dysfunction of the carnitine system in nontumor tissues, which is reversed by carnitine treatment, without affecting anticancer therapeutic efficacy. In conclusion, a more complex approach to mechanisms that underlie tumor growth, which takes into account the altered metabolic pathways in cancer disease, could represent a challenge for the future of cancer research. J. Cell. Physiol. 182:339–350, 2000.

Regulation by carnitine of myocardial fatty acid and carbohydrate metabolism under normal and pathological conditions

Abstract This review focuses on the regulation of myocardial fatty acids and glucose metabolism is physiological and pathological conditions, and the role of L-carnitine and of its derivative, propionyl-L-carnitine.Fatty acids are the major oxidation fuel for the heart, while glucose and lactate provide the remaining need. Fatty acids in cytoplasm are transformed to long-chain acyl-CoA and transferred into the mitochondrial matrix by the action of three carnitine dependent enzymes to produce acetyl-CoA through the β-oxidaton pathway. Another source of mitochondrial acetyl-CoA is from the oxidation of carbohydrates. The pyruvate dehydrogenase (PDH) complex, the key irreversible rate limiting step in carbohydrate oxidation, is modulated by the intra-mitochondrial ratio acetyl-CoA/CoA. An increased ratio results in the inhibition of PDH activity. A decreased ratio can relieve the inhibition of PDH as shown by the transfer of acetyl groups from acetyl-CoA to carnitine, forming acetylcarnitine, a reaction catalyzed by carnitine acetyl-transferase. This activity of L-carnitine in the modulation of the intramitochondrial acety-CoA/CoA ratio affects glucose oxidation.Myocardial substrate metabolism during ischemia is dependent upon the severity of ischemia. A very severe reduction of blood flow causes a decrease of substrate flux through PDH. When perfusion is only partially reduced there is an increase in the rate of glycolysis and a switch from lactate uptake to lactate production. Tissue levels of acyl-CoA and long-chain acylcarnitine increase with important functional consequences on cell membranes. During reperfusion fatty acids oxidation quickly recovers as the prevailing source of energy, while pyruvate oxidation is inhibited.A considerable body of experimental evidence suggests that L-carnitine exert a protective effect in in vitro and in vivo models of heart ischemia and hypertrophy. Clinical trials confirm these beneficial effects although controversial results are observed. The actions of L-carnitine and propionyl-L-carnitine cannot be explained as exclusively dependent on the stimulation of fatty acid oxidation but rather on a marked increase in glucose oxidation, via a relief of PDH inhibition caused by the elevated acetyl-CoA/CoA ratio. Enhanced pyruvate flux through PDH is beneficial for the cardial cells since less pyruvate is converted to lactate, a metabolic step resulting in the acidification of the intracellular compartment. In addition, L-carnitine decreases tissue levels of acyl moieties, a mechanism particularly important in the ischemic phase.

Protective actions of l-carnitine and acetyl-l-carnitine on the neurotoxicity evoked by mitochondrial uncoupling or inhibitors

The mechanism for the pathological increase in cell death in various disease states e.g. HIV immunodefficiency or even ageing or Alzheimers disease, occurs by complex and as yet undefined mechanism(s) related to immunological, virological or biochemical disturbances (i.e. energy depletion, oxidative stress, increased protein degradation). We have studied mitochondrial uncoupling or inhibitor toxicity on neurones at the cellular level and at the mitochondrial level using rhodamine (Rh123) and 10-nonylacridine orange (NAO) fluorescence with confocal microscopy. Blockade of the mitochondrial chain complexes at various points was studied. The possible protective effects of the compound L-carnitine, which plays a central role in mitochondrial function, was tested in this form of neurotoxicity. It appears that L-carnitine and its acetylated form, acetyl-L-carnitine, can attenuate the cell damage, as assessed by lactate dehydrogenase (LDH) release, evoked by the uncoupler, p-(trifluoromethoxy)phenylhdyrazone (FCCP), or by the inhibitors, 3-nitropropionic acid (3-NPA) or rotenone. Further, the FCCP-induced inhibition of Rh123 uptake was antagonized by the preincubation of cells with L-carnitine. Since such neurotoxic mechanisms may be operating in the various pathological forms of myotoxicity and neurotoxicity, these observations suggest potential for a therapeutic approach.