Elena Donetti

Inhibition of Class I Histone Deacetylases Unveils a Mitochondrial Signature and Enhances Oxidative Metabolism in Skeletal Muscle and Adipose Tissue

Chromatin modifications are sensitive to environmental and nutritional stimuli. Abnormalities in epigenetic regulation are associated with metabolic disorders such as obesity and diabetes that are often linked with defects in oxidative metabolism. Here, we evaluated the potential of class-specific synthetic inhibitors of histone deacetylases (HDACs), central chromatin-remodeling enzymes, to ameliorate metabolic dysfunction. Cultured myotubes and primary brown adipocytes treated with a class I–specific HDAC inhibitor showed higher expression of Pgc-1α, increased mitochondrial biogenesis, and augmented oxygen consumption. Treatment of obese diabetic mice with a class I– but not a class II–selective HDAC inhibitor enhanced oxidative metabolism in skeletal muscle and adipose tissue and promoted energy expenditure, thus reducing body weight and glucose and insulin levels. These effects can be ascribed to increased Pgc-1α action in skeletal muscle and enhanced PPARγ/PGC-1α signaling in adipose tissue. In vivo ChIP experiments indicated that inhibition of HDAC3 may account for the beneficial effect of the class I–selective HDAC inhibitor. These results suggest that class I HDAC inhibitors may provide a pharmacologic approach to treating type 2 diabetes.

An over-oxidized form of superoxide dismutase found in sporadic amyotrophic lateral sclerosis with bulbar onset shares a toxic mechanism with mutant SOD1

Recent studies suggest that Cu/Zn superoxide dismutase (SOD1) could be pathogenic in both familial and sporadic amyotrophic lateral sclerosis (ALS) through either inheritable or nonheritable modifications. The presence of a misfolded WT SOD1 in patients with sporadic ALS, along with the recently reported evidence that reducing SOD1 levels in astrocytes derived from sporadic patients inhibits astrocyte-mediated toxicity on motor neurons, suggest that WT SOD1 may acquire toxic properties similar to familial ALS-linked mutant SOD1, perhaps through posttranslational modifications. Using patients’ lymphoblasts, we show here that indeed WT SOD1 is modified posttranslationally in sporadic ALS and is iper-oxidized (i.e., above baseline oxidation levels) in a subset of patients with bulbar onset. Derivatization analysis of oxidized carbonyl compounds performed on immunoprecipitated SOD1 identified an iper-oxidized SOD1 that recapitulates mutant SOD1-like properties and damages mitochondria by forming a toxic complex with mitochondrial Bcl-2. This study conclusively demonstrates the existence of an iper-oxidized SOD1 with toxic properties in patient-derived cells and identifies a common SOD1-dependent toxicity between mutant SOD1-linked familial ALS and a subset of sporadic ALS, providing an opportunity to develop biomarkers to subclassify ALS and devise SOD1-based therapies that go beyond the small group of patients with mutant SOD1.

Non-Lipid-Related Effects of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors

With the increasing knowledge of the pathogenesis of atherosclerosis, it appears that in the future the prevention of cardiovascular disease will involve not only risk factor correction, but also direct pharmacological control of processes occurring in the arterial wall. Among these, a pivotal role is played by smooth muscle cell (SMC) migration and proliferation, which, together with lipid deposition, are prominent features of atherogenesis and restenosis after angioplasty. Mevalonate and other intermediates of cholesterol synthesis (isoprenoids) are essential for cell growth, hence drugs affecting this metabolic pathway are potential antiatherosclerotic agents. Recently, we provided in vitro and in vivo evidence that fluvastatin, simvastatin and lovastatin, but not pravastatin, decrease SMC migration and proliferation dose dependently, independently of their hypocholesterolemic properties. The in vitro inhibition of cell migration and proliferation induced by simvastatin and fluvastatin (70-90% decrease) was prevented completely by the addition of mevalonate, and partially prevented by farnesol and geranylgeraniol (80%), confirming the specific role of isoprenoid metabolites in regulating these cellular events, probably through prenylated protein(s). The in vivo antiproliferative activity of fluvastatin on neointimal hyperplasia in normocholesterolemic rabbits was also prevented fully by the local delivery of mevalonate, by means of an Alzet pump. Fluvastatin and simvastatin also inhibited cholesterol esterification and deposition induced by acetylated LDL in cultured macrophages. This effect was fully prevented by the addition of mevalonate or geranylgeraniol. Taken together, these results suggest that, beyond their effects on plasma lipids, HMG-CoA reductase inhibitors exert a direct antiatherosclerotic effect on the arterial wall, probably through local inhibition of isoprenoid biosynthesis.

Caseinphosphopeptide-induced calcium uptake in human intestinal cell lines HT-29 and Caco2 is correlated to cellular differentiation.

Caseinphosphopeptides (CPPs) are considered as mineral carriers because of their ability to bind and solubilize calcium ions, with the possible role, yet to be definitely assessed, of improving calcium absorption at the intestinal level. Previous works demonstrated that CPPs improve calcium uptake, with increasing intracellular calcium concentration, by human differentiated tumor HT-29 cells, and that this effect correlates with the supramolecular structure of CPPs in the presence of calcium ions. The aim of the present study was to establish whether the CPP effect on calcium uptake is specific for HT-29 cells and depends on the differentiated state of the cells. To this purpose, HT-29 and Caco2 cells, two models of intestinal cells, were differentiated following appropriate protocols, including treatment with 1,25-(OH)2 vitamin D3. The CPP-dependent intracellular calcium rises were monitored at the single-cell level through fura2-fluorescence assays, and cell differentiation was assessed by biochemical and morphological methods. Results clearly showed that the ability to take up extracellular calcium ions under CPP stimulation is exhibited by both HT-29 and Caco2 cells, but only upon cell differentiation. This evidence adds novel support to the notion that CPPs favour calcium absorption, thus possibly acting as cellular bio-modulators and carrying a nutraceutical potential.

Sperm Protein 17 Is Expressed in Human Somatic Ciliated Epithelia

It was once believed that sperm protein 17 (Sp17) was expressed exclusively in the testis and that its sole function was to bind to the oocyte during fertilization. However, immunohistochemistry of the human respiratory airways and reproductive systems show that it is abundant in ciliated cells but not in human cells with stereocilia and microvilli. The high degree of sequence conservation throughout its N-terminal half, and the presence of an A-kinase anchoring protein (AKAP)-binding motif within this region, suggest that Sp17 plays a regulatory role in a PKA-independent AKAP complex in both male germinal and ciliated somatic cells.

Casein phosphopeptide promotion of calcium uptake in HT‐29 cells − relationship between biological activity and supramolecular structure

Casein phosphopeptides (CPPs) form aggregated complexes with calcium phosphate and induce Ca2+ influx into HT‐29 cells that have been shown to be differentiated in culture. The relationship between the aggregation of CPPs assessed by laser light scattering and their biological effect was studied using the CPPs β‐CN(1–25)4P and αs1‐CN(59–79)5P, the commercial mixture CPP DMV, the ‘cluster sequence’ pentapeptide, typical of CPPs, and dephosphorylated β‐CN(1–25)4P, [β‐CN(1–25)0P]. The biological effect was found to be: (a) maximal with β‐CN(1–25)4P and null with the ‘cluster sequence’; (b) independent of the presence of inorganic phosphate; and (c) maximal at 4 mmol·L−1 Ca2+. The aggregation of CPP had the following features: (a) rapid occurrence; (b) maximal aggregation by β‐CN(1–25)4P with aggregates of 60 nm hydrodynamic radius; (c) need for the concomitant presence of Ca2+ and CPP for optimal aggregation; (d) lower aggregation in Ca2+‐free Krebs/Ringer/Hepes; (e) formation of bigger aggregates (150 nm radius) with β‐CN(1–25)0P. With both β‐CN(1–25)4P and CPP DMV, the maximum biological activity and degree of aggregation were reached at 4 mmol·L−1 Ca2+.

Inhibition of isoprenoid biosynthesis and arterial smooth-muscle cell proliferation.

Recently, we provided in vitro and in vivo evidence that several vastatins with different potencies decrease arterial smooth-muscle cell (SMC) proliferation independently of their hypocholesterolemic properties. In this study, the in vivo dose-dependent antiproliferative activity of fluvastatin on neointimal formation induced by the insertion of a collar around one carotid artery was investigated in normocholesterolemic rabbits (five animals per treatment group). Intraperitoneal fluvastatin treatment progressively inhibited intimal to medial tissue ratios (I/M) by 5, 48, and 64% versus controls at doses of 3, 5, or 10 mg/kg/day, respectively. Local arterial delivery by an Alzet pump of mevalonate (8 mg/kg/day) at the site of collar placement fully prevented a fluvastatin (5 mg/kg/day) inhibitory effect on both I/M and SMC proliferation, as assessed by direct incorporation of bromodeoxyuridine (BrdU) into replicating DNA. The results suggest that vastatins exert a direct antiproliferative effect on intimal myocytes beyond their effects on plasma lipids, probably through local inhibition of isoprenoid biosynthesis.

Effect of lercanidipine and its (R)-enantiomer on atherosclerotic lesions induced in hypercholesterolemic rabbits.

1 The in vivo antiatherogenic activity of the calcium antagonist lercanidipine and its (R)‐enantiomer was investigated in two different types of atherosclerotic lesions (hyperplastic and fatty‐streak lesions) in rabbits. 2 Lercanidipine (0.3, 1, and 3 mg kg−1 week−1) as well as its (R)‐enantiomer at 3 mg kg−1 week−1 were given by subcutaneous injection for 10 weeks to White New Zealand rabbits, with cholesterol feeding beginning at week 2. The hyperplastic lesion was obtained by positioning a hollow silastic collar around one carotid artery, while aortic fatty streak lesions were induced by cholesterol feeding. In untreated animals (n=5), 14 days after collar positioning an intimal hyperplasia was clearly detectable: the arteries without collar showed a intima/media (I/M) ratio of 0.03±0.02, whereas in carotids with a collar the ratio was 2±0.42. In lercanidipine‐treated animals a significant and dose‐dependent effect on intimal hyperplasia was observed. I/M ratios were 0.73±0.4, 0.42±0.1, 0.32±0.1 for 0.3, 1, and 3 mg kg−1 week−1, respectively (P<0.05). The lercanidipine enantiomer (3 mg kg−1 week−1) was as effective as the racemate (0.41±0.11). Proliferation of smooth muscle cells, assessed by incorporation of BrdU into DNA, was reduced by about 50%, 70%, 85%, and 80% by lercanidipine (0.3, 1, and 3 mg kg−1 week−1) and its (R)‐enantiomer, respectively. 3 The area of fatty‐streaks in the aorta (n=11–15) was significantly reduced by lercanidipine (3 mg kg−1 week−1, 16% vs 27%, P<0.05), a trend was observed also with lower doses. When different segments of the aorta were considered (arch, thoracic, abdominal) a significant and dose‐dependent effect in the thoracic and abdominal aorta was observed also at lower doses. The (R)‐enantiomer was as effective as lercanidipine. 4 These results suggest a direct antiatherosclerotic effect of lercanidipine, independent of modulation of risk factors such as hypercholesterolemia and/or hypertension as demonstrated by the absence of stereoselectivity.

Sperm protein 17 is expressed in the sperm fibrous sheath

BackgroundSperm protein 17 (Sp17) is a highly conserved mammalian protein characterized in rabbit, mouse, monkey, baboon, macaque, human testis and spermatozoa. mRNA encoding Sp17 has been detected in a range of murine and human somatic tissues. It was also recognized in two myeloma cell lines and in neoplastic cells from patients with multiple myeloma and ovarian carcinoma. These data all indicate that Sp17 is widely distributed in humans, expressed not only in germinal cells and in a variety of somatic tissues, but also in neoplastic cells of unrelated origin.MethodsSp17 expression was analyzed by immunocytochemistry and transmission electron microscopy on spermatozoa.ResultsHere, we demonstrate the ultrastructural localization of human Sp17 throughout the spermatozoa flagellar fibrous sheath, and its presence in spermatozoa during in vitro states from their ejaculation to the oocyte fertilization.ConclusionThese findings suggest a possible role of Sp17 in regulating sperm maturation, capacitation, acrosomal reaction and interactions with the oocyte zona pellucida during the fertilization process. Further, the high degree of sequence conservation throughout its N-terminal half, and the presence of an A-kinase anchoring protein (AKAP)-binding motif within this region, suggest that Sp17 might play a regulatory role in a protein kinase A-independent AKAP complex in both germinal and somatic cells.

Dual effects of the antioxidant agents probucol and carvedilol on proliferative and fatty lesions in hypercholesterolemic rabbits

The in vivo direct antiatherogenic activity of the antioxidant probucol (200 mg/kg per day) or the beta-blocker with antioxidant properties carvedilol (10 and 20 mg/kg per day) was tested in the same animal in two different types of atherosclerotic lesion (proliferative and fatty lesions) induced in cholesterol-fed rabbits (1%). Drugs were given daily mixed with standard diet for 8 weeks; body weight and plasma lipid profile were not different among groups throughout the study. Aortic fatty lesions were induced by cholesterol feeding (n = 25 in each group) and their extent expressed as % of aorta inner surface covered by plaques was significantly reduced by both drugs (28.2+/-9.6%, P <0.05, 19.9+/-6.2%, P <0.01 for low- and high-dose carvedilol, respectively; 22.3+/-7.6%, P <0.01 for probucol, versus 41.6+/-10.7% in control rabbits). Proliferative lesions were obtained by positioning a hollow silastic collar around one carotid artery 6 weeks after dietary and drug treatments started (n = 5 in each group). The neointimal formation, mostly composed by myocytes, was determined by measuring cross-sectional thickness ratio of intimal (I) and medial (M) tissue of fixed arteries. In untreated animals, collared arteries resulted in a significant neointimal cell accumulation compared to the sham (1.10+/-0.14 versus 0.02+/-0.01) without change in medial thickness. I/M ratio was reduced by about 50% in animals treated with probucol (0.51+/-0.1) and carvedilol (0.66+/-0.21 and 0.52+/-0.1 in the low- and high-dose group, respectively). Total plasma TBARS were more than 50% lower in both probucol- and high-dose carvedilol-treated rabbits. Results show that pharmacological pretreatment with antioxidants directly inhibits early atherogenic processes, representing a potentially useful approach in the prevention of atherosclerosis.